muramidase and Amyloidosis

Lysozyme amyloidosis is a non-neuropathic hereditary amyloidosis identified in 1993. About fifty cases of this rare, probably under-diagnosed disease are reported. Lysozyme amyloidosis has a very broad spectrum of clinical manifestations. Sicca syndrome is often the first symptom, preceding the diagnosis by several years. Every part of the digestive tract can be involved with different grades of severity. The hallmark of this amyloidosis is the usually life-threatening spontaneous hepatic rupture. Renal involvement is frequent and progresses towards end-stage renal failure and dialysis. Skin, lymph nodes, and spleen can also be affected. More recently, cardiac and pulmonary involvement was reported. Phenotypic heterogeneity and incomplete penetrance make the clinical diagnosis difficult. Amyloid deposits are revealed by Congo red staining with birefringence under polarized light. They can be limited or diffuse and lead to the progressive destruction of the architecture of an organ and its failure. Immunohistochemistry reveals the nature of the amyloid variant by identifying antilysozyme antibodies in the deposit. Up to know, eight pathologic mutations and one polymorphism involving exons 2, 3, and 4 of the lysozyme gene have been identified. The transmission is autosomal dominant, without any genotype-phenotype correlation. The therapeutic options are limited and based on symptomatic or supportive treatment. Renal and hepatic transplant has proved its benefits with a prolonged graft survival. A long term regular and multidisciplinary follow-up is required.

Topics: Amyloidosis; Diagnosis, Differential; Genetic Testing; Humans; Immunohistochemistry; Muramidase

Protein misfolding and aggregation are implicated in a wide range of increasingly prevalent human diseases ranging from dementia to diabetes. In this review we discuss the current experimental strategies that are being employed in the investigation of the pathogenesis of three important protein misfolding disorders. The first, Alzheimer's disease (AD), is the most prevalent neurodegenerative disease and is thought to be initiated by the aggregation of a natively unstructured peptide called amyloid beta (Abeta). We discuss methods for the characterization of the aggregation properties of Abeta in vitro and how the results of such experiments can be correlated with data from animal models of disease. We then consider another form of amyloidosis, where a systemic distribution of amyloid deposit is caused by aggregation and deposition of mutational variants of lysozyme. We describe how experiments in vitro, and more recently in vivo, have provided insights into the origins of this disease. Finally we outline the varied paradigms that have been employed in the study of the serpinopathies, and in particular, a dementia caused by neuroserpin polymerization.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Animals; Circular Dichroism; Humans; Microscopy, Electron, Transmission; Muramidase; Protein Conformation; Protein Folding

Autosomal dominant hereditary amyloidosis represents not 1 disease but a group of diseases, each the result of mutations in a specific protein. The most common form is transthyretin amyloidosis, which has been recognized clinically for over 50 years as a familial polyneuropathy. Nonneuropathic amyloidoses (Ostertag type amyloidosis) include those due to abnormalities in lysozyme, fibrinogen Aalpha-chain, and apolipoprotein A-I and A-II. The role of lysozyme in amyloid-related human disorders was first described in 1993; to date, there have been only 9 publications describing this disorder, which is a nonneuropathic form of hereditary amyloidosis. Reported cases have involved 7 unrelated families. We describe here our own experience with 4 families suffering from lysozyme amyloidosis: the first had prominent renal manifestations with sicca syndrome, the second and third had prominent gastrointestinal symptoms, and the fourth had a dramatic bleeding event due to rupture of abdominal lymph nodes. To our knowledge, this last symptom has not been reported previously, but is reminiscent of the hepatic hemorrhage seen in a previously reported case of a patient with lysozyme amyloidosis. To characterize the manifestations of this disorder, we performed an exhaustive literature review.Although hereditary amyloidosis is thought to be a rare disease, it is probably not as rare as we think and may well be underdiagnosed. Moreover, some cases of lysozyme amyloidosis are probably confused with acquired monoclonal immunoglobulin light-chain (AL) amyloidosis, formerly known as primary amyloidosis, which is the most frequent type of amyloidosis. Because treatment for each type of amyloidosis is different, and because therapy directed at 1 type may worsen symptoms of the other types, it is important to determine precisely the nature of the amyloid protein. Thus, hereditary lysozyme amyloidosis should be considered in all patients with systemic amyloidosis, particularly in patients who present with renal, gastrointestinal, or bleeding complications without evidence of AL or AA (secondary) amyloidoses.

Topics: Adult; Aged; Amyloidosis; Female; Gastrointestinal Diseases; Hemorrhage; Humans; Kidney Diseases; Lymph Nodes; Male; Middle Aged; Muramidase; Rupture

The ability of proteins to fold to their functional states following synthesis in the intracellular environment is one of the most remarkable features of biology. Substantial progress has recently been made towards understanding the fundamental nature of the mechanism of the folding process. This understanding has been achieved through the development and concerted application of a variety of novel experimental and theoretical approaches to this complex problem. The emerging view of folding is that it is a stochastic process, but one biased by the fact that native-like interactions between residues are, on average, more stable than non-native ones. The sequences of natural proteins have emerged through evolutionary processes such that their unique native states can be found very efficiently even in the complex environment inside a living cell. But under some conditions proteins fail to fold correctly, or to remain correctly folded, in living systems, and this failure can result in a wide range of diseases. One group of diseases, known as amyloidoses, which includes Alzheimer's disease and the transmissible spongiform encephalopathies, involves deposition of aggregated proteins in a variety of tissues. These diseases are particularly intriguing because evidence is accumulating that the formation of the highly organized amyloid aggregates is a generic property of polypeptides, and not simply a feature of the few proteins associated with recognized pathological conditions. That such aggregates are not normally found in properly functional biological systems is again a testament to evolution, in this case of a variety of mechanisms inhibiting their formation. Understanding the nature of such protective mechanisms is a crucial step in the development of strategies to prevent and treat these debilitating diseases.

Topics: Amyloid; Amyloidosis; Disease; Humans; Macromolecular Substances; Models, Molecular; Muramidase; Protein Folding

Topics: Amyloidosis; Apolipoproteins A; Cardiomyopathies; Dialysis; Fibrinogen; Humans; Kidney Diseases; Liver Diseases; Muramidase; Organ Transplantation; Prealbumin

The physiological metabolism of proteins guarantees that different cellular compartments contain the appropriate concentration of proteins to perform their biological functions and, after a variable period of wear and tear, mediates their natural catabolism. The equilibrium between protein synthesis and catabolism ensures an effective turnover, but hereditary or acquired abnormalities of protein structure can provoke a premature loss of biological function, an accelerated catabolism and diseases caused by the loss of an irreplaceable function. In certain proteins, abnormal structure and metabolism are associated with a strong tendency to self-aggregation into a polymeric fibrillar structure, and in these cases the disease is not principally caused by the loss of an irreplaceable function but by the action of this new biological entity. Amyloid fibrils are an apparently inert, insoluble, mainly extracellular protein polymer that kills the cell without tissue necrosis but by activation of the apoptotic mechanism. We analyzed the data reported so far on the structural and functional properties of four prototypic proteins with well-known biological functions (lysozyme, transthyretin, beta 2-microglobulin and apolipoprotein AI) that are able to create amyloid fibrils under certain conditions, with the perspective of evaluating whether the achievement of biological function favors or inhibits the process of fibril formation. Furthermore, studying the biological functions carried out by amyloid fibrils reveals new types of protein-protein interactions in the transmission of messages to cells and may provide new ideas for effective therapeutic strategies.

Topics: Alzheimer Disease; Amino Acid Substitution; Amyloid; Amyloidosis; Animals; Antigen-Presenting Cells; Apolipoprotein A-I; beta 2-Microglobulin; Biopolymers; Humans; Models, Molecular; Muramidase; Nerve Tissue Proteins; Prealbumin; Protein Conformation; Protein Folding; Renal Dialysis; Structure-Activity Relationship

The conformational change hypothesis postulates that tertiary structural changes under partially denaturing conditions convert one of 17 normally soluble and functional human proteins into an alternative conformation that subsequently undergoes self-assembly into an amyloid fibril, the putative causative agent in amyloid disease. This hypothesis is consistent with Anfinsen's view that the tertiary structure of a protein is determined both by its sequence and the aqueous environment; the latter does not always favor the normally folded state. Unlike sickle cell hemoglobin assembly, where owing to a surface mutation, hemoglobin polymerizes in its normally folded conformation, amyloid proteins self-assemble as a result of the formation of an alternative tertiary structure-a conformational intermediate formed under partially denaturing conditions. The pathway by which an amyloidogenic protein assembles into amyloid fibrils appears to involve quaternary structural intermediates that assemble into increasingly complex quaternary structures, including amyloid protofilaments, which ultimately assemble into amyloid fibrils. Several recent studies have discussed the multi-step assembly pathway(s) characterizing amyloid fibril formation.

Topics: Amyloid; Amyloid beta-Peptides; Amyloid Neuropathies; Amyloidosis; Humans; Muramidase; Prealbumin; Protein Conformation; Protein Folding; Protein Structure, Tertiary

Amyloidoses are diseases, including some currently prominent such as Alzheimer's disease, bovine spongiform encephalophaty (BSE) and Type II diabetes, in which soluble proteins are deposited in a specific, highly stable, fibrillar form. The amyloid fibrils are made up of protofilaments whose molecular structure is composed of pairs of beta-sheets in a helical form that allows them to be continuously hydrogen-bonded along the length of the fibril. The observation that similar fibrils are generated from different proteins indicates that fibril formation is accompanied by structural conversion. The transmissible spongiform encephalopathies, such as BSE and kuru, involve an infectious agent identified with the prion protein. The properties of the agent are more consistent with prion amyloid than the protein itself, suggesting infectivity in these diseases in equivalent to the 'seeding' of amyloid fibrils at a new site.

Topics: Amyloidosis; Animals; Cattle; Encephalopathy, Bovine Spongiform; Humans; Microscopy, Electron; Models, Molecular; Muramidase; Prealbumin; Protein Structure, Secondary; Scrapie; X-Ray Diffraction

Recent publications strongly support the hypothesis that conformational changes in amyloidogenic proteins lead to amyloid fibril formation and cause disease. Biophysical studies on several amyloidogenic proteins provide insights into the conformational changes required for fibrilogenesis. In addition, newly available moderate to high resolution structural studies are bringing us closer to understanding the structure of amyloid.

