Proteolytic Fragments Research Articles

Stromal remodeling regulates dendritic cell abundance and activity in the tumor microenvironment.

SUMMARYStimulatory type 1 conventional dendritic cells (cDC1s) engage in productive interactions with CD8+ effectors along tumor-stroma boundaries. The paradoxical accumulation of “poised” cDC1s within stromal sheets is unlikely to simply reflect passive exclusion from tumor cores. Drawing parallels with embryonic morphogenesis, we hypothesized that invasive margin stromal remodeling generates developmentally conserved cell fate cues that regulate cDC1 behavior. We find that, in human T cell-inflamed tumors, CD8+ T cells penetrate tumor nests, whereas cDC1s are confined within adjacent stroma that recurrently displays site-specific proteolysis of the matrix proteoglycan versican (VCAN), an essential organ-sculpting modification in development. VCAN is necessary, and its proteolytic fragment (matrikine) versikine is sufficient for cDC1 accumulation. Versikine does not influence tumor-seeding pre-DC differentiation; rather, it orchestrates a distinctive cDC1 activation program conferring exquisite sensitivity to DNA sensing, supported by atypical innate lymphoid cells. Thus, peritumoral stroma mimicking embryonic provisional matrix remodeling regulates cDC1 abundance and activity to elicit T cell-inflamed tumor microenvironments.

Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity.

The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.

Altered Balance of Reelin Proteolytic Fragments in the Cerebrospinal Fluid of Alzheimer's Disease Patients.

Reelin binds to the apolipoprotein E receptor apoER2 to activate an intracellular signaling cascade. The proteolytic cleavage of reelin follows receptor binding but can also occur independently of its binding to receptors. This study assesses whether reelin proteolytic fragments are differentially affected in the cerebrospinal fluid (CSF) of Alzheimer’s disease (AD) subjects. CSF reelin species were analyzed by Western blotting, employing antibodies against the N- and C-terminal domains. In AD patients, we found a decrease in the 420 kDa full-length reelin compared with controls. In these patients, we also found an increase in the N-terminal 310 kDa fragment resulting from the cleavage at the so-called C-t site, whereas the 180 kDa fragment originated from the N-t site remained unchanged. Regarding the C-terminal proteolytic fragments, the 100 kDa fragment resulting from the cleavage at the C-t site also displayed increased levels, whilst the one resulting from the N-t site, the 250 kDa fragment, decreased. We also detected the presence of an aberrant reelin species with a molecular mass of around 500 kDa present in AD samples (34 of 43 cases), while it was absent in the 14 control cases analyzed. These 500 kDa species were only immunoreactive to N-terminal antibodies. We validated the occurrence of these aberrant reelin species in an Aβ42-treated reelin-overexpressing cell model. When we compared the AD samples from APOE genotype subgroups, we only found minor differences in the levels of reelin fragments associated to the APOE genotype, but interestingly, the levels of fragments of apoER2 were lower in APOE ε4 carriers with regards to APOE ε3/ε3. The altered proportion of reelin/apoER2 fragments and the occurrence of reelin aberrant species suggest a complex regulation of the reelin signaling pathway, which results impaired in AD subjects.

Proteolysis: a key post-translational modification regulating proteoglycans.

Proteoglycans are composite molecules comprising a protein backbone, i.e., the core protein, with covalently attached glycosaminoglycan chains of distinct chemical types. Most proteoglycans are secreted or attached to the cell membrane. Their specialized structures, binding properties, and biophysical attributes underlie diverse biological roles, which include modulation of tissue mechanics, cell adhesion, and the sequestration and regulated release of morphogens, growth factors, and cytokines. As an irreversible post-translational modification, proteolysis has a profound impact on proteoglycan function, abundance, and localization. Proteolysis is required for molecular maturation of some proteoglycans, clearance of extracellular matrix proteoglycans during tissue remodeling, generation of bioactive fragments from proteoglycans, and ectodomain shedding of cell-surface proteoglycans. Genetic evidence shows that proteoglycan core protein proteolysis is essential for diverse morphogenetic events during embryonic development. In contrast, dysregulated proteoglycan proteolysis contributes to osteoarthritis, cardiovascular disorders, cancer, and inflammation. Proteolytic fragments of perlecan, versican, aggrecan, brevican, collagen XVIII, and other proteoglycans are associated with independent biological activities as so-called matrikines. Yet, proteoglycan proteolysis has been investigated to only a limited extent to date. Here, we review the actions of proteases on proteoglycans and illustrate their functional impact with several examples. We discuss the applications and limitations of strategies used to define cleavage sites in proteoglycans and explain how proteoglycanome-wide proteolytic mapping, which is desirable to fully understand the impact of proteolysis on proteoglycans, can be facilitated by integrating classical proteoglycan isolation methods with mass spectrometry-based proteomics.

