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1.
Adv Exp Med Biol ; 1221: 631-645, 2020.
Article in English | MEDLINE | ID: mdl-32274729

ABSTRACT

Amyloidosis refers to a group of diseases characterized by abnormal deposition of denatured endogenous proteins, termed amyloid, in the affected organs. Analysis of biopsy and autopsy tissues from patients revealed the presence of heparan sulfate proteoglycans (HSPGs) along with amyloid proteins in the deposits. For a long time, HSPGs were believed to occur in the deposits as an innocent bystander. Yet, the consistent presence of HSPGs in various deposits, regardless of the amyloid species, led to the hypothesis that these macromolecular glycoconjugates might play functional roles in the pathological process of amyloidosis. In vitro studies have revealed that HSPGs, or more precisely, the heparan sulfate (HS) side chains interact with amyloid peptides, thus promoting amyloid fibrillization. Although information on the mechanisms of HS participation in amyloid deposition is limited, recent studies involving a transgenic mouse model of Alzheimer's disease point to an active role of HS in amyloid formation. Heparanase cleavage alters the molecular structure of HS, and thus modulates the functional roles of HS in homeostasis, as well as in diseases, including amyloidosis. The heparanase transgenic mice have provided models for unveiling the effects of heparanase, through cleavage of HS, in various amyloidosis conditions.


Subject(s)
Amyloidosis/metabolism , Glucuronidase/metabolism , Heparitin Sulfate/metabolism , Alzheimer Disease/enzymology , Alzheimer Disease/metabolism , Amyloidosis/enzymology , Animals , Heparan Sulfate Proteoglycans , Heparitin Sulfate/chemistry , Humans
2.
J Biol Chem ; 287(44): 37206-18, 2012 Oct 26.
Article in English | MEDLINE | ID: mdl-22918834

ABSTRACT

The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.


Subject(s)
Amyloid/physiology , Chromatin/metabolism , Neutrophils/pathology , Acetophenones/pharmacology , Amyloid/chemistry , Amyloid/genetics , Amyloid Neuropathies, Familial/enzymology , Amyloid Neuropathies, Familial/genetics , Amyloid Neuropathies, Familial/pathology , Amyloidosis/enzymology , Amyloidosis/metabolism , Amyloidosis/pathology , Animals , Biomarkers/metabolism , Cell Nucleus/metabolism , Cell Survival/drug effects , Chromatin/enzymology , Cricetinae , Extracellular Space/enzymology , Extracellular Space/metabolism , Hep G2 Cells , Humans , Immunoglobulin Light-chain Amyloidosis , Lung/enzymology , Lung/metabolism , Lung/pathology , Mutation, Missense , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/metabolism , Neutrophils/enzymology , Neutrophils/metabolism , Onium Compounds/pharmacology , Pancreatic Elastase , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Peptide Fragments/physiology , Prealbumin/chemistry , Prealbumin/genetics , Prealbumin/physiology , Protein Structure, Quaternary , Proteolysis , Reactive Oxygen Species/metabolism , Skin/enzymology , Skin/metabolism , Skin/pathology , alpha-Synuclein/chemistry , alpha-Synuclein/genetics , alpha-Synuclein/physiology
3.
FASEB J ; 26(1): 192-202, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21965601

ABSTRACT

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.


Subject(s)
Amyloidosis/enzymology , Eye Abnormalities/enzymology , Muramidase/metabolism , Unfolded Protein Response/physiology , Amyloidosis/pathology , Animals , Animals, Genetically Modified , DNA-Binding Proteins/genetics , Disease Models, Animal , Drosophila Proteins/genetics , Drosophila melanogaster , Endoplasmic Reticulum Stress/physiology , Eye Abnormalities/genetics , Eye Abnormalities/pathology , Female , Green Fluorescent Proteins/genetics , Hemolymph/enzymology , Humans , Male , Metamorphosis, Biological/physiology , Microscopy, Electron, Scanning , Muramidase/genetics , Photoreceptor Cells, Invertebrate/enzymology , Photoreceptor Cells, Invertebrate/pathology , Photoreceptor Cells, Invertebrate/ultrastructure , Solubility
4.
Curr Protein Pept Sci ; 22(2): 170-189, 2021.
Article in English | MEDLINE | ID: mdl-33292151