Topics: Adult; Aged; Aged, 80 and over; Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Amyloid Neuropathies; Amyloidosis; Animals; Humans; Immunoglobulin Light Chains; Models, Molecular; Muramidase; Prealbumin; Protein Conformation; Protein Folding; Structure-Activity Relationship

Topics: Amyloidosis; Bromelains; Chymotrypsin; Dimethyl Sulfoxide; Humans; Intestinal Absorption; Molecular Weight; Muramidase; Nervous System Diseases

Topics: Amyloidosis; Blood Vessels; Diabetes Insipidus; Humans; Hypercalcemia; Hyponatremia; Kidney; Kidney Diseases; Lactates; Leukemia; Lymphoma; Muramidase; Nephrotic Syndrome; Uric Acid; Urinary Tract

Topics: Amyloidosis; Blood Protein Disorders; Bone Neoplasms; gamma-Globulins; Humans; Karyotyping; Kidney Diseases; Kinetics; Leukemia; Leukemia, Lymphoid; Leukemia, Plasma Cell; Lung Neoplasms; Lymph Nodes; Lymphoma; Microscopy, Electron; Multiple Myeloma; Muramidase; Plasmacytoma

Curcumin has attracted more attention because of its inhibition efficacy on protein amyloid fibrillation. However, the inhibition mechanism was still ambiguous and the clinical application of curcumin was greatly limited because of its poor stability at physiological conditions for the presence of β-diketone moiety. In this paper, a new mono-ketone-containing curcumin analogue (MDHC) was designed and synthesized to realize the possible inhibition mechanism and unveil the important role of β-diketone moiety of curcumin in the inhibition process of amyloid fibrillation using hen egg white lysozyme (HEWL) as model protein. Although all experiment results (ThT, CR, ANS and TEM) showed that the inhibitory capacity of curcumin was better than MDHC, MDHC still could show obvious inhibition effect. Molecular docking showed that both curcumin and MDHC could bind with HEWL by hydrogen bond of phenloic hydroxyl and the binding energy of MDHC was higher than that of curcumin. All the findings inferred that β-diketone group was one of great important groups in the inhibition process of HEWL amyloid fibrillation, which provided more room to construct novel inhibition reagents.

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Animals; Chick Embryo; Curcumin; Egg White; Molecular Docking Simulation; Muramidase

Protein folding is crucial for biological activity. Proteins' failure to fold correctly underlies various pathological processes, including amyloidosis, the aggregation of insoluble proteins (e.g., lysozymes) in organs. The exact conditions that trigger the structural transition of amyloids into β-sheet-rich aggregates are poorly understood, as is the case for the amyloidogenic self-assembly pathway. Ultrasound is routinely used to destabilize a protein's structure and enhance amyloid growth. Here, we report on an unexpected ultrasound effect on lysozyme amyloid species at different stages of aggregation: ultrasound-induced structural perturbation gives rise to nonamyloidogenic folds. Our infrared and X-ray analyses of the chemical, mechanical, and thermal effects of sound on lysozyme's structure found, in addition to the expected ultrasound-induced damage, evidence of irreversible disruption of the β-sheet fold of fibrillar lysozyme resulting in their structural transformation into monomers with no β-sheets. This structural transition is reflected in changes in the kinetics of protein self-assembly, namely, either prolonged nucleation or accelerated fibril growth. Using solution X-ray scattering, we determined the structure, the mass fraction of lysozyme monomer, and the morphology of its filamentous assemblies formed under different sound parameters. A nanomechanical analysis of ultrasound-modified protein assemblies revealed a correlation between the β-sheet content and elastic modulus of the protein material. Suppressing one of the ultrasound-derived effects allowed us to control the structural transformations of lysozyme. Overall, our comprehensive investigation establishes the boundary conditions under which ultrasound damages protein structure and fold. This knowledge can be utilized to impose medically desirable structural modifications on amyloid β-sheet-rich proteins.

Topics: Amyloid; Amyloid beta-Peptides; Amyloidosis; Humans; Muramidase; Protein Folding

Numerous pathophysiological conditions known as amyloidosis, have been connected to protein misfolding leading to aggregation of proteins. Inhibition of cytotoxic aggregates or disaggregation of the preformed fibrils is thus one of the important strategies in the prevention of such diseases. Growing interest and exploration of identification of small molecules mainly natural compounds can prevent or delay amyloid fibril formation. We examined the mechanism of interaction and inhibition of human lysozyme (HL) aggregates with luteolin (LT). Biophysical and computational approaches have been employed to study the effect of LT on HL amyloid aggregation. Transmission Electronic Microscopy, Thioflavin T fluorescence, UV-vis spectroscopy, and RLS demonstrates that LT inhibit HL fibril formation. ANS fluorescence and hemolytic assay was also employed to examine the effect of the LT on toxicity of HL aggregation. Docking and molecular dynamics results showed that LT interacted with HL via hydrophobic and hydrogen interactions, thus reducing fibrillation levels. These findings highlight the benefit of polyphenols as safe therapy for preventing amyloid related diseases.

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Humans; Luteolin; Muramidase

Lysozyme amyloidosis is a systemic non-neuropathic disease caused by the accumulation of amyloids of mutant lysozyme. Presently, therapeutic interventions targeting lysozyme amyloidosis, remain elusive with only therapy available for lysozyme amyloidosis being supportive management. In this work, we examined the effects of moxifloxacin, a synthetic fluoroquinolone antibiotic on the amyloid formation of human lysozyme. The ability of moxifloxacin to interfere with lysozyme amyloid aggregation was examined using various biophysical methods like Rayleigh light scattering, Thioflavin T fluorescence assay, transmission electron microscopy and docking method. The reduction in scattering and ThT fluorescence along with extended lag phase in presence of moxifloxacin, suggest that the antibiotic inhibits and impedes the lysozyme fibrillation in concentration dependent manner. From ANS experiment, we deduce that moxifloxacin is able to decrease the hydrophobicity of the protein molecule thereby preventing aggregation. Our CD and DLS results show that moxifloxacin stabilizes the protein in its native monomeric structure, thus also showing retention of lytic activity upto 69% and inhibition of cytotoxicity at highest concentration of moxifloxacin. The molecular docking showed that moxifloxacin forms a stable complex of -7.6 kcal/mol binding energy and binds to the aggregation prone region of lysozyme thereby stabilising it and preventing aggregation. Moxifloxacin also showed disaggregase potential by disrupting fibrils and decreasing the β-sheet content of the fibrils. Our current study, thus highlight the anti-amyloid and disaggregase property of an antibiotic moxifloxacin and hence sheds light on the future of antibiotics against protein aggregation, a hallmark event in many neurodegenerative diseases.

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Anti-Bacterial Agents; Humans; Molecular Docking Simulation; Moxifloxacin; Muramidase

Systemic AA amyloidosis is a world-wide occurring protein misfolding disease in humans and animals that arises from the formation of amyloid fibrils from serum amyloid A (SAA) protein and their deposition in multiple organs.. To identify new agents that prevent fibril formation from SAA protein and to determine their mode of action.. We used a cell model for the formation of amyloid deposits from SAA protein to screen a library of peptides and small proteins, which were purified from human hemofiltrate. To clarify the inhibitory mechanism the obtained inhibitors were characterised in cell-free fibril formation assays and other biochemical methods.. We identified lysozyme as an inhibitor of SAA fibril formation. Lysozyme antagonised fibril formation both in the cell model as well as in cell-free fibril formation assays. The protein binds SAA with a dissociation constant of 16.5 ± 0.6 µM, while the binding site on SAA is formed by segments of positively charged amino acids.. Our data imply that lysozyme acts in a chaperone-like fashion and prevents the aggregation of SAA protein through direct, physical interactions.

Topics: Amyloid; Amyloidosis; Animals; Humans; Immunoglobulin Light-chain Amyloidosis; Muramidase; Serum Amyloid A Protein

Lysozyme amyloidosis is caused by an amino acid substitution in the sequence of this protein. In our study, we described a clinical case of lysozyme amyloidosis in a Russian family. In our work, we described in detail the histological changes in tissues that appeared as a result of massive deposition of amyloid aggregates that affected almost all organ systems, with the exception of the central nervous system. We determined the type of amyloidosis and mutations using mass spectrometry. Using mass spectrometry, the protein composition of tissue samples of patient 1 (autopsy material) and patient 2 (biopsy material) with histologically confirmed amyloid deposits were analyzed. Amino acid substitutions p.F21L/T88N in the lysozyme sequence were identified in both sets of samples and confirmed by sequencing of the lysozyme gene of members of this family. We have shown the inheritance of these mutations in the lysozyme gene in members of the described family. For the first time, we discovered a mutation in the first exon p.F21L of the lysozyme gene, which, together with p.T88N amino acid substitution, led to amyloidosis in members of the studied family.

Topics: Amino Acid Substitution; Amyloid; Amyloidosis; Humans; Muramidase; Mutation

Aggregation of unfolded or misfolded proteins into amyloid fibrils can cause various diseases in humans. However, the fibrils synthesized in vitro can be developed toward useful biomaterials under some physicochemical conditions. In this study, atomistic molecular dynamics simulations were performed to address the mechanism of beta-sheet formation of the unfolded hen egg-white lysozyme (HEWL) under a high temperature and low pH. Simulations of the protonated HEWL at pH 2 and the non-protonated HEWL at pH 7 were performed at the highly elevated temperature of 450 K to accelerate the unfolding, followed by the 333 K temperature to emulate some previous in vitro studies. The simulations showed that HEWL unfolded faster, and higher beta-strand contents were observed at pH 2. In addition, one of the simulation replicas at pH 2 showed that the beta-strand forming sequence was consistent with the 'K-peptide', proposed as the core region for amyloidosis in previous experimental studies. Beta-strand formation mechanisms at the earlier stage of amyloidosis were explained in terms of the radial distribution of the amino acids. The separation between groups of positively charged sidechains from the hydrophobic core corresponded to the clustering of the hydrophobic residues and beta-strand formation.

Topics: Amino Acids; Amyloid; Amyloidosis; Animals; Chickens; Egg White; Hydrogen-Ion Concentration; Molecular Dynamics Simulation; Muramidase; Protein Conformation, beta-Strand

Lysozyme-derived (ALys) amyloidosis is a rare type of hereditary amyloidosis. Nine amyloidogenic variants and ∼30 affected families have been described worldwide. The most common manifestations are renal dysfunction, gastrointestinal tract symptoms, and sicca syndrome. We report on the clinical course of ten patients from six families representing one of the largest cohorts published so far. Seven patients carried the W64R variant showing the whole spectrum of ALys-associated symptoms. Two patients-a mother-son pair-carried a novel lysozyme variant, which was associated with nephropathy and peripheral polyneuropathy. In accordance with previous findings, the phenotype resembled within these families but did not correlate with the genotype. To gain insights into the effect of the variants at the molecular level, we analysed the structure of lysozyme and performed comparative computational predictions on aggregation propensity and conformational stability. Our study supports that decreased conformational stability is a key factor for lysozyme variants to be prone to aggregation. In summary, ALys amyloidosis is a very rare, but still heterogeneous disease that can manifest at an early age. Our newly identified lysozyme variant is associated with nephropathy and peripheral polyneuropathy. Further research is needed to understand its pathogenesis and to enable the development of new treatments.

Topics: Amyloidosis; Amyloidosis, Familial; Gastrointestinal Diseases; Humans; Kidney Diseases; Muramidase; Polyneuropathies

Amyloid fibril formation is associated with several amyloidoses, including neurodegenerative Alzheimer's or Parkinson's diseases. The process of such fibrillar structure formation is still not fully understood, with new mechanistic insights appearing on a regular basis. This, in turn, has limited the development of potential anti-amyloid compounds, with only a handful of effective cures or treatment modalities available. One of the multiple amyloid aggregation factors that requires further examination is the ability of proteins to form multiple, structurally distinct aggregates, based on the environmental conditions. In this work, we examine how the initial folding state affects the fibrilization of lysozyme-an amyloidogenic protein, often used in protein aggregation studies. We show that there is a correlation between the initial state of the protein and the aggregate formation lag time, rate of elongation, resulting aggregate structural variability and dye-binding properties, as well as formation lag time and rate of elongation.