Abstract 150: Prevalent PKC-ι/ζ signaling is crucial for apoptosis inhibition and invasiveness of glioblastoma through upregulation of Pdk1/Akt1/14-3-3 cascade and Smad2/3

Abstract Glioblastoma (GB) is an aggressive form of cancer derived from astrocytes and often found in brain or spinal cord. Poor prognosis of GB has limitations in current therapeutics mainly due to the development of resistance. Our results demonstrated that atypical protein kinase C-iota and zeta (aPKC-ι/ζ) levels were over-expressed in GB cells (U-87 and T-98) and tissue samples compared to normal brain cells/tissues which is correlated with increased invasiveness. The present study shows the downstream effects of siRNA knockdown of PKC-ι/ζ on the expression of aPKCs, E-cadherin, Vimentin, p-Vimentin, 14-3-3 and p-14-3-3, BAD, p-BAD, Pdk1, Akt1, c-Raf, Tak1, Smad2/3 and p-Smad2/3. Downregulation of Vimentin, p-Vimentin (S33 and S56) and increased E-cadherin indicated the downregulation of epithelial-mesenchymal transition (EMT). Proteolytic E-cadherin fragment of 80 kDa indicates the reduction of cancer progression. Result also indicated that SNAIL1, SLUG and PRRX1 transcriptional activities were downregulated as a result of aPKC diminution. These transcription factors are known to induce and promote EMT. 14-3-3 is known to bind Pdk1, BAD, Akt1 and c-Raf and perform various anti-apoptotic tasks. Our results indicated that total 14-3-3 levels were significantly increased as a result of aPKC attenuation along with dephosphorylated BAD. In addition, p-14-3-3, p-BAD, pAkt1, Pdk1 levels were significantly decreased. Unphosphorylated 14-3-3 does not bind with their partners: phosphorylation on conserved S259 and S621 through Tak1 which is essential for its activation. Our results indicated that Tak1 levels significantly decreased as a result of aPKC diminution. In-vitro migration/invasion assays, immunoprecipitation (IP), immunofluorescence and immuno-gold transmission electron microscopic techniques, real time qPCR and Western blot techniques are being used in this study to further analyze role of aPKCs in GB malignancy. Our IP and kinase activity assay data confirmed that PKC-ι heavily interacts with Pdk1 and PKC-ζ interacts with Akt1, thereby activating those two kinases via phosphorylation. Data confirmed that aPKC attenuation triggers multiple pro apoptotic signaling cascades through downregulation of Akt1, Pdk1, 14-3-3 and Smad2/3.We have used a novel PKC-ι specific inhibitor 5-amino-1-(2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S) and a PKC-ζ specific inhibitor 8-hydroxy-1,3,6-naphthalenetrisulfonic acid (ζ-Stat) to conduct in-vivo experiments on murine models. Excised tumors will be analyzed for pathways observed in in-vitro experiments. Overall results suggest that aPKCs are crucial for increased invasiveness in GB. Results suggested that aPKC can be targeted to develop customized, tailored therapies for Glioblastoma which merit further research. Citation Format: Wishrawana Sarathi Ratnayake, Khandker M. Khalid, Sloan Breedy, Luke Lajmi, Tracess Smalley, Christopher A. Apostolatos, Robert Hill, James Fackrell, Mildred Acevedo-Duncan. Prevalent PKC-ι/ζ signaling is crucial for apoptosis inhibition and invasiveness of glioblastoma through upregulation of Pdk1/Akt1/14-3-3 cascade and Smad2/3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 150.

Selective isolation of hyaluronan by solid phase adsorption to silica

A solid phase adsorption method for selective isolation of hyaluronan (HA) from biological samples is presented. Following enzymatic degradation of protein, HA can be separated from sulfated glycosaminoglycans, other unsulfated glycosaminoglycans, nucleic acids, and proteolytic fragments by adsorption to amorphous silica at specific salt concentrations. The adsorbed HA can be released from silica using neutral and basic aqueous solutions. HA ranging in size from ∼9 kDa to MDa polymers has been purified by this method from human serum and conditioned medium of cultured cells.

