Purines and Pyrimidines: Metabolism, Function and Potential as Therapeutic Options in Neurodegenerative Diseases.

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.

Implications of Heparan Sulfate and Heparanase in Amyloid Diseases.

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.

Herbalome of Chandraprabha vati, a polyherbal formulation of Ayurveda prevents fibrillation of lysozyme by stabilizing aggregation-prone intermediate state.

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.

The E3 ubiquitin ligase Idol controls brain LDL receptor expression, ApoE clearance, and Aß amyloidosis.

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.

Trientine reduces BACE1 activity and mitigates amyloidosis via the AGE/RAGE/NF-κB pathway in a transgenic mouse model of Alzheimer's disease.

AIMS: There is mounting evidence that the transition metal copper may play an important role in the pathophysiology of Alzheimer's disease (AD). Triethylene tetramine dihydrochloride (trientine), a CuII-selective chelator, is a commonly used treatment for Wilson's disease to decrease accumulated copper, and thereby decreases oxidative stress. In the present study, we evaluated the effects of a 3-month treatment course of trientine (Trien) on amyloidosis in 7-month-old ß-amyloid (Aß) precursor protein and presenilin-1 (APP/PS1) double transgenic (Tg) AD model mice. RESULTS: We observed that Trien reduced the level of advanced glycation end products (AGEs), and decreased Aß deposition and synapse loss in brain of APP/PS1 mice. Importantly, we found that Trien blocked the receptor for AGEs (RAGE), downregulated ß-site APP cleaving enzyme 1 (BACE1), inhibited amyloidogenic APP cleavage, and subsequently reduced Aß levels. In vitro, in SH-SY5Y cells overexpressing Swedish mutant APP, Trien-mediated downregulation of BACE1 occurred via inhibition of the NF-κB signaling pathway. INNOVATION: In this study, we demonstrated for the first time that Trien inhibited amyloidogenic pathway including targeting the downregulation of RAGE and NF-κB. CONCLUSION: Trien might mitigate amyloidosis in AD by inhibiting the RAGE/NF-κB/BACE1 pathway. Our study demonstrates that Trien may be a viable therapeutic strategy for the intervention and treatment of AD and other AD-like pathologies.

Amyloid fibrils trigger the release of neutrophil extracellular traps (NETs), causing fibril fragmentation by NET-associated elastase.

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.

Accelerated resolution of AA amyloid in heparanase knockout mice is associated with matrix metalloproteases.

AA-amyloidosis is a disease characterized by abnormal deposition of serum A amyloid (SAA) peptide along with other components in various organs. The disease is a complication of inflammatory conditions that cause persistent high levels of the acute phase reactant SAA in plasma. In experimental animal models, the deposited amyloid is resolved when the inflammation is stopped, suggesting that there is an efficient clearance mechanism for the amyloid. As heparan sulfate (HS) is one of the major components in the amyloid, its metabolism is expected to affect the pathology of AA amyloidosis. In this study, we investigated the effect of heparanase, a HS degradation enzyme, in resolution of the AA amyloid. The transgenic mice deficient in heparanase (Hpa-KO) produced a similar level of SAA in plasma as the wildtype control (Ctr) mice upon induction by injection of AEF (amyloid enhancing factor) and inflammatory stimuli. The induction resulted in formation of SAA amyloid 7-days post treatment in the spleen that displayed a comparable degree of amyloid load in both groups. The amyloid became significantly less in the Hpa-KO spleen than in the Ctr spleen 10-days post treatment, and was completely resolved in the Hpa-KO spleen on day 21 post induction, while a substantial amount was still detected in the Ctr spleen. The rapid clearance of the amyloid in the Hpa-KO mice can be ascribed to upregulated matrix metalloproteases (MMPs) that are believed to contribute to degradation of the protein components in the AA amyloid. The results indicate that both heparanase and MMPs play important parts in the pathological process of AA amyloidosis.

A preclinical assessment of neural stem cells as delivery vehicles for anti-amyloid therapeutics.