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Antiviral Agents; Dermatologic Agents; Humans; Muramidase; Protein Aggregates; Protein Folding

Some natural variants of human lysozyme are associated with systemic non-neurological amyloidosis that leads to amyloid protein fibril deposition in different tissues. Inhibition of amyloid fibrillation by nanomaterials is considered to be an effective approach to treating amyloidosis. Here, we prepared a targeted, highly loaded curcumin lysozyme-imprinted nanosphere (CUR-MIMS) that could effectively inhibit the aggregation of lysozyme with lysozyme adsorption capacity of 193.57 mg g

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Curcumin; Humans; Muramidase; Nanospheres

The effect of two widely used polyphenols, curcumin and EGCG was investigated on the amyloid fibrillogenesis of the well-characterized model protein human lysozyme (HuL), associated with non-neuropathic systemic amyloidosis, towards exploring their efficacy as modulators of HuL amyloid aggregation and toxicity and unravelling their mechanism of action. Curcumin exerts its inhibitory influence towards HuL fibrillation by interacting with the prefibrillar and fibrillar intermediates resulting in complete suppression of fibrillation at ∼200 µM and effectively disaggregates preformed fibrils of HuL. EGCG on the other hand suppresses fibrillation only upto 70% at ∼400 µM, modulates the pathway towards large, β-sheet rich amyloid fibril-like aggregates and modifies the preformed fibrils into similar type of large, clustered aggregate assemblies. The overall surface hydrophobicity and cytotoxicity of HuL is significantly reduced not only in the presence of curcumin but also EGCG, despite the latter forming large agglomerates, which could be accounted for by the dense and highly clustered nature of aggregates rendering their surface less exposed and thus less amenable to interact with cellular entities thereby causing reduced cellular toxicity. This study highlights the differential mechanisms employed by curcumin and ECCG in modulating the fibrillation pathway of HuL and illustrates the importance of overall modulation of fibrillation towards a general reduction in toxicity, rather than specifically focusing only on inhibition of fibrillation. This study also demonstrates how two widely different polyphenols employ disparate mechanisms to modulate the fibrillation pathway of a single protein and yet converge towards a common effect of alleviation of cytotoxicity.

Topics: Amyloid; Amyloidosis; Curcumin; Humans; Muramidase; Polyphenols

Renal amyloidosis typically manifests albuminuria, nephrotic-range proteinuria, and ultimately progresses to end-stage renal failure if diagnosed late. Different types of renal amyloidosis have completely different treatments and outcomes. Therefore, amyloidosis typing is essential for disease prognosis, genetic counseling and treatment. Thirty-six distinct proteins currently known to cause amyloidosis that have been described as amyloidogenic precursors, immunohistochemistry (IHC) or immunofluorescence (IF), can be challenging for amyloidosis typing especially in rare or hereditary amyloidosis in clinical practice. We made a pilot study that optimized the proteomics pre-processing procedures for trace renal amyloidosis formalin-fixed paraffin-embedded (FFPE) tissue samples, combined with statistical and bioinformatics analysis to screen out the amyloidosis-related proteins to accurately type or subtype renal amyloidosis in order to achieve individual treatment. A sensitive, specific and reliable FFPE-based proteomics analysis for trace sample manipulation was developed for amyloidosis typing. Our results not only underlined the great promise of traditional proteomics and bioinformatics analysis using FFPE tissues for amyloidosis typing, but also proved that retrospective diagnosis and analysis of previous cases laid a solid foundation for personalized treatment.

Topics: Amyloidosis; Base Sequence; Case-Control Studies; Formaldehyde; Humans; Kidney; Mass Spectrometry; Muramidase; Paraffin Embedding; Pilot Projects; Proteomics; Tissue Fixation

Several human diseases like Parkinson's, Alzheimer's disease, and systemic amyloidosis are associated with the misfolding and aggregation of protein molecules.. The present study demonstrated the comparison of 4-methyl coumarin and 4-methylthiocoumarin derivative for their anti-amyloidogenic and disaggregation activities. The hen egg-white lysozyme is used as a model system to study protein aggregation and disaggregation under in vitro conditions.. Techniques used in the study were Thioflavin T fluorescence assay, intrinsic fluorescence assay, circular dichroism, transmission electron microscopy, and molecular dynamics.. Fifteen compounds were screened for their anti-amyloidogenic and disaggregation potential. Six compounds significantly inhibited the fibril formation, whereas ten compounds showed disaggregation property of pre-formed fibrils. Under in vitro conditions, the compound C3 and C7 showed significant inhibition of fibril formation in a concentration-dependent manner as compared to control. C3 and C7 demonstrated 93% and 76% inhibition of fibril formation, respectively. Furthermore, C3 and C7 exhibited 83% and 76% disaggregation activity, respectively, of pre-formed HEWL fibrils at their highest concentration. These anti-amyloidogenic and disaggregation potential of C3 and C7 were validated by intrinsic fluorescence, CD, molecular dynamics, and TEM study.. 4-methylthiocoumarins derivatives have shown better anti-amyloidogenic activity as compared to 4-methylcoumarin derivatives for both amyloid formation as well as disaggregation of preformed amyloid fibrils. Structurally, the derivatives of 4-methylthiocoumarins (C3 and C7) contain thio group on 2nd position that might be responsible for anti-amyloidogenic activity as compared to 4- methylcoumarin derivatives (C2 and C4).. C3 and C7 are novel 4-methylthiocoumarin derivatives that can be used as a lead for alleviation and symptoms associated with protein aggregation disorders.

Topics: Amyloid; Amyloidosis; Animals; Circular Dichroism; Coumarins; Dose-Response Relationship, Drug; Humans; Molecular Docking Simulation; Muramidase; Protein Structure, Secondary

Lysozyme amyloidosis (ALys) is caused by the deposition of amyloid-like fibrils of lysozyme in the tissues of the gastrointestinal tract, liver and kidneys. The treatment/prevention of ALys is not known yet. Therefore, searching for therapeutic agents for amyloidosis is of great value. In this study, we have examined the ability of the aqueous extract of herbalome (thirty herbal components) of Chandraprabha vati (EHCV), a polyherbal Ayurvedic formulation, to prevent fibrillation of lysozyme. Transmission electron microscopy and multiple biophysical techniques were used to examine the processes. We found complete inhibition of the fibrillation by EHCV, whereas none of the thirty ingredients of EHCV was able to prevent the reaction, solely. We also found the EHCV induced and stabilized secondary structures of aggregation-prone state (APS) of lysozyme. Moreover, an increase in the secondary structure and stability of APS were found to correlate with the inhibition reaction. We conclude that EHCV modulates the structure and stability of APS and converts it into an aggregation resistant state (ARS). We hypothesized that herbal components of Ayurvedic formulation may provide a combination of molecules, which could efficiently prevent aggregation reaction.

Topics: Amyloid; Amyloidosis; Drug Compounding; Egg Proteins; Medicine, Ayurvedic; Minerals; Muramidase; Plant Extracts; Plants, Medicinal; Protein Aggregates; Protein Structure, Secondary

Many conflicting reports about the involvement of serum amyloid P component (SAP) in amyloid diseases have been presented over the years; SAP is known to be a universal component of amyloid aggregates but it has been suggested that it can both induce and suppress amyloid formation. By using our Drosophila model of systemic lysozyme amyloidosis, SAP has previously been shown to reduce the toxicity induced by the expression of the disease-associated lysozyme variant, F57I, in the Drosophila central nervous system. This study further investigates the involvement of SAP in modulating lysozyme toxicity using histochemistry and spectral analyses on the double transgenic WT and F57I lysozyme flies to probe; i) formation of aggregates, ii) morphological differences of the aggregated lysozyme species formed in the presence or absence of SAP, iii) location of lysozyme and iv) co-localisation of lysozyme and SAP in the fly brain. We found that SAP can counteract the toxicity (measured by the reduction in the median survival time) induced by F57I lysozyme by converting toxic F57I species into less toxic amyloid-like structures, as reflected by the spectral changes that p-FTAA undergoes when bound to lysozyme deposits in F57I-F57I-SAP flies as compared to F57I-F57I flies. Indeed, when SAP was introduced to in vitro lysozyme fibril formation, the endpoint fibrils had enhanced ThT fluorescence intensity as compared to lysozyme fibrils alone. This suggests that a general mechanism for SAP's role in amyloid diseases may be to promote the formation of stable, amyloid-like fibrils, thus decreasing the impact of toxic species formed along the aggregation pathway.

Topics: Amyloid; Amyloidosis; Animals; Animals, Genetically Modified; Disease Models, Animal; Drosophila melanogaster; Drosophila Proteins; Humans; Muramidase; Protein Aggregates; Protein Aggregation, Pathological; Serum Amyloid P-Component

Polyamines can induce protein aggregation that can be related to the physiology of the cellular function. Polyamines have been implicated in protein aggregation which may lead to neuropathic and non neuropathic amyloidosis.. Change in the level of polyamine concentration has been associated with ageing and neurodegeneration such as Parkinson's disease, Alzheimer's disease. Lysozyme aggregation in the presence of polyamines leads to non neuropathic amyloidosis. Polyamine analogues can suppress or inhibit protein aggregation suggesting their efficacy against amyloidogenic protein aggregates.. In this study we report the comparative interactions of lysozyme with the polyamine analogue, 1-naphthyl acetyl spermine in comparison with the biogenic polyamines through spectroscopy, calorimetry, imaging and docking techniques. The findings revealed that the affinity of binding varied as spermidine > 1-naphthyl acetyl spermine > spermine. The biogenic polyamines accelerated the rate of fibrillation significantly, whereas the analogue inhibited the rate of fibrillation to a considerable extent. The polyamines bind near the catalytic diad residues viz. Glu35 and Asp52, and in close proximity of Trp62 residue. However, the analogue showed dual nature of interaction where its alkyl amine region bind in same way as the biogenic polyamines bind to the catalytic site, while the naphthyl group makes hydrophobic contacts with Trp62 and Trp63, thereby suggesting its direct influence on fibrillation.. This study, thus, potentiates, the development of a polyamine analogue that can perform as an effective inhibitor targeted towards aggregation of amyloidogenic proteins.

Topics: Amyloid; Amyloidosis; Animals; Avian Proteins; Biogenic Polyamines; Chickens; Hydrophobic and Hydrophilic Interactions; Molecular Docking Simulation; Muramidase; Protein Aggregates; Protein Aggregation, Pathological; Spermidine; Spermine

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Humans; Muramidase; Proteomics

There are many reports demonstrating that various derivatives of carbon nanoparticles are effective inhibitors of protein aggregation. As surface structural features of nanoparticles play a key role on modulating amyloid fibrillation process, in the present in vitro study, bovine insulin and hen egg white lysozyme (HEWL) were selected as two model proteins to investigate the reducing effect of graphene oxide quantum dots (GOQDs) on their assembly under amyloidogenic conditions. GOQDs were prepared through direct pyrolysis of citric acid, and the reduction step was carried out using ascorbic acid. The prepared nanoparticles were characterized by UV-Vis, X-ray photoelectron, and FT-IR spectroscopies, transmission electron and atomic force microscopies, zeta potential measurement, and Nile red fluorescence assay. They showed the tendencies to modulate the assembly of the proteins through different mechanisms. While GOQDs appeared to have the capacity to inhibit fibrillation, the presence of reduced GOQDs (rGOQDs) was found to promote protein assembly via shortening the nucleation phase, as suggested by ThT fluorescence data. Moreover, the structures produced in the presence of GOQDs or rGOQDs were totally nontoxic. We suggest that surface properties of these particles may be part of the differences in their mechanism(s) of action.