Towards Bacterial Expression of Unstable Mutants of a Mitochondrial Enzyme

Mutations in the human AARS2 gene, which codes for the mitochondrial alanyl‐tRNA synthetase, are linked with severe disease, including infantile cardiomyopathy and leukodystrophy. Our laboratory is interested in understanding, on a molecular level, why different mutations result in different disease phenotypes. Attempts to produce some mutants of AARS2 using a bacterial expression system have resulted in poor expression of proteins contaminated with proteolytic fragments. To improve expression of these mutants, we have used solubility tags to facilitate the purification of full‐length mutant protein. Addition of an MBP solubility tag greatly improved expression and purification of the wildtype and mutant enzymes. We are currently investigating strategies for solubility tag removal and characterization of wildtype and mutant thermal stability.

Chimeric Fusion between Clostridium Ramosum IgA Protease and IgG Fc Provides Long-Lasting Clearance of IgA Deposits in Mouse Models of IgA Nephropathy.

IgA nephropathy is a common primary glomerulonephritis caused by mesangial deposition of poly-IgA complexes. The disease follows a variable course of clinical progression, with a high risk of kidney failure. Although no specific therapy is available, enzymatic strategies to clear IgA deposits are being considered for the treatment of rapidly progressive IgA nephropathy. We chose an IgA protease of commensal bacterium Clostridium ramosum, termed AK183, as the template for constructing a recombinant biologic. To extend the t1/2 in blood, we fused AK183 to the Fc segment of human IgG1. Activities of this Fc-AK183 fusion protein toward the cleavage and subsequent clearance of IgA were tested in mouse models. First, we discovered an autocleavage activity of AK183 that separates the N-terminal protease from its C-terminal autotransporter β domain. Therefore, we grafted Fc to the N terminus of AK183 and demonstrated its week-long enzymatic activity in mice. In addition, the proteolytic fragments of IgA generated in the reaction with Fc-AK183 were effectively removed from circulation via kidney filtration. The combined actions of Fc-AK183-mediated cleavage and subsequent renal clearance of IgA resulted in a lasting obliteration of blood IgA, as demonstrated in a human IgA-injection model and in a humanized α1KI transgenic model. Fc-AK183 was also able to remove chronic IgA and associated complement C3 deposits in the glomerulus. We constructed a chimeric fusion of IgA protease with Fc and demonstrated its long-lasting efficacy as a promising targeted therapy for IgA nephropathy in mouse models.

Insights into Site‐1 Protease Recognition of a Sigma Regulator Involved in Cell Surface Signaling During Pseudomonas capeferrumPseudobactin BN7/8 Import