Transplantation of neural stems cells (NSCs) could be a useful means to deliver biologic therapeutics for late-stage Alzheimer's disease (AD). In this study, we conducted a small preclinical investigation of whether NSCs could be modified to express metalloproteinase 9 (MMP9), a secreted protease reported to degrade aggregated Aß peptides that are the major constituents of the senile plaques. Our findings illuminated three issues with using NSCs as delivery vehicles for this particular application. First, transplanted NSCs generally failed to migrate to amyloid plaques, instead tending to colonize white matter tracts. Second, the final destination of these cells was highly influenced by how they were delivered. We found that our injection methods led to cells largely distributing to white matter tracts, which are anisotropic conduits for fluids that facilitate rapid distribution within the CNS. Third, with regard to MMP9 as a therapeutic to remove senile plaques, we observed high concentrations of endogenous metalloproteinases around amyloid plaques in the mouse models used for these preclinical tests with no evidence that the NSC-delivered enzymes elevated these activities or had any impact. Interestingly, MMP9-expressing NSCs formed substantially larger grafts. Overall, we observed long-term survival of NSCs in the brains of mice with high amyloid burden. Therefore, we conclude that such cells may have potential in therapeutic applications in AD but improved targeting of these cells to disease-specific lesions may be required to enhance efficacy.

Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster.

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.

Alanine-to-threonine substitutions and amyloid diseases: butyrylcholinesterase as a case study.

Alanine-to-threonine (A to T) substitutions caused by single nucleotide polymorphisms (SNPs) occur in diverse proteins, and in certain cases these substitutions induce self-aggregation into amyloid fibrils or aggregation in other amyloidogenic proteins. This is compatible with the inverse preferences of alanine to form helices and of threonine to support beta-sheet structures, which are crucial for amyloid fibrils formation. Our interest in these mutations was initiated by studying the potential effects of the A539T substitution in the butyrylcholinesterase BChE-K variant on amyloid fibrils formation in Alzheimer's disease. Other examples are, Parkinson's disease (PD), where A53T alpha-synuclein occurs in Lewy bodies and familial amyloid polyneuropathy (FAP), where an A25T substitution appears in transthyretin (TTR). In peripheral organs, an A34T substitution is found in the light chain immunoglobulin genes of patients with systemic amyloidosis and in familial hypercholesterolemia, an A370T substitution occurs in the LDLR regulator of cholesterol homeostasis. That such substitutions appear in proteins with important cellular functions suggests that they confer antagonistic pleiotropy, providing added value at an earlier age but causing damages and inducing amyloid diseases later on. This, in turn, may explain the evolutionary selection and preservation of these substitutions. The structural effect of residue substitutions and in particular A to T substitutions in amyloidogenic diseases thus merits further attention.

Neprilysin deficiency-dependent impairment of cognitive functions in a mouse model of amyloidosis.

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.

Is the urinary protein excretion pattern compatible with renal morphological findings in renal amyloidosis?

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.

Excessive fibrinolysis in AL-amyloidosis is induced by urokinae-type plasminogen activator from bone marrow plasma cells.

Activation of fibrinolysis system and excessive fibrinolysis are observed in monoclonal antibody light chain (AL)-amyloidosis. However, the mechanisms by which activation of fibrinolysis occurs in AL-amyloidosis have not been fully elucidated. To determine whether urokinase type-plasminogen activator (uPA), an important activator of fibrinolytic system, contributes to the activation of fibrinolytic system in AL-amyloidosis, we immunohistologically examined uPA in bone marrow plasma cells. More than 90% of bone marrow plasma cells from five different AL-amyloidosis patients were uPA-positive as examined with immunohistochemical staining. All the bone marrow plasma cells from seven different patients with multiple myeloma were uPA-negative. A patient with AL-amyloidosis, who had bleeding diathesis and excessive fibrinolysis with hypofibrinogenemia, was treated with nafamostat mesilate, a potential inhibitor of uPA. After the administration of nafamostat mesilate, the bleeding diathesis disappeared, and excessive fibrinolysis and hypofibrinogenemia were improved. The present data suggested that uPA expressed in plasma cells may have contributed to the pathogenesis of excessive fibrinolysis.