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Animals; Cattle; Graphite; Insulin; Models, Biological; Muramidase; Nanoparticles; Oxygen; Protein Aggregates; Quantum Dots; Surface Properties

Increasing evidence proposed that amyloid deposition by proteins play a crucial role in an array of neurotoxic and degenerative disorders like Parkinson's disease, systemic amyloidosis etc, that could be controlled by anti-aggregation methodologies which either inhibit or disaggregate such toxic aggregates. The present work targets the amyloid inhibiting and disaggregating potential of promethazine (PRM) against human insulin (HI) and human lysozyme (HL) fibrillogenesis. Biophysical techniques like Rayleigh scattering measurements (RLS), Thioflavin T (ThT) and 8-Anilinonaphthalene-1-sulfonic acid (ANS) fluorescence measurement, circular dichroism (CD) and dynamic light scattering (DLS) measurements illustrated the inhibitory action of PRM. The half maximal inhibitory concentration (IC

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Anilino Naphthalenesulfonates; Benzothiazoles; Circular Dichroism; Dynamic Light Scattering; Fluorescence; Humans; Insulin; Muramidase; Parkinson Disease; Promethazine; Protein Aggregates; Protein Aggregation, Pathological; Thiazoles

Topics: Acarbose; Amyloidosis; Circular Dichroism; Drug Repositioning; Glycoside Hydrolase Inhibitors; Humans; Molecular Docking Simulation; Molecular Structure; Muramidase; Protein Binding; Thermodynamics

The 129-residue lysozyme has been shown to form amyloid fibrils in vitro. While methylene blue (MB), a compound in the phenothiazinium family, has been shown to dissemble tau fibril formation, its anti-fibrillogenic effect has not been thoroughly characterized in other proteins/peptides. This study examines the effects of MB on the in vitro fibrillogenesis of lysozyme at pH 2.0 and 55 °C. Our results demonstrated that, upon 7-day incubation, the plateau ThT fluorescence of the sample was found to be ~8.69% or ~2.98% of the control when the molar ratio of lysozyme to MB was at 1:1.11 or 1:3.33, respectively, indicating that the inhibitory potency of MB against lysozyme fibrillogenesis is positively correlated with its concentration. We also found that MB is able to destabilize the preformed lysozyme fibrils. Moreover, molecular docking and molecular dynamics simulations results revealed that MB's mechanism of fibril formation inhibition may be triggered by binding with lysozyme's aggregation-prone region. Results reported here provide solid support for MB's effect on amyloid fibrillogenesis. We believe the additional insights gained herein may pave way to the discovery of other small molecules that may have similar action toward amyloid fibril formation and its associated diseases.

Topics: Amyloid; Amyloidosis; Methylene Blue; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Muramidase; Protein Aggregates; Protein Aggregation, Pathological; Protein Binding; Spectrum Analysis; Structure-Activity Relationship

Protein amyloid aggregation is an important pathological feature of a group of different degenerative human diseases called amyloidosis. We tested effect of two phospholipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) on amyloid aggregation of hen egg white (HEW) lysozyme in vitro.. Effect of phospholipids was investigated using spectroscopic techniques (fluorescence and CD spectroscopy), atomic force microscopy and image analysis.. Phospholipids DMPC and DHPC are able dose-dependently inhibit lysozyme fibril formation. The length of the phospholipid tails and different structural arrangement of the phospholipid molecules affect inhibitory activity; long-chain DMPC inhibits fibrillization more efficiently. Interestingly, interference of DMPC with lysozyme amyloid fibrils has no effect on their morphology or amount.. Phospholipid molecules have significant effect on lysozyme amyloid fibrillization. We suggest that inhibitory activity is due to the interference of phospholipids with lysozyme leading to the blocking of the intermolecular protein interactions important for formation of the cross-β structure within the core of the fibrils. The higher inhibitory activity of DMPC is probably due to adsorption of protein molecules on the liposome surfaces which caused decrease of species needed for fibrillization. Interaction of the phospholipids with formed fibrils is not sufficient enough to interrupt the bonds in β-sheets which are required for destroying of amyloid fibrils.. The obtained results contribute to a better understanding of the effect of phospholipids on amyloid fibrillization of the lysozyme. The data suggest that DMPC and DHPC phospholipids represent agents able to modulate lysozyme amyloid aggregation.

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Animals; Chickens; Dimyristoylphosphatidylcholine; Humans; Microscopy, Atomic Force; Muramidase; Phosphatidylcholines; Phospholipid Ethers; Phospholipids; Phosphorylcholine; Protein Aggregation, Pathological

Heparin, a biopolymer possessing high negative charge density, is known to accelerate amyloid fibrillation by various proteins. Using hen egg white lysozyme, we studied the effects of heparin on protein aggregation at low pH, raised temperature, and applied ultrasonic irradiation, conditions under which amyloid fibrillation was promoted. Heparin exhibited complex bimodal concentration-dependent effects, either accelerating or inhibiting fibrillation at pH 2.0 and 60 °C. At concentrations lower than 20 μg/ml, heparin accelerated fibrillation through transient formation of hetero-oligomeric aggregates. Between 0.1 and 10 mg/ml, heparin rapidly induced amorphous heteroaggregation with little to no accompanying fibril formation. Above 10 mg/ml, heparin again induced fibrillation after a long lag time preceded by oligomeric aggregate formation. Compared with studies performed using monovalent and divalent anions, the results suggest two distinct mechanisms of heparin-induced fibrillation. At low heparin concentrations, initial hen egg white lysozyme cluster formation and subsequent fibrillation is promoted by counter ion binding and screening of repulsive charges. At high heparin concentrations, fibrillation is caused by a combination of salting out and macromolecular crowding effects probably independent of protein net charge. Both fibrillation mechanisms compete against amorphous aggregation, producing a complex heparin concentration-dependent phase diagram. Moreover, the results suggest an active role for amorphous oligomeric aggregates in triggering fibrillation, whereby breakdown of supersaturation takes place through heterogeneous nucleation of amyloid on amorphous aggregates.

Topics: Amyloid; Amyloidogenic Proteins; Amyloidosis; Animals; Egg White; Heparin; Hydrogen-Ion Concentration; Muramidase; Protein Aggregates

In spite of the fact that amyloid related neurodegenerative illnesses and non-neuropathic systemic amyloidosis have allured the research endeavors, as no cure has been announced yet apart from symptomatic treatment. Therapeutic agents which can reduce or disaggregate those toxic oligomers and fibrillar species have been studied with more compounds are on their way. The current research work describes comprehensive biophysical, computational and microscopic studies which reveal that L-3, 4-dihydroxyphenylalanine (L-Dopa) have indisputable efficacy to hinder the heat induced amyloid fibrillation of the human lysozyme (HL) and also preserve the fibril disaggregating potential. The IC

Topics: Amyloidosis; Cell Line, Tumor; Cell Survival; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Inhibitory Concentration 50; Levodopa; Molecular Docking Simulation; Muramidase; Parkinsonian Disorders; Protein Multimerization; Protein Structure, Secondary

Lysozyme amyloidosis is a hereditary disease in which mutations in the gene coding for lysozyme leads to misfolding and consequently accumulation of amyloid material. To improve understanding of the processes involved we expressed human wild type (WT) lysozyme and the disease-associated variant F57I in the central nervous system (CNS) of a Drosophila melanogaster model of lysozyme amyloidosis, with and without co-expression of serum amyloid p component (SAP). SAP is known to be a universal constituent of amyloid deposits and to associate with lysozyme fibrils. There are clear indications that SAP may play an important role in lysozyme amyloidosis, which requires further elucidation. We found that flies expressing the amyloidogenic variant F57I in the CNS have a shorter lifespan than flies expressing WT lysozyme. We also identified apoptotic cells in the brains of F57I flies demonstrating that the flies' neurological functions are impaired when F57I is expressed in the nerve cells. However, co-expression of SAP in the CNS prevented cell death and restored the F57I flies' lifespan. Thus, SAP has the apparent ability to protect nerve cells from damage caused by F57I. Furthermore, it was found that co-expression of SAP prevented accumulation of insoluble forms of lysozyme in both WT- and F57I-expressing flies. Our findings suggest that the F57I mutation affects the aggregation process of lysozyme resulting in the formation of cytotoxic species and that SAP is able to prevent cell death in the F57I flies by preventing accumulation of toxic F57I structures.

Topics: Amyloidosis; Animals; Animals, Genetically Modified; Apoptosis; Central Nervous System; Disease Models, Animal; Drosophila melanogaster; Gene Expression; Humans; Longevity; Muramidase; Mutation; Neurons; Plaque, Amyloid; Protective Factors; Protein Aggregation, Pathological; Serum Amyloid P-Component; Transgenes

Misfolded β-sheet-rich protein aggregates termed amyloid, deposit in vivo leading to debilitating diseases such as Alzheimer's, prion and renal amyloidosis diseases etc. Strikingly, amyloid can induce conversion of their natively folded monomers into similarly aggregated conformation via 'seeding'. The specificity of seeding is well documented in vivo for prions, where prion-variants arising from conformationally altered amyloids of the same protein, faithfully seed monomers into amyloid displaying the original variant's conformation. Thus far, amyloid variant formation is reported only for a few non-prion proteins like Alzheimer's Aβ42-peptide and β-2 microglobulin, however, their conformational cross-seeding capabilities are unexplored. While mutant human lysozyme causes renal amyloidosis, the hen egg white lysozyme (HEWL) has been extensively investigated in vitro as a model amyloid protein. Here we investigated if wild-type HEWL could form self-seeding amyloid variants to examine if variant formation is more wide-spread. We found that HEWL aggregates formed under quiescent versus agitated conditions, displayed different particle sizes, detergent stabilities & β-sheet content, and they only seeded monomeric HEWL under similar incubation conditions, but not under swapped incubation conditions thereby showing amyloid variant formation by HEWL analogous to prion variants. This may have implications to the amyloidosis caused by different mutants of human lysozyme.