Gram‐negative CSS pathways are composed of three key components, an outer membrane transducer that senses extracellular stimuli, an inner membrane sigma regulator responsible for transducing a signal from the periplasm to the cytoplasm, and an extracytoplasmic function (ECF) sigma factor which activates transcription of target genes (1). In our model system, the Pseudomonas capeferrumpseudobactin BN7/8 (Pup) import system, these proteins are PupB, PupR, and PupI, respectively. Our recent insights into this CSS system indicate the C‐terminal cell‐surface signaling domain (CCSSD) of PupR and the N‐terminal signaling domain (NTSD) of PupB form a stable complex in the periplasm (2). This complex stabilizes the PupR CCSSD prior to signal transduction (2). Subsequently, CSS signal transduction is activated by regulated intramembrane proteolysis (RIP) of the sigma regulator by an initial site‐1 protease followed by a site‐2 protease (3, 4). Recent data has implicated the C‐terminal processing protease, Prc (or Tsp), as the site‐1 protease in PseudomonasCSS (4). Therefore, our objective is to investigate the recognition and degradation of the PupR CCSSD by Prc in the presence and absence of the PupB NTSD utilizing both active protease and catalytically inactive mutants, Prc‐His6 S485A and Prc‐His6 K510A. We hypothesize that the site‐1 protease, Prc, will recognize and cleave the PupR CCSSD unless shielded by PupB NTSD in non‐signaling conditions. Here we present the 2.0 Å structure of Prc‐His6 S485A solved by X‐ray crystallography and additional structural information of Prc‐His6 K510A by size exclusion chromatography small angle X‐ray scattering. Furthermore, we characterize the interaction of Prc‐His6 K510A with MBP‐PupR CCSSD in the presence and absence of PupB NTSD by affinity pull‐down assays and isothermal titration calorimetry, verifying the tighter affinity of the PupR CCSSD for the PupB and supporting its potential shielding role. Additionally, we show proteolytic degradation of MBP‐PupR CCSSD by active Prc‐His6 and analyze the proteolytic fragments by SDS‐PAGE and mass spectrometry. Together these data provide new structural insights into how CSS is activated by RIP following extracellular stimulus. Thus, we support a new CCS activation model wherein the sigma regulator is shielded from Prc by interacting with the transducer NTSD until an event alters this interaction allowing RIP.1. N. Noinaj, et al., TonB‐Dependent Transporters: Regulation, Structure, and Function. Annual Review of Microbiology 64, 43‐60 (2010).2. J. L. Jensen, et al., Structural basis of cell surface signaling by a conserved sigma regulator in Gram‐negative bacteria. Journal of Biological Chemistry (2020).3. R. C. Draper, et al., Differential proteolysis of sigma regulators controls cell‐surface signalling in Pseudomonas aeruginosa. Mol Microbiol 82, 1444‐1453 (2011).4. K. C. Bastiaansen, et al., The Prc and RseP proteases control bacterial cell‐surface signalling activity. Environ Microbiol 16, 2433‐2443 (2014).

Charge and redox states modulate granulin—TDP-43 coacervation toward phase separation or aggregation

Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. To study the factors that influence the dynamics between these cytoplasmic foci, we investigated the effects of cysteine-rich granulins (GRNs 1–7), which are the proteolytic products of progranulin, a protein implicated in FTLD, on TDP-43. We show that extracellular GRNs, typically generated during inflammation, internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show less likelihood of their presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Using charge patterning and conserved cysteines among the wild-type GRNs as guides, along with specifically engineered mutants, we discover that the negative charges on GRNs drive LLPS while the positive charges and the redox state of cysteines modulate these phase transitions. Furthermore, RNA and GRNs compete and expel one another from PrLD condensates, providing a basis for GRN’s absence in SGs. Together, the results help uncover potential modulatory mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize and modulate cytoplasmic TDP-43 inclusions in proteinopathies.

BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

SummaryNeurodegeneration is associated with the aggregation of proteins bearing solvent-exposed hydrophobicity as a result of their misfolding and/or proteolytic cleavage. An understanding of the cellular protein quality control mechanisms which prevent protein aggregation is fundamental to understanding the etiology of neurodegeneration. By examining the metabolism of disease-linked C-terminal fragments of the TAR DNA-binding protein 43 (TDP43), we found that the Bcl-2 associated athanogene 6 (BAG6) functions as a sensor of proteolytic fragments bearing exposed hydrophobicity and prevents their intracellular aggregation. In addition, BAG6 facilitates the ubiquitylation of TDP43 fragments by recruiting the Ub-ligase, Ring finger protein 126 (RNF126). Authenticating its role in preventing aggregation, we found that TDP43 fragments form intracellular aggregates in the absence of BAG6. Finally, we found that BAG6 could interact with and solubilize additional neurodegeneration-associated proteolytic fragments. Therefore, BAG6 plays a general role in preventing intracellular aggregation associated with neurodegeneration.

Mitochondrial and Neuronal Dysfunctions in L1 Mutant Mice.

Adhesion molecules regulate cell proliferation, migration, survival, neuritogenesis, synapse formation and synaptic plasticity during the nervous system’s development and in the adult. Among such molecules, the neural cell adhesion molecule L1 contributes to these functions during development, and in synapse formation, synaptic plasticity and regeneration after trauma. Proteolytic cleavage of L1 by different proteases is essential for these functions. A proteolytic fragment of 70 kDa (abbreviated L1-70) comprising part of the extracellular domain and the transmembrane and intracellular domains was shown to interact with mitochondrial proteins and is suggested to be involved in mitochondrial functions. To further determine the role of L1-70 in mitochondria, we generated two lines of gene-edited mice expressing full-length L1, but no or only low levels of L1-70. We showed that in the absence of L1-70, mitochondria in cultured cerebellar neurons move more retrogradely and exhibit reduced mitochondrial membrane potential, impaired Complex I activity and lower ATP levels compared to wild-type littermates. Neither neuronal migration, neuronal survival nor neuritogenesis in these mutants were stimulated with a function-triggering L1 antibody or with small agonistic L1 mimetics. These results suggest that L1-70 is important for mitochondrial homeostasis and that its absence contributes to the L1 syndrome phenotypes.