ABSTRACT

Various neurodegenerative disorders have various molecular origins but some common molecular mechanisms. In the current scenario, there are very few treatment regimens present for advanced neurodegenerative diseases. In this context, there is an urgent need for alternate options in the form of natural compounds with an ameliorating effect on patients. There have been individual scattered experiments trying to identify potential values of various intracellular metabolites. Purines and Pyrimidines, which are vital molecules governing various aspects of cellular biochemical reactions, have been long sought as crucial candidates for the same, but there are still many questions that go unanswered. Some critical functions of these molecules associated with neuromodulation activities have been identified. They are also known to play a role in foetal neurodevelopment, but there is a lacuna in understanding their mechanisms. In this review, we have tried to assemble and identify the importance of purines and pyrimidines, connecting them with the prevalence of neurodegenerative diseases. The leading cause of this class of diseases is protein misfolding and the formation of amyloids. A direct correlation between loss of balance in cellular homeostasis and amyloidosis is yet an unexplored area. This review aims at bringing the current literature available under one umbrella serving as a foundation for further extensive research in this field of drug development in neurodegenerative diseases.


Subject(s)
Gene Expression Regulation/drug effects , Metabolic Networks and Pathways/genetics , Purines/therapeutic use , Pyrimidines/therapeutic use , Alzheimer Disease/drug therapy , Alzheimer Disease/enzymology , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloidosis/drug therapy , Amyloidosis/enzymology , Amyloidosis/genetics , Amyloidosis/pathology , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Humans , Huntington Disease/drug therapy , Huntington Disease/enzymology , Huntington Disease/genetics , Huntington Disease/pathology , Hypoxanthine Phosphoribosyltransferase/genetics , Hypoxanthine Phosphoribosyltransferase/metabolism , Multiple Sclerosis/drug therapy , Multiple Sclerosis/enzymology , Multiple Sclerosis/genetics , Multiple Sclerosis/pathology , Neurofibrillary Tangles/drug effects , Neurofibrillary Tangles/enzymology , Neurofibrillary Tangles/genetics , Neurofibrillary Tangles/pathology , Neurons/drug effects , Neurons/enzymology , Neurons/pathology , Parkinson Disease/drug therapy , Parkinson Disease/enzymology , Parkinson Disease/genetics , Parkinson Disease/pathology , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Purines/metabolism , Pyrimidines/metabolism , Synapses/drug effects , Thymidine Phosphorylase/genetics , Thymidine Phosphorylase/metabolism
5.
Int J Biol Macromol ; 148: 102-109, 2020 Apr 01.
Article in English | MEDLINE | ID: mdl-31945445

ABSTRACT

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.


Subject(s)
Amyloid/chemistry , Amyloidosis/enzymology , Minerals/chemistry , Muramidase/chemistry , Plant Extracts/chemistry , Protein Aggregates/drug effects , Drug Compounding , Egg Proteins/chemistry , Medicine, Ayurvedic , Plants, Medicinal , Protein Structure, Secondary
6.
Neurochem Res ; 34(4): 717-26, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19199031