Topics: Amyloid; Amyloidosis; Animals; Chickens; Detergents; Female; In Vitro Techniques; Muramidase; Particle Size; Protein Aggregates; Protein Conformation

Although the cure of amyloid related neurodegenerative diseases, non-neuropathic amyloidogenic diseases and non-neuropathic systemic amyloidosis are appealing energetic research attempts, beneficial medication is still to be discovered. There is a need to explore intensely stable therapeutic compounds, potent enough to restrict, disrupt or wipe out such toxic aggregates. We had performed a comprehensive biophysical, computational and cell based assay, that shows Nordihydroguaiaretic acid (NA) not only significantly inhibits heat induced hen egg white lysozyme (HEWL) fibrillation but also disaggregates preformed HEWL fibrils and reduces the cytoxicity of amyloid fibrils as well as disaggregated fibrillar species. The inhibitory potency of NA was determined by an IC

Topics: Amyloid; Amyloidosis; Benzothiazoles; Cell Line, Tumor; Hot Temperature; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Immunoglobulin Light-chain Amyloidosis; Inhibitory Concentration 50; Kinetics; Light; Masoprocol; Microscopy, Fluorescence; Molecular Docking Simulation; Muramidase; Nephelometry and Turbidimetry; Protein Aggregates; Protein Binding; Protein Conformation; Scattering, Radiation; Spectrometry, Fluorescence; Thiazoles

Amyloid protein depositions play crucial roles in a variety of degenerative disorders composing amyloidosis. There is a great interest in developing small molecule inhibitors of amyloidogenic processes. We examined the inhibitory effects of echinacoside (ECH) with different concentrations and at different fiber-forming stages in vitro utilizing the hen egg-white lysozyme (HEWL) model system. We also evaluated the antioxidant capacity of ECH by using elimination tests for the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl (HO) free radicals. We investigated the protection provided by ECH against neurotoxicity induced by β-amyloid protein (Aβ). Through spectroscopic analyses, electron microscopy, cell viability assay, and hemolysis assay, we found that ECH dose dependently inhibited HEWL aggregation, and this inhibition occurred in different fiber-forming stages. ECH could also scavenge the DPPH and OH free radicals in a concentration-dependent manner. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2',7'-dichlorodihydrofluoresceindiacetate (DCFH-DA) fluorescent measurement results indicated that ECH could increase viability of rat pheochromocytoma PC12 cells injured by Aβ and suppress the increase in intracellular reactive oxygen species (ROS) triggered by Aβ. The present study findings facilitate a better understanding of the interaction between ECH and amyloid-forming proteins and also shed light on the protection of ECH against amyloid fibril-induced neuronal cell death.

Topics: Amyloid beta-Peptides; Amyloidosis; Animals; Antioxidants; Biphenyl Compounds; Cell Death; Cell Survival; Glycosides; Humans; Hydroxyl Radical; Muramidase; PC12 Cells; Picrates; Protective Agents; Rats; Reactive Oxygen Species

Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using β2-microglobulin (β2m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the deleterious effects of preformed amyloid aggregates and suggest why endocytic trafficking through acidic compartments may be a key factor in amyloid disease.

Topics: Amyloid; Amyloidosis; Benzothiazoles; beta 2-Microglobulin; Endosomes; HSP70 Heat-Shock Proteins; Humans; Hydrogen-Ion Concentration; Kinetics; Lysosomes; Monocytes; Muramidase; Protein Binding; Recombinant Proteins; Spectrometry, Fluorescence; Thiazoles

The question of how an aggregating protein can influence aggregation of other proteins located in its vicinity is particularly significant because many proteins coexist in cells. We demonstrate in vitro coaggregation and cross-seeding of lysozyme, bovine serum albumin, insulin, and cytochrome c during their amyloid formation. The coaggregation process seems to be more dependent on the temperature-induced intermediate species of these proteins and less dependent on their sequence identities. Because amyloid-linked inclusions and plaques are recognized as multicomponent entities originating from aggregation of the associated protein, these findings may add new insights into the mechanistic understanding of amyloid-related pathologies.

Topics: Amino Acid Sequence; Amyloid; Amyloidosis; Animals; Cattle; Circular Dichroism; Cytochromes c; Humans; Insulin; Kinetics; Microscopy, Electron, Transmission; Molecular Sequence Data; Muramidase; Protein Aggregates; Protein Aggregation, Pathological; Sequence Homology, Amino Acid; Serum Albumin, Bovine; Spectrometry, Fluorescence

While amyloid-related diseases are at the center of intense research efforts, no feasible cure is currently available for these diseases. The experimental and computational techniques were used to study the ability of glyco-acridines to prevent lysozyme amyloid fibrillization in vitro. Fluorescence spectroscopy and atomic force microscopy have shown that glyco-acridines inhibit amyloid aggregation of lysozyme; the inhibition efficiency characterized by the half-maximal inhibition concentration IC50 was affected by the structure and concentration of the derivative. We next investigated relationship between the binding affinity and the inhibitory activity of the compounds. The semiempirical quantum PM6-DH+ method provided a good correlation pointing to the importance of quantum effects on the binding of glyco-acridine derivatives to lysozyme. The contribution of linkers may be explained by the valence bond theory. Our data provide a basis for the development of new small molecule inhibitors effective in therapy of amyloid-related diseases.

Topics: Acridines; Amyloid; Amyloid beta-Peptides; Amyloidosis; Humans; Muramidase; Protein Conformation; Structure-Activity Relationship

Sarcoidosis is a multisystem inflammatory disorder of unknown cause which can affect any organ system. Autosomal dominant lysozyme amyloidosis is a very rare form of hereditary amyloidosis. The Arg64 variant is extraordinarily rare with each family showing a particular pattern of organ involvement, however while Sicca syndrome, gastrointestinal involvement and renal failure are common, lymph node involvement is very rare. In this case report we describe the first reported case of sarcoidosis in association with hereditary lysozyme amyloidosis.

Topics: Amyloidosis; Amyloidosis, Familial; Humans; Muramidase; Sarcoidosis; Sarcoidosis, Pulmonary

Inflammation occurs in many amyloidoses, but its underlying mechanisms remain enigmatic. Here we show that amyloid fibrils of human lysozyme, which are associated with severe systemic amyloidoses, induce the secretion of pro-inflammatory cytokines through activation of the NLRP3 (NLR, pyrin domain containing 3) inflammasome and the Toll-like receptor 2, two innate immune receptors that may be involved in immune responses associated to amyloidoses. More importantly, our data clearly suggest that the induction of inflammatory responses by amyloid fibrils is linked to their intrinsic structure, because the monomeric form and a non-fibrillar type of lysozyme aggregates are both unable to trigger cytokine secretion. These lysozyme species lack the so-called cross-β structure, a characteristic structural motif common to all amyloid fibrils irrespective of their origin. Since fibrils of other bacterial and endogenous proteins have been shown to trigger immunological responses, our observations suggest that the cross-β structural signature might be recognized as a generic danger signal by the immune system.

Topics: Amyloid; Amyloidosis; Animals; Carrier Proteins; Cell Line; HEK293 Cells; Humans; Immunity, Innate; Inflammasomes; Inflammation; Interleukin-1beta; Mice; Mice, Inbred C57BL; Muramidase; NLR Family, Pyrin Domain-Containing 3 Protein; Protein Structure, Secondary; Toll-Like Receptor 2

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.

Topics: Amyloidosis; Animals; Animals, Genetically Modified; Disease Models, Animal; DNA-Binding Proteins; Drosophila melanogaster; Drosophila Proteins; Endoplasmic Reticulum Stress; Eye Abnormalities; Female; Green Fluorescent Proteins; Hemolymph; Humans; Male; Metamorphosis, Biological; Microscopy, Electron, Scanning; Muramidase; Photoreceptor Cells, Invertebrate; Solubility; Unfolded Protein Response

Amyloid depositions of proteins play crucial roles in a wide variety of degenerative disorders called amyloidosis. In the present study, we used hen egg white lysozyme (HEWL), as an in vitro model system, to induce fibrillation under high temperatures and acidic pH conditions, and investigated the inhibitory and disruptive effects of two salen-manganese complexes, namely EUK-8 and EUK-134, with aromatic structures, against fibrilization. Results of this study showed that EUK-8 and EUK-134 in a dose-dependent manner inhibited the HEWL aggregation. Similar results were obtained when these compounds were added to pre-formed amyloid fibrils. Docking results also demonstrated that the aromatic rings of EUK-8 and EUK-134 interact with the hydrophobic region of lysozyme via Van der Waals interactions. Results of MTT assay indicated that addition of pre-formed fibrils treated with EUK-8 and EUK-134 at doses 1:1 and 5:1mM; drug to protein, to SK-N-MC cells significantly increased the viability of cells, compared to the fibril sample alone. Based on these results, it might be concluded that in addition to inherent hydrophobicity associated with the ligand section of each of the derivatives, electron density around the central metal ion of the derivatives contributes to lower lysozyme fibril accumulation.

Topics: Amyloid; Amyloidosis; Animals; Cell Line, Tumor; Chemistry; Chickens; Humans; Hydrogen-Ion Concentration; Ions; Manganese; Metals; Muramidase; Protein Binding; Protein Structure, Secondary; Solvents; Temperature

Accumulation of intra- and/or extracellular misfolded proteins as amyloid fibrils is a key hallmark in more than 20 amyloid-related diseases. In that respect, blocking or reversing amyloid aggregation via the use of small compounds is considered as two useful approaches in hampering the development of these diseases. In this research, we have studied the ability of different manganese-salen derivatives to inhibit amyloid self-assembly as well as to dissolve amyloid aggregates of hen egg-white lysozyme, as an in vitro model system, with the aim of investigating their structure-activity relationships. By coupling several techniques such as thioflavin T and anilinonaphthalene-8-sulfonic acid fluorescence, congo red absorbance, far-UV circular dichroism, and transmission electron microscopy, we demonstrated that all compounds possessed anti-amyloidogenic activities and were capable of dispersing the fibrillar aggregates. In addition, MTT assay of the treated SK-N-MC cells with the preformed fibrils formed in the presence of compounds at a drug-to-protein molar ratio of 5:1, indicated a significant increase in the viability of cells, compared to the fibrils formed in the absence of each of the compounds. Our spectroscopy, electron microscopy, and cellular studies indicated that EUK-15, with a methoxy group at the para position (group R(5)), had higher activity to either inhibit or disrupt the β-sheet structures relative to other compounds. On the basis of these results, it can be concluded that in addition to aromatic rings of each of the derivatives, the type and position of the side group(s) contribute to lower lysozyme fibril accumulation.

Topics: Amyloid; Amyloidosis; Animals; Antioxidants; Cell Line, Tumor; Cell Survival; Chickens; Ethylenediamines; Humans; Manganese; Muramidase; Protein Conformation; Structure-Activity Relationship

More than twenty-seven human proteins can fold abnormally to form amyloid deposits associated with a number of degenerative diseases. The research reported here is aimed at exploring the connection between curcumin's thermostability and its inhibitory activity toward the amyloid fibrillation of hen egg-white lysozyme (HEWL).. ThT fluorescence spectroscopy, equilibrium thermal denaturation analysis, and transmission electron microscopy were employed for structural characterization. MTT reduction and flow cytometric analyses were used to examine cell viability.. The addition of thermally pre-treated curcumin was found to attenuate the formation of HEWL fibrils and the observed fibrillation inhibition was dependent upon the pre-incubation temperature of curcumin. Our results also demonstrated that the cytotoxic effects of fibrillar HEWL species on PC 12 and SH-SY5Y cells were decreased and negatively correlated with curcumin's thermostability. Next, an enhanced stability of HEWL was perceived upon the addition of curcumin pre-incubated at lower temperature. Furthermore, we found that the alteration of curcumin's thermostability was associated with its inhibitory potency against HEWL fibrillation.. We believe that the results from this research may contribute to the development of effective therapeutics for amyloidoses.