An MMP-degraded and cross-linked fragment of type III collagen as a non-invasive biomarker of hepatic fibrosis resolution.

Background and AimsLiver fibrosis results from a prolonged wound healing response to continued injury with excessive production of extracellular proteins. In patients with chronic liver disease, the monitoring of liver fibrosis dynamics is of high interest. Whilst markers of fibrogenesis exist, markers of hepatic fibrosis resolution remain an unmet clinical need. Thus, we sought to develop an assay quantifying a circulating proteolytic fragment of cross‐linked type III collagen as a biomarker of fibrolysis, testing its utility in two clinical cohorts of liver fibrosis of distinct aetiology and regressing endotypeMethodsWe used a monoclonal antibody targeting the C‐telopeptide of type III collagen following C‐proteinase cleavage to develop and validate a neo‐epitope‐specific enzyme‐linked immunosorbent assay (CTX‐III). A potential fibrosis resolution marker, CTX‐III, was measured in two clinical cohorts of patients with obesity‐associated non‐alcoholic fatty liver disease undergoing bariatric surgery or hepatitis C virus infection from a clinical trial study evaluating the anti‐fibrotic effect of farglitazar.ResultsCTX‐III was robust and specific for the targeted neo‐epitope with good reproducibility in EDTA plasma. We assessed type III collagen remodelling using a panel of biomarkers, including a type III collagen formation marker (PRO‐C3), degradation (C3M), and CTX‐III (fibrolysis). Net fibrolysis was increased in patients with non‐alcoholic fatty liver disease following bariatric surgery (p < .001). Moreover, net fibrolysis identified spontaneous fibrotic regressors from stable and progressors (p < .05 and p < .001) among hepatitis C virus infection patients.ConclusionCirculating CTX‐III as a marker of fibrolysis indicates the biomarker's beneficial use in assessing hepatic fibrosis resolution.

Cytoskeletal protein degradation in brain death donor kidneys associates with adverse posttransplant outcomes

In brain death, cerebral injury contributes to systemic biological dysregulation, causing significant cellular stress in donor kidneys adversely impacting the quality of grafts. Here, we hypothesized that donation after brain death (DBD) kidneys undergo proteolytic processes that may deem grafts susceptible to posttransplant dysfunction. Using mass spectrometry and immunoblotting, we mapped degradation profiles of cytoskeletal proteins in deceased and living donor kidney biopsies. We found that key cytoskeletal proteins in DBD kidneys were proteolytically cleaved, generating peptide fragments, predominantly in grafts with suboptimal posttransplant function. Interestingly, α‐actinin‐4 and talin‐1 proteolytic fragments were detected in brain death but not in circulatory death or living donor kidneys with similar donor characteristics. As talin‐1 is a specific proteolytic target of calpain‐1, we investigated a potential trigger of calpain activation and talin‐1 degradation using human ex vivo precision‐cut kidney slices and in vitro podocytes. Notably, we showed that activation of calpain‐1 by transforming growth factor‐β generated proteolytic fragments of talin‐1 that matched the degradation fragments detected in DBD preimplantation kidneys, also causing dysregulation of the actin cytoskeleton in human podocytes; events that were reversed by calpain‐1 inhibition. Our data provide initial evidence that brain death donor kidneys are more susceptible to cytoskeletal protein degradation. Correlation to posttransplant outcomes may be established by future studies.

Granulins modulate liquid-liquid phase separation or aggregation of prion-like domain of TDP-43 by electrostatic interactions and the redox state of cysteines

The cytoplasmic inclusions of TDP-43 and its proteolytic fragments are the pathological hallmarks of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The aberrant TDP-43 fragments span the c-terminal region of the protein (C35, C25 and C18) that contain a prion-like domain (PrLD; C18) with a low complexity sequence. The full-length TDP-43 partitions into the cytoplasmic membraneless stress granules (SGs) under stress. We investigated the role of cysteine-rich modules called granulins (GRNs, 1-7) that are proteolytic products of progranulin (PGRN), a risk factor for FTLD.