ABSTRACT

Alzheimer's disease, responsible for the vast majority of dementia cases in the elderly population, is caused by accumulation of toxic levels of amyloid beta peptide (A beta) in the brain. Neprilysin is a major enzyme responsible for the degradation of A beta in vivo. We have previously shown that elevation of neprilysin levels in the brain delays the deposition of A beta-plaques in a mouse model of amyloidosis and that lack of neprilysin leads to increased A beta generation and to signs of incipient neurodegeneration in mouse brains. This study was designed to test whether low brain levels of neprilysin affect the amyloid pathology or perturb the learning and memory performance of mice. Double-mutated mice carrying a targeted depletion of one allele of Mme, the gene encoding neprilysin, and over-expressing human amyloid precursor protein (APP), exhibited a reinforced amyloid pathology in comparison with their APP transgenic littermates. Moreover, in contrast to their parental lines, these mice were impaired in the Morris water maze learning and memory paradigm and showed facilitated extinction in the conditioned taste aversion test. These data suggest that even a partial neprilysin deficiency, as is found during aging, exacerbates amyloid pathology and may impair cognitive functions.


Subject(s)
Amyloid beta-Peptides/metabolism , Amyloidosis/enzymology , Cognition Disorders/enzymology , Neprilysin/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloidosis/pathology , Amyloidosis/psychology , Animals , Avoidance Learning , Behavior, Animal , Cognition Disorders/pathology , Cognition Disorders/psychology , Hippocampus/physiopathology , Maze Learning , Mice , Mice, Transgenic , Neprilysin/genetics , Plaque, Amyloid/pathology
7.
Ren Fail ; 31(1): 13-7, 2009.
Article in English | MEDLINE | ID: mdl-19142804

ABSTRACT

The aims of this study are to compare urinary protein excretion pattern with renal morphological findings and to find out whether urinary protein excretion pattern is a prognostic indicator of renal amyloidosis. Fifteen children with renal amyloidosis secondary to familial Mediterranean fever were included in the study. The patients were classified into three groups according to the degree of tubulointerstitial injury in renal biopsy (group 1, <25%; group 2, 25-50%; and group 3, >50%). In all patients, urinary protein electrophoresis were performed. Levels of urinary beta(2)-microglobulin, retinol binding protein, and beta.N-acetyl-D glucosaminidase were measured as markers for tubular injury, and urinary excretions of protein and albumin and plasma albumin levels were measured as markers of glomerular injury. While urinary excretions of protein and albumin and plasma albumin levels were not different between groups, higher urinary beta(2-)microglobulin and retinol binding protein values and lower creatinine clearance values were found in group 3 than in groups 1 and 2 (p < 0.05). We concluded that analysis of urinary protein excretion pattern is a non-invasive and reliable method to detect the degree of tubulointerstitial injury as the most important prognostic factor in renal amyloidosis and may be used to determine the changes during the follow-up period of the patients.


Subject(s)
Amyloidosis/pathology , Amyloidosis/urine , Kidney Diseases/pathology , Kidney Diseases/urine , Proteinuria/pathology , Acetylglucosaminidase/metabolism , Adolescent , Amyloidosis/enzymology , Child , Cohort Studies , Creatinine/metabolism , Female , Humans , Kidney Diseases/enzymology , Male , Predictive Value of Tests , Proteinuria/blood , Proteinuria/enzymology , Reproducibility of Results , Retinol-Binding Proteins/metabolism , Serum Albumin/metabolism , beta 2-Microglobulin/metabolism
8.
Nephrology (Carlton) ; 13(2): 104-8, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18275497