Topics: Amyloid; Amyloidosis; Animals; Cell Survival; Curcumin; Flow Cytometry; Muramidase; PC12 Cells; Protein Folding; Rats; Spectrophotometry, Ultraviolet; Temperature; Thermodynamics

Topics: Amino Acid Sequence; Amyloid; Amyloidosis; Animals; Chickens; Humans; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Muramidase; Point Mutation; Protein Unfolding; Sequence Alignment; Thermodynamics

Peptide amyloid aggregation is a hallmark of several human pathologies termed amyloid diseases. We have investigated the effect of electrostatically stabilized magnetic nanoparticles of Fe(3)O(4) on the amyloid aggregation of lysozyme, as a prototypical amyloidogenic protein. Thioflavin T fluorescence assay and atomic force microscopy were used for monitoring the inhibiting and disassembly activity of magnetic nanoparticles of Fe(3)O(4). We have found that magnetic Fe(3)O(4) nanoparticles are able to interact with lysozyme amyloids in vitro leading to a reduction of the amyloid aggregates, thus promoting depolymerization; the studied nanoparticles also inhibit lysozyme amyloid aggregation. The ability to inhibit lysozyme amyloid formation and promote lysozyme amyloid disassembly exhibit concentration-dependent characteristics with IC50 = 0.65 mg ml(-1) and DC50 = 0.16 mg ml(-1) indicating that nanoparticles interfere with lysozyme aggregation already at stoichiometric concentrations. These features make Fe(3)O(4) nanoparticles of potential interest as therapeutic agents against amyloid diseases and their non-risk exploitation in nanomedicine and nanodiagnostics.

Topics: Amyloidosis; Animals; Chickens; Ferrosoferric Oxide; Humans; Magnetics; Muramidase; Nanoparticles; Protein Conformation; Protein Folding; Solubility

To understand the role of ATP underlying the enhanced amyloidosis of hen egg white lysozyme (HEWL), the synchrotron radiation circular dichroism, combined with tryptophan fluorescence, dynamic light-scattering, and differential scanning calorimetry, is used to examine the alterations of the conformation and thermal unfolding pathway of the HEWL in the presence of ATP, Mg(2+)-ATP, ADP, AMP, etc. It is revealed that the binding of ATP to HEWL through strong electrostatic interaction changes the secondary structures of HEWL and makes the exposed residue W62 move into hydrophobic environments. This alteration of W62 decreases the beta-domain stability of HEWL, induces a noncooperative unfolding of the secondary structures, and produces a partially unfolded intermediate. This intermediate containing relatively rich alpha-helix and less beta-sheet structures has a great tendency to aggregate. The results imply that the ease of aggregating of HEWL is related to the extent of denaturation of the amyloidogenic region, rather than the electrostatic neutralizing effect or monomeric beta-sheet enriched intermediate.

Topics: Adenosine Triphosphate; Amyloidosis; Animals; Muramidase; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Static Electricity; Tryptophan

We have studied the interaction of hen egg white lysozyme (HEWL) amyloid fibrils with human erythrocytes and lipid vesicles. The fibrils caused extensive aggregation of human erythrocytes and lipid vesicles without any significant lysis. The membrane activity of HEWL fibrils suggests that the interaction of lysozyme fibrils with cellular membranes could be a contributing factor under disease conditions.

Topics: Amyloid; Amyloidosis; Animals; Cell Membrane; Chickens; Erythrocyte Aggregation; Female; Humans; Liposomes; Muramidase; Protein Binding

Hereditary amyloidoses are caused by germline mutations, which increase the propensity of a protein to form cross-beta aggregates and deposit as amyloid. Hereditary amyloidoses are particularly interesting as they help to understand how changes in the primary structure of an otherwise non-amyloidogenic protein contribute to amyloidogenesis. Here we report on a novel form of systemic ALys amyloidosis, caused by compound heterozygosity in exon 2 (p.T70N) and exon 4 (p.W112R) of the lysozyme gene (LYZ), with both mutations being present on the same allele. This type of hereditary ALys amyloidosis is characterized by extended amyloid deposits in the upper gastrointestinal tract, entire colon, and kidney, leading to gastrointestinal bleeding. Both mutations are probably effective in disease manifestation. The novel mutation at position 112 in the mature protein is located within the alpha-helical domain of the protein and therefore outside the cluster of residues that has so far been implicated in ALys amyloidosis. Taken together with the p.T70N mutation, this results in a lysozyme species where the correct folding of various protein domains is probably impaired and increases the propensity of amyloid fibril formation. Interestingly, this form of ALys amyloidosis is also characterized by the occurrence of proteolytic fragments of lysozyme in the amyloid deposits.

Topics: Adult; Amino Acid Sequence; Amyloid; Amyloidosis; Blotting, Western; Colon; DNA Mutational Analysis; Duodenum; Electrophoresis, Polyacrylamide Gel; Exons; Heterozygote; Humans; Immunohistochemistry; Male; Molecular Sequence Data; Muramidase; Protein Structure, Secondary

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild-type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200- and 30-fold lower, respectively, than that of wild-type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins, under controlled conditions, can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post-transcriptional processes, and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly, our results show that the human disease-associated mutations do not have levels of expression that are out of line with destabilizing mutations at other sites. These findings indicate that a complex interplay between reduced native-state stability, lower secretion levels, and protein aggregation propensity influences the types of mutation that give rise to familial forms of amyloid disease.

Topics: Amyloidosis; Enzyme Stability; Humans; Isoenzymes; Models, Molecular; Muramidase; Pichia; Protein Denaturation; Protein Structure, Tertiary; RNA, Messenger

In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-32. However, soluble amyloid oligomers of lysozyme caused death of all these cell types, as estimated by flow-cytometry counting dead cells stained with ethidium bromide. The primary cell cultures appeared to be more sensitive to amyloid than neuroblastoma cell line IMR-32. Amyloid cytotoxicity depends on the size of oligomeric particles: samples containing 20-mers formed at pH 4.5 were more toxic than tetramers and octamers present in the solution at pH 2.0. Soluble amyloid oligomers can self-assemble into doughnut-like structures; however, no correlation was observed between the amount of the doughnut-like structures in the sample and its cytotoxicity. The fact that the intermediate oligomers of such an abundant protein as lysozyme display cytotoxicity confirms a hypothesis that cytotoxicity is a common feature of protein amyloid. Inhibition of intermediate oligomer formation is crucial in preventing amyloid pathogeneses.

Topics: Amyloid; Amyloid beta-Peptides; Amyloidosis; Animals; Benzothiazoles; Cell Line, Tumor; Cell Survival; Cells, Cultured; Congo Red; Dimerization; Female; Horses; Humans; Hydrogen-Ion Concentration; Kinetics; Macromolecular Substances; Mice; Mice, Inbred BALB C; Microscopy, Atomic Force; Models, Molecular; Muramidase; Protein Structure, Secondary; Thiazoles

In amyloid diseases, it is not evident which protein aggregates induce cell death via specific molecular mechanisms and which cause damage because of their mass accumulation and mechanical properties. We showed that equine lysozyme assembles into soluble amyloid oligomers and protofilaments at pH 2.0 and 4.5, 57 degrees C. They bind thioflavin-T and Congo red similar to common amyloid structures, and their morphology was monitored by atomic force microscopy. Molecular volume evaluation from microscopic measurements allowed us to identify distinct types of oligomers, ranging from tetramer to octamer and 20-mer. Monomeric lysozyme and protofilaments are not cytotoxic, whereas the oligomers induce cell death in primary neuronal cells, primary fibroblasts, and the neuroblastoma IMR-32 cell line. Cytotoxicity was accessed by ethidium bromide staining, MTT reduction, and TUNEL assays. Primary cultures were more susceptible to the toxic effect induced by soluble amyloid oligomers than the neuroblastoma cell line. The cytotoxicity correlates with the size of oligomers; the sample incubated at pH 4.5 and containing larger oligomers, including 20-mer, appears to be more cytotoxic than the lysozyme sample kept at pH 2.0, in which only tetramers and octamers were found. Soluble amyloid oligomers may assemble into rings; however, there was no correlation between the quantity of rings in the sample and its toxicity. The cytotoxicity of transient oligomeric species of the ubiquitous protein lysozyme indicates that this is an intrinsic feature of protein amyloid aggregation, and therefore soluble amyloid oligomers can be used as a primary therapeutic target and marker of amyloid disease.

Topics: Amyloid; Amyloidosis; Animals; Cell Death; Cell Line, Tumor; Cells, Cultured; Dimerization; Fibroblasts; Horses; Hydrogen-Ion Concentration; Mice; Mice, Inbred BALB C; Microscopy, Atomic Force; Muramidase; Neuroblastoma; Neurons

Topics: Amyloidosis; Diagnosis, Differential; Female; Humans; Middle Aged; Muramidase

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5A. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70-74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo.

Topics: Amyloidosis; Crystallography, X-Ray; Humans; Models, Molecular; Muramidase; Mutation; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation

Variant forms of lysozyme, a ubiquitous bacteriolytic enzyme, are known to lead to hereditary non-neuropathic renal amyloidosis and, so far, three different mutations of the lysozyme gene have been reported. In this study, we report a novel lysozyme variant, Phe57Ile, associated with renal amyloidosis in three patients in one Italian Canadian family.. The proband was a 52-year-old woman who developed renal failure at the age of 42 years. Renal biopsy demonstrated replacement of glomeruli by amyloid. Her younger sister and her younger daughter who underwent renal transplantation also had renal amyloidosis. The proband's older daughter and her niece were in good health. To elucidate pathogenesis of this hereditary renal amyloidosis, DNA analyses of the lysozyme gene, including single strand confirmation polymorphism, direct DNA sequence, and restriction fragment length polymorphism analyses, were performed.. DNA analyses of the lysozyme gene revealed a T to A transversion at the first position of codon 57 of the lysozyme gene in the proband, her sister, and her affected and unaffected daughters, indicating a replacement of Phe by Ile at residue 57. In addition, DNA sequencing demonstrated a C to A transversion at the second position of codon 70, denoting a replacement of Thr by Asn at residue 70, in the proband's sister and her niece. Thus, the proband's sister is compound heterozygous for the Phe57Ile and Thr70Asn alleles.. Distinctive clinical features in patients of this family are nephropathy due to renal amyloidosis. Our results indicate that the novel lysozyme variant Phe57Ile is associated with renal amyloidosis in this family. From our results, a clear relation between the Thr70Asn polymorphism and renal amyloidosis could not be demonstrated.