Anti-Excitotoxic Effects of N-Butylidenephthalide Revealed by Chemically Insulted Purkinje Progenitor Cells Derived from SCA3 iPSCs.

Spinocerebellar ataxia type 3 (SCA3) is characterized by the over-repetitive CAG codon in the ataxin-3 gene (ATXN3), which encodes the mutant ATXN3 protein. The pathological defects of SCA3 such as the impaired aggresomes, autophagy, and the proteasome have been reported previously. To date, no effective treatment is available for SCA3 disease. This study aimed to study anti-excitotoxic effects of n-butylidenephthalide by chemically insulted Purkinje progenitor cells derived from SCA3 iPSCs. We successfully generated Purkinje progenitor cells (PPs) from SCA3 patient-derived iPSCs. The PPs, expressing both neural and Purkinje progenitor’s markers, were acquired after 35 days of differentiation. In comparison with the PPs derived from control iPSCs, SCA3 iPSCs-derived PPs were more sensitive to the excitotoxicity induced by quinolinic acid (QA). The observations of QA-treated SCA3 PPs showing neural degeneration including neurite shrinkage and cell number decrease could be used to quickly and efficiently identify drug candidates. Given that the QA-induced neural cell death of SCA3 PPs was established, the activity of calpain in SCA3 PPs was revealed. Furthermore, the expression of cleaved poly (ADP-ribose) polymerase 1 (PARP1), a marker of apoptotic pathway, and the accumulation of ATXN3 proteolytic fragments were observed. When SCA3 PPs were treated with n-butylidenephthalide (n-BP), upregulated expression of calpain 2 and concurrent decreased level of calpastatin could be reversed, and the overall calpain activity was accordingly suppressed. Such findings reveal that n-BP could not only inhibit the cleavage of ATXN3 but also protect the QA-induced excitotoxicity from the Purkinje progenitor loss.

Lysosomal gene Hexb displays haploinsufficiency in a knock-in mouse model of Alzheimer’s disease

Lysosomal network abnormalities are an increasingly recognised feature of Alzheimer’s disease (AD), which appear early and are progressive in nature. Sandhoff disease and Tay-Sachs disease (neurological lysosomal storage diseases caused by mutations in genes that code for critical subunits of β-hexosaminidase) result in accumulation of amyloid-β (Aβ) and related proteolytic fragments in the brain. However, experiments that determine whether mutations in genes that code for β-hexosaminidase are risk factors for AD are currently lacking. To determine the relationship between β-hexosaminidase and AD, we investigated whether a heterozygous deletion of Hexb, the gene that encodes the beta subunit of β-hexosaminidase, modifies the behavioural phenotype and appearance of disease lesions in AppNL-G-F/NL-G-F(AppKI/KI) mice. AppKI/KI and Hexb+/- mice were crossed and evaluated in a behavioural test battery. Neuropathological hallmarks of AD and ganglioside levels in the brain were also examined. Heterozygosity of Hexb in AppKI/KI mice reduced learning flexibility during the Reversal Phase of the Morris water maze. Contrary to expectation, heterozygosity of Hexb caused a small but significant decrease in amyloid beta deposition and an increase in the microglial marker IBA1 that was region- and age-specific. Hexb heterozygosity caused detectable changes in the brain and in the behaviour of an AD model mouse, consistent with previous reports that described a biochemical relationship between HEXB and AD. This study reveals that the lysosomal enzyme gene Hexb is not haplosufficient in the mouse AD brain.

UNC5C Receptor Proteolytic Cleavage by Active AEP Promotes Dopaminergic Neuronal Degeneration in Parkinson's Disease.