ABSTRACT

BACKGROUND: It is recognized that matrix metalloproteinase-3 (MMP-3) is abundantly expressed in active rheumatoid synovium, and that serum level of MMP-3 is a useful marker for diagnosis of rheumatoid arthritis and for evaluation of prognosis in joint destruction. Little is known about serum MMP-3 levels in haemodialysis (HD) patients, and thus, the association between serum MMP-3 and dialysis-related amyloidosis (DRA) has yet to be elucidated. METHODS: Serum levels of MMP-3 were measured by enzyme immunoassay in 150 HD patients, 90 without DRA and 60 with DRA, before HD. Simple regression analysis was performed to investigate the relationship between serum level of MMP-3 and clinical parameters, including age, HD duration, C-reactive protein and beta2 microglobulin (BMG). RESULTS: Serum levels of MMP-3 were significantly higher in HD patients with DRA than in HD patients without DRA (258.2 +/- 118.1 vs 201.5 +/- 98.4 pg/mL, P = 0.0017), and both levels were significantly higher than those of healthy subjects (45.6 +/- 13.4 pg/mL, P < 0.0001). Serum MMP-3 levels significantly correlated with serum levels of BMG (r = 0.197, P = 0.0164) and HD duration (r = 0.168, P = 0.0427). Moreover, serum MMP-3 levels significantly correlated with serum BMG levels in HD patients without DRA (r = 0.341, P = 0.0012), but not in HD patients with DRA. CONCLUSION: Our results suggest that matrix metalloproteinase activity increases in HD patients, which may be associated with BMG and DRA.


Subject(s)
Amyloidosis/enzymology , Kidney Failure, Chronic/therapy , Matrix Metalloproteinase 3/blood , Renal Dialysis/adverse effects , Aged , Amyloidosis/etiology , Biomarkers/blood , C-Reactive Protein/metabolism , Female , Humans , Kidney Failure, Chronic/enzymology , Male , Middle Aged , Time Factors , Treatment Outcome , Up-Regulation , beta 2-Microglobulin/blood
9.
Hum Mutat ; 27(1): 119-20, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16329101

ABSTRACT

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.


Subject(s)
Amyloidosis/enzymology , Amyloidosis/genetics , Exons/genetics , Heterozygote , Muramidase/genetics , Adult , Amino Acid Sequence , Amyloid/metabolism , Blotting, Western , Colon/pathology , DNA Mutational Analysis , Duodenum/pathology , Electrophoresis, Polyacrylamide Gel , Humans , Immunohistochemistry , Male , Molecular Sequence Data , Muramidase/chemistry , Protein Structure, Secondary
10.
Medicine (Baltimore) ; 85(1): 66-73, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16523055

ABSTRACT

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.


Subject(s)
Amyloidosis/complications , Amyloidosis/physiopathology , Muramidase/metabolism , Adult , Aged , Amyloidosis/enzymology , Female , Gastrointestinal Diseases/etiology , Hemorrhage , Humans , Kidney Diseases/etiology , Lymph Nodes/pathology , Male , Middle Aged , Rupture
11.
FEBS J ; 273(4): 711-20, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16441658

ABSTRACT

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.


Subject(s)
Isoenzymes/chemistry , Muramidase/chemistry , Pichia/metabolism , Amyloidosis/enzymology , Enzyme Stability , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Models, Molecular , Muramidase/genetics , Muramidase/metabolism , Pichia/genetics , Protein Denaturation , Protein Structure, Tertiary , RNA, Messenger/metabolism
12.
Sci Transl Med ; 7(314): 314ra184, 2015 Nov 18.
Article in English | MEDLINE | ID: mdl-26582899

ABSTRACT

Apolipoprotein E (ApoE) is an important modifier of Alzheimer's disease (AD) pathogenesis, and its abundance has been linked to the clearance of ß-amyloid (Aß) in the brain. The pathways that control the clearance of ApoE in the brain are incompletely understood. We report that Idol, an E3 ubiquitin ligase that targets the low-density lipoprotein receptor (LDLR) for degradation, is a critical determinant of brain ApoE metabolism and Aß plaque biogenesis. Previous work has shown that Idol contributes minimally to the regulation of hepatic LDLR expression in mice. By contrast, we demonstrate that Idol is a primary physiological regulator of LDLR protein in the brain, controlling the clearance of both ApoE-containing high-density lipoprotein (HDL) particles and Aß. We studied the consequences of loss of Idol expression in a transgenic mouse model of Aß amyloidosis. Idol deficiency increased brain LDLR, decreased ApoE, decreased soluble and insoluble Aß, reduced amyloid plaque burden, and ameliorated neuroinflammation. These findings identify Idol as a gatekeeper of LDLR-dependent ApoE and Aß clearance in the brain and a potential enzyme target for therapeutic intervention in AD.