Topics: Amyloidosis; Family Health; Female; Humans; Kidney Diseases; Middle Aged; Muramidase; Pedigree; Point Mutation; Polymorphism, Restriction Fragment Length; Polymorphism, Single-Stranded Conformational; White People

Definition of the transition mechanism from the native globular protein into fibrillar polymer was greatly improved by the biochemical and biophysical studies carried out on the two amyloidogenic variants of human lysozyme, I56T and D67H. Here we report thermodynamic and kinetic data on folding as well as structural features of a naturally occurring variant of human lysozyme, T70N, which is present in the British population at an allele frequency of 5% and, according to clinical and histopathological data, is not amyloidogenic. This variant is less stable than the wild-type protein by 3.7 kcal/mol, but more stable than the pathological, amyloidogenic variants. Unfolding kinetics in guanidine are six times faster than in the wild-type, but three and twenty times slower than in the amyloidogenic variants. Enzyme catalytic parameters, such as maximal velocity and affinity, are reduced in comparison to the wild-type. The solution structure, determined by 1H NMR and modeling calculations, exhibits a more compact arrangement at the interface between the beta-sheet domain and the subsequent loop on one side and part of the alpha domain on the other side, compared with the wild-type protein. This is the opposite of the conformational variation shown by the amyloidogenic variant D67H, but it accounts for the reduced stability and catalytic performance of T70N.

Topics: Alleles; Amyloid; Amyloidosis; Catalysis; Circular Dichroism; Crystallography, X-Ray; Dose-Response Relationship, Drug; Genotype; Guanidine; Humans; Kinetics; Magnetic Resonance Spectroscopy; Models, Molecular; Muramidase; Protein Conformation; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Temperature; Thermodynamics; Time Factors; Ultraviolet Rays

Amyloid diseases are characterized by an aberrant assembly of a specific protein or protein fragment into fibrils and plaques that are deposited in various organs and tissues, often with serious pathological consequences. Non-neuropathic systemic amyloidosis is associated with single point mutations in the gene coding for human lysozyme. Here we report that a single-domain fragment of a camelid antibody raised against wild-type human lysozyme inhibits the in vitro aggregation of its amyloidogenic variant, D67H. Our structural studies reveal that the epitope includes neither the site of mutation nor most residues in the region of the protein structure that is destabilized by the mutation. Instead, the binding of the antibody fragment achieves its effect by restoring the structural cooperativity characteristic of the wild-type protein. This appears to occur at least in part through the transmission of long-range conformational effects to the interface between the two structural domains of the protein. Thus, reducing the ability of an amyloidogenic protein to form partly unfolded species can be an effective method of preventing its aggregation, suggesting approaches to the rational design of therapeutic agents directed against protein deposition diseases.

Topics: Amyloidosis; Animals; Camelids, New World; Circular Dichroism; Epitopes; Humans; Immunoglobulin Fab Fragments; Magnetic Resonance Spectroscopy; Models, Molecular; Muramidase; Mutation; Protein Denaturation; Protein Structure, Tertiary; X-Ray Diffraction

Hereditary nonneuropathic systemic lysozyme amyloidosis is a very rare form of amyloidosis, and only 4 families with this condition have been detailed until now in the literature. Clinical manifestations of lysozyme amyloidosis observed until now mainly concerned the kidneys, liver, and digestive tract. We report here a new family with hereditary lysozyme amyloidosis who presented predominantly with gastrointestinal involvement. The proband, a middle-aged woman, underwent partial gastrectomy for a hemorrhagic "gastric peptic ulcer" in 1984. Gastrointestinal amyloidosis was diagnosed in 1998 on biopsies performed on the gastroduodenal anastomosis, which appeared to be very congestive at presentation. Immunohistochemical stainings in tissue sections were positive for lysozyme. Amyloid was also observed in the colonic mucosa. The patient had a mutation in the lysozyme gene characterized by substitution of the amino acid at position 64 in the mature protein from tryptophan to arginine, previously described in only 1 French family with prominent nephropathy. It is interesting to note that her father had died many years before with an uncharacterized digestive amyloidosis. Our observation shows that a search for gastrointestinal amyloidosis is important, particularly when physicians are faced with congestive mucosa, unexplained abdominal hemorrhage, or abdominal symptoms. When gastrointestinal amyloidosis is diagnosed, it is important to determine with precision the nature of the amyloid fibril proteins, because various types of amyloidosis can involve the gastrointestinal tract.

Topics: Amyloidosis; Family Health; Female; Gastrointestinal Diseases; Humans; Middle Aged; Muramidase; Phenotype

Hemodialysis membranes eliminate by filtration low-molecular-weight toxic metabolites (urea and creatinine) with minimum interactions between blood components and the membrane itself. However, the ability of a membrane to adsorb specific proteins could be beneficial if the accumulation of these same proteins is implicated in the genesis of a pathological condition. Beta-amyloidosis which accompanies the elevation of beta2-microglobulin (11.8 kDa) in the plasma of dialysed patients is one such condition (Biochem. Biophys. Res. Commun. 129 (3) (1985) 701-706: Lancet 1 (1986) 1240-1311). To determine whether increases in plasma beta2-microglobulin levels were due to differences in filtration efficacy of the membrane used and/or to certain characteristics of this protein, e.g. its charge (pI 5.7) the adsorption and filtration of [3H] beta2-microglobulin and [3H] lysozyme of similar MW 14.5 kDa, but pI: 10.8 were compared on different membranes. It was found that, neither [3H] beta2-microglobulin nor [3H] lysozyme are removed by cuprophan, whereas over 75% of beta2-microglobulin is removed by filtration on polyacrylonitrile, polyacrylonitrile-polyethyleneimine, polysulfone and >95% by adsorption to polymethylmethacrylate-BK. For lysozyme, removal by adsorption is >95% on polyacrylonitrile and polyacrylonitrile-polyethyleneimine, 72% on polymethylmethacrylate-BK and by filtration is 95% on polysulfone. Hemodialysis membranes must therefore not simply be considered as filters of low-molecular-weight metabolites but should be equally assessed for their capacity to eliminate potentially deleterious low-molecular-weight plasma proteins.

Topics: Adsorption; Amyloidosis; beta 2-Microglobulin; Biocompatible Materials; Dialysis; Filtration; Humans; Kinetics; Membranes, Artificial; Molecular Weight; Muramidase; Proteins; Renal Dialysis; Structure-Activity Relationship

The number of proteins with mutations resulting in amyloidosis has continued to increase. Five proteins--transthyretin, fibrinogen alpha-A chain, apolipoprotein AI, lysozyme, apolipoprotein AII, cystatin C and gelsolin--can be associated with hereditary amyloidosis involving the kidney.. A French family with a history of autosomal dominant hereditary amyloidosis with early sicca syndrome and nephropathy leading to renal failure after the fifth to the seventh decade was studied. Several tissue specimens obtained from the proband and his relatives were examined. Immunohistochemistry was performed on paraffin embedded sections using the indirect immunoperoxidase technique. We searched for mutations in the five exons and flanking introns of the lysozyme gene.. Amyloid deposits from the bowel, labial salivary gland and kidney were intensively stained by anti-lysozyme antibody. Sequence analysis of lysozyme exon 2 from the affected individuals revealed a nucleotide substitution predicting a substitution of the amino acid at position 64 in the mature protein from tryptophane, an aromatic residue to the cationic residue arginine (W64R).. We report a novel mutation (W64R) of the lysozyme that is associated with hereditary amyloidosis and prominent nephropathy. Since the treatment of hereditary amyloidosis greatly varies with the nature of the amyloid protein, thorough characterization of the latter is crucial for the management of the disease.

Topics: Aged; Amino Acid Substitution; Amyloid; Amyloidosis; Base Sequence; France; Genetic Variation; Humans; Immunohistochemistry; Kidney Diseases; Male; Muramidase; Pedigree; Tissue Distribution

Hereditary, autosomal dominant amyloidosis, caused by mutations in the genes encoding transthyretin, fibrinogen A alpha-chain, lysozyme, or apolipoprotein A-I, is thought to be extremely rare and is not routinely included in the differential diagnosis of systemic amyloidosis unless there is a family history.. We studied 350 patients with systemic amyloidosis, in whom a diagnosis of the light-chain (AL) type of the disorder had been suggested by clinical and laboratory findings and by the absence of a family history, to assess whether they had amyloidogenic mutations.. Amyloidogenic mutations were present in 34 of the 350 patients (9.7 percent), most often in the genes encoding fibrinogen A alpha-chain (18 patients) and transthyretin (13 patients). In all 34 of these patients, the diagnosis of hereditary amyloidosis was confirmed by additional investigations. A low-grade monoclonal gammopathy was detected in 8 of the 34 patients (24 percent).. A genetic cause should be sought in all patients with amyloidosis that is not the reactive systemic amyloid A type and in whom confirmation of the AL type cannot be obtained.

Topics: Adult; Aged; Amyloidosis; Amyloidosis, Familial; Apolipoprotein A-I; Diagnostic Errors; DNA Mutational Analysis; Female; Fibrinogen; Genotype; Heterozygote; Humans; Kidney; Middle Aged; Muramidase; Paraproteinemias; Point Mutation; Prealbumin; Radionuclide Imaging

Cryo-electron microscopy studies are presented on amyloid fibrils isolated from amyloidotic organs of two patients with different forms of hereditary non-neuropathic systemic amyloidosis, caused, respectively, by Leu60Arg apolipoprotein AI and Asp67His lysozyme. Although ex vivo amyloid fibrils were thought to be more uniform in structure than those assembled in vitro, our findings show that these fibrils are also quite variable in structure. Structural disorder and variability of the fibrils have precluded three-dimensional reconstruction, but averaged cryo-electron microscopy images suggest models for protofilament packing in the lysozyme fibrils. We conclude that ex vivo amyloid fibrils, although variable, assemble as characteristic structures according to the identity of the precursor protein.

Topics: Amino Acid Substitution; Amyloidosis; Apolipoprotein A-I; Biopolymers; Cryoelectron Microscopy; Humans; Models, Molecular; Muramidase; Mutation, Missense; Protein Structure, Quaternary; Spleen

Wild-type human lysozyme and its two stable amyloidogenic variants have been found to form partially folded states at low pH. These states are characterized by extensive disruption of tertiary interactions and partial loss of secondary structure. Incubation of the proteins at pH 2.0 and 37 degrees C (Ile56Thr and Asp67His variants) or 57 degrees C (wild-type) results in the formation of large numbers of fibrils over several days of incubation. Smaller numbers of fibrils could be observed under other conditions, including neutral pH. These fibrils were analyzed by electron microscopy, Congo red birefringence, thioflavine-T binding, and X-ray fiber diffraction, which unequivocally show their amyloid character. These data demonstrate that amyloidogenicity is an intrinsic property of human lysozyme and does not require the presence of specific mutations in its primary structure. The amyloid fibril formation is greatly facilitated, however, by the introduction of "seeds" of preformed fibrils to the solutions of the variant proteins, suggesting that seeding effects could be important in the development of systemic amyloidosis. Fibril formation by wild-type human lysozyme is greatly accelerated by fibrils of the variant proteins and vice versa, showing that seeding is not specific to a given protein. The fact that wild-type lysozyme has not been found in ex vivo deposits from patients suffering from this disease is likely to be related to the much lower population of incompletely folded states for the wild-type protein compared to its amyloidogenic variants under physiological conditions. These results support the concept that the ability to form amyloid is a generic property of proteins, but one that is mitigated against in a normally functioning organism.