[kappa-light chain amyloidosis of the liver, a rare cause of liver enzyme elevation]. / Leichtkettenamyloidose der Leber vom kappa-Typ. Seltene Ursache einer cholestatischen Leberenzymkonstellation.

HISTORY AND ADMISSION FINDINGS: A 69-year-old man was admitted to the department of gastroenterology having for months had persistently elevated liver enzymes after discontinuing systemic antimycotic therapy. He reported loosing five kilogram of body weight in the past six months. No macroscopic or microscopic abnormalities had been found on esophago-gastroduodenoscopy. INVESTIGATIONS: Congo-red staining of the liver biopsy revealed massive sinusoidal amyloidosis of the liver. Immunoelectrophoresis of the urine and serum, as well as bone marrow biopsy, ruled out multiple myeloma or Waldenström's disease. Immunohistochemical staining identified the amyloid protein as a IgG kappa light chain (KLC). The free light chain (FLC) test confirmed KLC monoclonal gammopathy with an abnormal free kappa to lambda chain (KLLC) ratio. TREATMENT AND COURSE: Systemic KLC amyloidosis in this patient older than 65 years was given chemotherapy with melaphalan and dexamethasone (M-Dex). After three courses of M-Dex the renal clearance deteriorated and the serum N-terminal probrain natriuretic peptide (T-proBNP) had increased. COURSE: The patient was included in a phase II clinical trial which evaluates the use of bortezomib in patients with amyloidosis. Normalization of the free KLLC ratio and the NT-proBNP level will serve as important prognostic indicators. CONCLUSION: KLC amyloidosis is a rare cause of elevated liver enzymes. The nonspecific symptoms often delay the diagnosis. FLC testing is a helpful tool in identifying monoclonal gammopathies, even when immunoelectrophoretic tests are normals.

Overexpression of neprilysin reduces alzheimer amyloid-beta42 (Abeta42)-induced neuron loss and intraneuronal Abeta42 deposits but causes a reduction in cAMP-responsive element-binding protein-mediated transcription, age-dependent axon pathology, and premature death in Drosophila.

The amyloid-beta42 (Abeta42) peptide has been suggested to play a causative role in Alzheimer disease (AD). Neprilysin (NEP) is one of the rate-limiting Abeta-degrading enzymes, and its enhancement ameliorates extracellular amyloid pathology, synaptic dysfunction, and memory defects in mouse models of Abeta amyloidosis. In addition to the extracellular Abeta, intraneuronal Abeta42 may contribute to AD pathogenesis. However, the protective effects of neuronal NEP expression on intraneuronal Abeta42 accumulation and neurodegeneration remain elusive. In contrast, sustained NEP activation may be detrimental because NEP can degrade many physiological peptides, but its consequences in the brain are not fully understood. Using transgenic Drosophila expressing human NEP and Abeta42, we demonstrated that NEP efficiently suppressed the formation of intraneuronal Abeta42 deposits and Abeta42-induced neuron loss. However, neuronal NEP overexpression reduced cAMP-responsive element-binding protein-mediated transcription, caused age-dependent axon degeneration, and shortened the life span of the flies. Interestingly, the mRNA levels of endogenous fly NEP genes and phosphoramidon-sensitive NEP activity declined during aging in fly brains, as observed in mammals. Taken together, these data suggest both the protective and detrimental effects of chronically high NEP activity in the brain. Down-regulation of NEP activity in aging brains may be an evolutionarily conserved phenomenon, which could predispose humans to developing late-onset AD.

Increased levels of serum matrix metalloproteinase-3 in haemodialysis patients with dialysis-related amyloidosis.

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.

Lysozyme amyloidosis: report of 4 cases and a review of the literature.

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.

Impact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris.

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.

ALys amyloidosis caused by compound heterozygosity in exon 2 (Thr70Asn) and exon 4 (Trp112Arg) of the lysozyme gene.

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.