Subject(s)
Alzheimer Disease/enzymology , Amyloid beta-Peptides/metabolism , Amyloidosis/enzymology , Apolipoproteins E/metabolism , Brain/enzymology , Receptors, LDL/metabolism , Ubiquitin-Protein Ligases/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Alzheimer Disease/therapy , Amyloid beta-Peptides/genetics , Amyloidosis/genetics , Amyloidosis/pathology , Amyloidosis/therapy , Animals , Brain/pathology , Cell Line , Disease Models, Animal , Female , Genetic Predisposition to Disease , Male , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Plaque, Amyloid , Proteolysis , Time Factors , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics
13.
Curr Drug Targets ; 3(2): 155-73, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11958298

ABSTRACT

The aspartic proteinases are a family of enzymes involved in a number of important biological processes. In animals the enzyme renin has a hypertensive action through its role in the renin-angiotensin system. The retroviral aspartic proteinases, such as the HIV proteinase, are essential for maturation of the virus particle and inhibitors have a proven therapeutic record in the treatment of AIDS. The lysosomal aspartic proteinase cathepsin D has been implicated in tumorigenesis and the stomach enzyme pepsin, which plays a major physiological role in hydrolysis of acid-denatured proteins, is responsible for much of the tissue damage in peptic ulcer disease. Since aspartic proteinases also play major roles in amyloid disease, malaria and common fungal infections such as candidiasis, inhibitors to these enzymes are much sought after as potential therapeutic agents. In all aspartic proteinases, the catalytic aspartate residues are involved in an intricate arrangement of hydrogen bonds involving a solvent molecule which is presumed to be water. The catalytic mechanism is thought to involve nucleophilic attack of the active site water molecule on the scissile bond carbonyl generating a tetrahedral gem-diol intermediate. The design of inhibitors generally involves the use of short oligopeptides containing a transition state analogue which mimic this tetrahedral intermediate. The application of structure-based drug design to members of the aspartic proteinase family is the main subject of this review.


Subject(s)
Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/physiology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , Amyloidosis/drug therapy , Amyloidosis/enzymology , Animals , Aspartic Acid Endopeptidases/antagonists & inhibitors , HIV Infections/drug therapy , HIV Infections/enzymology , Humans , Hypertension/drug therapy , Hypertension/enzymology , Technology, Pharmaceutical/methods
14.
Am J Clin Pathol ; 98(5): 534-41, 1992 Nov.
Article in English | MEDLINE | ID: mdl-1485607

ABSTRACT

Severe bleeding resulting from excessive fibrinolysis has been observed in patients with primary amyloidosis. The authors studied a patient with this hemostatic disorder before and during therapy with epsilon-aminocaproic acid. Excessive fibrinolysis was associated with depressed plasma concentrations of coagulation Factors XII, XI, high-molecular-weight kininogen, and Factors VIII and V; and plasminogen and alpha-2-plasmin inhibitor. These deficiencies were corrected with treatment. The functional and antigenic concentrations of tissue plasminogen activator and plasminogen activator inhibitor in the patient's plasma were normal. Urokinase-type activator activity and antigen were three to five times elevated in the patient's plasma. Results of immunoprecipitation showed that single-chain urokinase-type activator was the primary urokinase-type activator species in the patient's plasma. Excessive fibrinolysis in patients with amyloidosis results from increased plasma single-chain urokinase-type activator activity.