Topics: Amyloid; Amyloidosis; Circular Dichroism; Genetic Variation; Humans; Hydrogen-Ion Concentration; Kinetics; Microscopy, Electron; Muramidase; Point Mutation; Protein Conformation; Protein Folding; Temperature

The unfolding and refolding properties of human lysozyme and two amyloidogenic variants (Ile56Thr and Asp67His) have been studied by stopped-flow fluorescence and hydrogen exchange pulse labeling coupled with mass spectrometry. The unfolding of each protein in 5.4 M guanidine hydrochloride (GuHCl) is well described as a two-state process, but the rates of unfolding of the Ile56Thr variant and the Asp67His variant in 5.4 M GuHCl are ca. 30 and 160 times greater, respectively, than that of the wild type. The refolding of all three proteins in 0.54 M GuHCl at pH 5.0 proceeds through persistent intermediates, revealed by multistep kinetics in fluorescence experiments and by the detection of well-defined populations in quenched-flow hydrogen exchange experiments. These findings are consistent with a predominant mechanism for refolding of human lysozyme in which one of the structural domains (the alpha-domain) is formed in two distinct steps and is followed by the folding of the other domain (the beta-domain) prior to the assembly of the two domains to form the native structure. The refolding kinetics of the Asp67His variant are closely similar to those of the wild-type protein, consistent with the location of this mutation in an outer loop of the beta-domain which gains native structure only toward the end of the refolding process. By contrast, the Ile56Thr mutation is located at the base of the beta-domain and is involved in the domain interface. The refolding of the alpha-domain is unaffected by this substitution, but the latter has the effect of dramatically slowing the folding of the beta-domain and the final assembly of the native structure. These studies suggest that the amyloidogenic nature of the lysozyme variants arises from a decrease in the stability of the native fold relative to partially folded intermediates. The origin of this instability is different in the two variants, being caused in one case primarily by a reduction in the folding rate and in the other by an increase in the unfolding rate. In both cases this results in a low population of soluble partially folded species that can aggregate in a slow and controlled manner to form amyloid fibrils.

Topics: Amyloid; Amyloidosis; Aspartic Acid; Histidine; Humans; Isoleucine; Kinetics; Mass Spectrometry; Models, Molecular; Muramidase; Protein Folding; Protons; Recombinant Proteins; Spectrometry, Fluorescence; Threonine

Two kindreds with hereditary systemic amyloidosis caused by the first two mutations to be described in the human lysozyme gene were discovered recently and study of the variant lysozyme has been powerfully informative about mechanisms of amyloid fibrillogenesis. However, the clinical manifestations in these families, additional members of which have lately been identified, have not previously been reported in detail.. The proband presented with proteinuria aged 50 and a family history of amyloidosis, and underwent renal biopsy, whole-body serum amyloid P component (SAP) scintigraphy, and sequencing of the lysozyme gene. Her family history and the phenotype of hereditary lysozyme amyloidosis were thoroughly documented and compared with the presentation and natural history of all other known patients with this condition.. The proband belonged to an extended English family other members of which were known to have hereditary lysozyme amyloidosis. Those with amyloid in previous generations presented with renal involvement, frequently developed complications due to gastrointestinal amyloid, and died before age 60. All amyloid deposits were composed of lysozyme and complete concordance was established between amyloid and heterozygosity for a point mutation in the lysozyme gene, encoding the previously reported Asp67His substitution in the mature protein.. The phenotype, reported for the first time in this extended kindred, contrasts with that of an apparently unrelated family carrying the same mutation who presented with spontaneous hepatic haemorrhage and rupture, and with the manifestations in a family with the lysozyme Ile56Thr variant who presented with dermal petechiae before proceeding to fatal visceral amyloidosis. A remarkably wide spectrum of disease can be caused by the same amyloid fibril protein, although renal involvement predominates in all cases except those dying of hepatic rupture.

Topics: Amyloidosis; England; Female; Genetic Variation; Humans; Immunohistochemistry; Kidney; Kidney Diseases; Male; Middle Aged; Muramidase; Mutation; Pedigree; Radionuclide Imaging; Serum Amyloid P-Component

Topics: Alzheimer Disease; Amyloid; Amyloidosis; Brain; Humans; Muramidase; Point Mutation; Prion Diseases; Prions; Protein Conformation

Tissue deposition of soluble proteins as amyloid fibrils underlies a range of fatal diseases. The two naturally occurring human lysozyme variants are both amyloidogenic, and are shown here to be unstable. They aggregate to form amyloid fibrils with transformation of the mainly helical native fold, observed in crystal structures, to the amyloid fibril cross-beta fold. Biophysical studies suggest that partly folded intermediates are involved in fibrillogenesis, and this may be relevant to amyloidosis generally.

Topics: Amyloid; Amyloidosis; Circular Dichroism; Cloning, Molecular; Crystallography, X-Ray; Enzyme Stability; Hot Temperature; Humans; Liver Diseases; Models, Molecular; Muramidase; Point Mutation; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Recombinant Proteins

Amyloid deposits regress when the supply of fibril precursor proteins is sufficiently reduced, indicating that amyloid fibrils are degradable in vivo. Serum amyloid P component (SAP), a universal constituent of amyloid deposits, efficiently protects amyloid fibrils from proteolysis in vitro, and may contribute to persistence of amyloid in vivo. Drugs that prevent binding of SAP to amyloid fibrils in vivo should therefore promote regression of amyloid and we are actively seeking such agents. A complementary strategy is identification of critical molecular processes in fibrillogenesis as targets for pharmacological intervention. All amyloidogenic variants of apolipoprotein AI contain an additional positive charge in the N-terminal fibrillogenic region of the protein. This is unlikely to be a coincidence and should be informative about amyloidogenesis by this protein. The two amyloidogenic variants of human lysozyme, caused by the first natural mutations found in its gene, provide a particularly powerful model system because both the crystal structure and folding pathways of wild-type lysozyme are so well characterized. The amyloidogenic variant lysozymes have similar 3D crystal structures to the wild type, but are notably less thermostable. They unfold on heating, lose enzymic activity, and aggregate to form amyloid fibrils in vitro.

Topics: Amyloid; Amyloidosis; Animals; Apolipoprotein A-I; Drug Design; Humans; Mice; Muramidase; Mutation; Serum Amyloid P-Component

The physicochemical properties of an amyloidogenic mutant human lysozyme (Ile56Thr) were examined in order to elucidate the mechanism of amyloid formation. The crystal structure of the mutant protein was the same as the wild-type structure, except that the hydroxyl group of the introduced Thr56 formed a hydrogen bond with a water molecule in the interior of the protein. The other physicochemical properties of the mutant protein in the native state were not different from those of the wild-type protein. However, the equilibrium and kinetic stabilities of the mutant protein were remarkably decreased due to the introduction of a polar residue (Thr) in the interior of the molecule. It can be concluded that the amyloid formation of the mutant human lysozyme is due to a tendency to favor (partly or/and completely) denatured structures.

Topics: Amyloidosis; Crystallography, X-Ray; Humans; Models, Molecular; Muramidase; Mutagenesis; Protein Conformation; Protein Denaturation; Thermodynamics

Hereditary non-neuropathic systemic amyloidosis (Ostertag-type) is a rare autosomal dominant disease in which amyloid deposition in the viscera is usually fatal by the fifth decade. In some families it is caused by mutations in the apolipoprotein AI gene but in two unrelated English families under our care the amyloid deposits did not contain apoAI, despite a report that this may have been the case in one of them. Lysozyme is a ubiquitous bacteriolytic enzyme present in external secretions and in polymorphs and macrophages, but its physiological role is not always clear. Here we report that in these two families, lysozyme is the amyloid fibril protein. Affected individuals are heterozygous for point mutations in the lysozyme gene that cause substitution of highly conserved residues, namely threonine for isoleucine at position 56 in one family, and histidine for aspartic acid at residue 67 in the other. Amyloid fibrils from one individual were composed of the full-length Thr-56 variant lysozyme molecule. To our knowledge, this is the first report of naturally occurring variants of human lysozyme and of lysozyme-associated disease. As the structures of human and hen egg-white lysozyme are known to atomic resolution and their folding and structure-function relationships have been exhaustively analysed, our observations should provide a powerful model for understanding amyloidogenesis.

Topics: Amino Acid Sequence; Amyloidosis; Base Sequence; Heterozygote; Humans; Immunohistochemistry; Male; Models, Molecular; Molecular Sequence Data; Muramidase; Pedigree; Point Mutation

Topics: Amyloidosis; Animals; Glycoproteins; Inflammation; Mice; Muramidase

The effects of 4 proteolytic enzymes, alpha-chymotrypsin, bromeline, collagenase, and lysozyme on amyloid tissue sections from a patient with familial amyloidotic polyneuropathy (FAP) were evaluated. Degradation of amyloid fibrils was significant with alpha-chymotrypsin, moderate with bromeline and collagenase, and slight with lysozyme. All of these proteases except collagenase are used as oral mucolytics in humans. The possibility of their clinical usefulness in the treatment or prevention of the development of FAP is discussed.

Topics: Amyloid; Amyloidosis; Bromelains; Chymotrypsin; Humans; In Vitro Techniques; Kidney; Microbial Collagenase; Muramidase; Peptide Hydrolases; Peripheral Nervous System Diseases

Spontaneous amyloidosis was noted in a significant number of germfree mice in comparison with their conventional contemporaries. The adjusted prevalence of this disease was increased in both groups by whole-body exposure at 6 weeks of age to 700 rad of ionizing radiation. The germfree groups demonstrated persistent hypogammaglobulinemia throughout their lifespans and no evidence of significant inflammatory processes at necropsy. The possible interpretation of these observations is discussed and it is concluded that defective or deficient immunoglobulin production may be the essential prerequisite for the development of amyloidosis.

Topics: Agammaglobulinemia; Age Factors; Amyloid; Amyloidosis; Animals; Aspartate Aminotransferases; Blood Chemical Analysis; Blood Proteins; Blood Urea Nitrogen; Cobalt Isotopes; Disease Models, Animal; Female; Germ-Free Life; Immunosuppression Therapy; Kidney; L-Lactate Dehydrogenase; Lymphoma, Non-Hodgkin; Mice; Muramidase; Radiation Effects; Serum Albumin; Serum Globulins; Thymus Neoplasms

Topics: Adolescent; Adult; Aged; Aging; Amyloidosis; Animals; Antibody Formation; Arteritis; Autoantibodies; Blood Group Antigens; Cattle; Cell Nucleus; Child; Child, Preschool; Cricetinae; Dental Caries; Dogs; Erythrocytes; Fishes; Haplorhini; Humans; Immunity; Infant; Infant, Newborn; Isoantibodies; Kidney; Lymphatic System; Lymphoma; Mice; Middle Aged; Muramidase; Rheumatoid Factor; Serum Globulins; Stomach; Temperature; Thyroid Gland

Topics: Amyloidosis; Animals; Antigens; Caseins; Dextrans; Freund's Adjuvant; Mice; Muramidase; Serum Albumin, Bovine; Splenic Diseases

Topics: Aging; Amyloidosis; Animals; Appendix; Autoantibodies; Azathioprine; Cricetinae; Fishes; Hemolysin Proteins; Immune Tolerance; Kidney; Lymph Nodes; Lymphoid Tissue; Male; Mice; Muramidase; Organ Size; Parabiosis; Radiation Effects; Spleen; Transplantation Immunology