Subject(s)
Amyloidosis/enzymology , Amyloidosis/physiopathology , Fibrinolysis/physiology , Urokinase-Type Plasminogen Activator/blood , Amyloidosis/blood , Factor V/analysis , Factor VII/analysis , Factor XI/analysis , Factor XII/analysis , Humans , Kininogens/analysis , Male , Middle Aged , Plasminogen Activators/blood , Plasminogen Inactivators/blood , Precipitin Tests
15.
J Biochem ; 120(6): 1216-23, 1996 Dec.
Article in English | MEDLINE | ID: mdl-9010773

ABSTRACT

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.


Subject(s)
Amyloidosis/enzymology , Muramidase/genetics , Amyloidosis/genetics , Crystallography, X-Ray , Humans , Models, Molecular , Mutagenesis , Protein Conformation , Protein Denaturation , Thermodynamics
16.
Virchows Arch ; 428(1): 37-46, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8646367

ABSTRACT

Immunolocalization of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in periarticular tissues of beta 2-microglobulin amyloidosis patients was investigated. MMP-1 (interstitial collagenase) the most strongly expressed of the MMPs, was localized in the synovial lining cells, mesenchymal cells in granulation tissue and nodular amyloid deposits, and chondrocytes within areas of cartilage erosion. Expression of MMP-1 was correlated with the degree of macrophage infiltration and synovial cell hyperplasia, but it was not correlated with the degree of amyloid deposition or haemodialysis period. Expression of MMP-1 appeared more intense than that of TIMP-1 and TIMP-2 in highly inflammatory cases. MMP-2 was mildly expressed in the interstitial fibroblasts and MMP-3 was faintly stained in the extracellular matrix of the synovial membrane. MMP-9 (gelatinase B) was found to be strongly positive in the osteoclasts which increased in the progressing osteolytic lesion from the destructive arthropathy. These results suggest involvement of MMPs in inflammation with an imbalance between expression of MMPs and TIMPs being closely related to pathogenesis of the destructive arthropathy.


Subject(s)
Amyloidosis/etiology , Amyloidosis/pathology , Cartilage, Articular/pathology , Metalloendopeptidases/adverse effects , Metalloendopeptidases/biosynthesis , Renal Dialysis/adverse effects , beta 2-Microglobulin/metabolism , Amyloidosis/enzymology , Arthritis, Rheumatoid/enzymology , Arthritis, Rheumatoid/etiology , Arthritis, Rheumatoid/pathology , Cartilage, Articular/enzymology , Glycoproteins/metabolism , Humans , Metalloendopeptidases/antagonists & inhibitors , Protease Inhibitors/metabolism , Synovitis/enzymology , Synovitis/etiology , Synovitis/pathology , Tissue Inhibitor of Metalloproteinases
17.
Brain Res ; 513(2): 181-92, 1990 Apr 16.
Article in English | MEDLINE | ID: mdl-2350688

ABSTRACT

To investigate the role of proteolysis in amyloid formation, we studied the localization of the proteolytic enzymes, cathepsin D and cathepsin B, in the prefrontal cerebral cortex and hippocampus of human postmortem brains from patients with Alzheimer's disease and from individuals free of neurological disease. In control and Alzheimer brains, cathepsin immunoreactivity within cells was localized to lysosome-related structures, which were particularly abundant in neuronal perikarya. In Alzheimer brain, cathepsin immunoreactivity was also heavily concentrated extracellularly within senile plaques. Cathepsin immunoreactivity associated with plaques was not confined to lysosomes and was distributed throughout the plaque. Isolated amyloid cores, however, were not immunostained. Cathepsin-laden perikarya of degenerating neurons were frequently seen within senile plaques and, in the more advanced stages of degeneration, cathepsin immunoreactivity was present throughout the cytoplasm. Other identified constituents of senile plaques appeared to be less significant sources of cathepsin immunoreactivity, including astrocytes, degenerating neurites, microglia and macrophages. These results demonstrate that lysosomal proteinases are major constituents of the senile plaque and that degenerating neuronal perikarya are a principal source of the cathepsin immunoreactivity. We propose that the unregulated action of extracellular cathepsins liberated from degenerating neurons may lead to abnormal processing of the amyloid precursor protein and to the formation of amyloid locally within senile plaques in Alzheimer's disease.


Subject(s)
Alzheimer Disease/enzymology , Amyloidosis/enzymology , Cathepsin B/metabolism , Cathepsin D/metabolism , Cerebral Cortex/enzymology , Hippocampus/enzymology , Lysosomes/enzymology , Aged , Aged, 80 and over , Alzheimer Disease/pathology , Amyloidosis/pathology , Cerebral Cortex/pathology , Hippocampus/pathology , Humans , Immunohistochemistry , Middle Aged
18.
J Neurol Sci ; 84(2-3): 295-9, 1988 Apr.
Article in English | MEDLINE | ID: mdl-2837542

ABSTRACT

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.


Subject(s)
Amyloid/metabolism , Amyloidosis/enzymology , Kidney/enzymology , Peptide Hydrolases/metabolism , Peripheral Nervous System Diseases/enzymology , Amyloidosis/genetics , Bromelains/metabolism , Chymotrypsin/metabolism , Humans , In Vitro Techniques , Microbial Collagenase/metabolism , Muramidase/metabolism , Peripheral Nervous System Diseases/genetics
19.
J Neurol Sci ; 81(1): 79-84, 1987 Oct.
Article in English | MEDLINE | ID: mdl-3316509

ABSTRACT

Serum levels of 6 protease inhibitors, alpha 1-antitrypsin, Cl inactivator, alpha 2-macroglobulin, antithrombin-3, alpha 1-antichymotrypsin and inter-alpha-trypsin inhibitor were measured in patients with familial amyloidotic polyneuropathy (FAP) and a control group without neurologic disease. No significant differences were observed between the 2 groups. The proteolytic effect of brinase, an enzyme from Aspergillus oryzae, on amyloid tissue sections from patients with FAP was also evaluated. Amyloid fibrils were degraded by brinase, while the tissue structure remained fairly intact.


Subject(s)
Amyloid/metabolism , Amyloidosis/enzymology , Brinolase/pharmacology , Peptide Hydrolases/pharmacology , Protease Inhibitors/blood , Amyloidosis/genetics , Humans , Kidney/drug effects , Kidney/metabolism
20.
Vestn Ross Akad Med Nauk ; (2): 58-61, 1999.
Article in Russian | MEDLINE | ID: mdl-10204025

ABSTRACT

To estimate the individual role of the plasminogen activators (PA) urokinase (u-PA) and tissue (t-PA) in the development of two renal diseases (the nephrotic forms of chronic glomerulonephritis (CGN) and amyloidosis, the baseline plasma and urine levels of u-PA and t-PA antigens, their functional activity (FPAA), and changes in these parameters were determined after protein loading test (0.7 g/kg). In healthy individuals and patients with amyloidosis, the baseline FPAA changes from 0 to the maximum were caused only by the alterations of u-PA levels, in those with CGN, they were induced by the changes in the content of u-PA and t-AP antigens. The functional loading test revealed PA reserves solely in patients having a high baseline FPAA for both nephropathies: u-PA in amyloidosis and t-PA in CGN. In all the patients, the urine levels of u-PA antigens were 20-40 times more than those of t-PA antigens and 5-6 times less than those plasma u-PA. The findings suggest that urokinase may be regarded as the major plasminogen activator involved in CGN and amyloidosis.


Subject(s)
Amyloidosis/enzymology , Glomerulonephritis/enzymology , Plasminogen Activators/metabolism , Urokinase-Type Plasminogen Activator/metabolism , Amyloidosis/blood , Amyloidosis/urine , Biomarkers/blood , Biomarkers/urine , Chronic Disease , Enzyme-Linked Immunosorbent Assay , Follow-Up Studies , Glomerulonephritis/blood , Glomerulonephritis/urine , Humans , Prognosis
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