Netrin‐1 is a chemotropic cue mediating axon growth and neural migration in neuronal development, and its receptors deletion in colorectal cancer and UNC5s act as dependence receptors regulating neuronal apoptosis. Asparagine endopeptidase (AEP) is an age‐dependent protease that cuts human alpha‐synuclein (α‐Syn) at N103 and triggers its aggregation and neurotoxicity. In the current study, it is reported that UNC5C receptor is cleaved by AEP in Parkinson's disease (PD) and facilitates dopaminergic neuronal loss. UNC5C is truncated by active AEP in human α‐SNCA transgenic mice in an age‐dependent manner or induced by neurotoxin rotenone. Moreover, UNC5C is fragmented by AEP in PD brains, inversely correlated with reduced netrin‐1 levels. Netrin‐1 deprivation in primary cultures induces AEP and caspase‐3 activation, triggering UNC5C proteolytic fragmentation and enhancing neuronal loss. Noticeably, blocking UNC5C cleavage by AEP attenuates netrin‐1 deprivation‐elicited neuronal death and motor disorders in netrin flox/flox mice. Overexpression of AEP‐truncated UNC5C intracellular fragment strongly elicits α‐Syn aggregation and dopaminergic loss, locomotor deficits in α‐SNCA transgenic mice. Hence, the findings demonstrate that netrin‐1 reduction and UNC5C truncation by AEP contribute to PD pathogenesis.

Уровень протеолитических фрагментов IgG как дополнительный прогностический биомаркер рака предстательной железы

Introduction. Prostate cancer (PC) leads the world structure of male cancer mortality. Understanding the prognosis for PC patients is vitally important and requires diagnostic enhancements such as developing biomarker panels. The studyaimed to estimate sensitivity, specificity, and prognostic significance of the level of immunoglobulin G (IgG) proteolysis products in the blood serum depending on PC Gleason score and detect how the levels of IgG proteolytic fragments were associated with the diagnostic marker expression. Materials and methods. The study included 90 PC patients. We used the Gleason score to evaluate tumor grade. The level of the IgG proteolysis products and total prostate-specific antigen (PSA) in the serum was detected with ELISA. We performed an immunohistochemical assay with Ki-67, plasminogen-binding protein enolase-1 (ENO-1), and prostate-specific membrane antigen (PSMA). Results. ROC analysis demonstrated 100% specificity of the IgG proteolysis products test regardless of Gleason score, while the sensitivity reached 72.2%, 57.5%, 52.6%, and 46.2% for ISUP stages 1, 2, 3, and 4, respectively. An intergroup comparison showed significant differences between patient groups according to Gleason score, preoperative PSA and IgG proteolysis product serum levels and Ki-67 index. We detected a positive correlation between the level of IgG proteolysis products in the PC patients’ serum and ENO-1 expression and a negative correlation with the Gleason score and PSMA expression. Conclusion. Preoperative detection of high levels of proteolytic IgG fragments in the serum of PC patients even with low Gleason score and PSMA expression may help predict a more aggressive disease course and choose the management strategy. Keywords: prostate cancer, plasminogen activation system, proteolytic fragments of immunoglobulin G, oncomarkers

A new look at Alzheimer’s genetics: APP dampens neuronal excitability by regulating the size and cellular location of the axon initial segment

Early-onset forms of familial Alzheimer's disease (FAD) are usually caused by mutations in three known disease genes: the amyloid precursor protein (APP), and two presenilin genes (PSEN1 and PSEN2). APP is a single-transmembrane protein whose normal biological function is largely unknown. The congruence of FAD genetics and the presence of amyloid plaque pathology has focused attention on the APP proteolytic fragment Aβ, even while the normal function of APP remains largely unexplored. To fill this gap in our understanding, have studied the axon initial segment (AIS), a specialized region of a neuron that initiates an action potential.Immunocytochemistry of the two AIS scaffold proteins, Ankyrin G and βIV-spectrin, was performed on sections from the R1.40 mouse model of AD (B6.129-Tg(APPSw)40Btla/Mmjax) and from human frontal cortex samples (Braak Stage V-VI). We measured the length of the AIS and the intensity of their immunostaining. APP-overexpressing cultures will be recorded on the multielectrode-array (MEA) wells for electrophysiology studies RESULT: In frontal cortex of human AD cases and R1.40 mice, the AIS was shorter than in controls. In vitro, the AIS of cultured mouse neurons transfected with wild type or Swedish APP (APPSwe ) was shorter and also shifted distally along the axon. Both changes were greater with APPSwe than with wild type APP. Significantly, the addition of either Aβ oligomers or fibrils elicited no response. By transfecting neurons cultured in MEA wells, we showed that the observed shifts in the AIS altered neuronal firing patterns. We observed an overall decrease in mean spike number but not in spike amplitude.Our data suggest that the APP protein, but not the Aβ peptide, regulates neuronal activity through its effects on the position and length AIS. As APP is a neuronal stress response protein, our hypothesis is that age, trauma or genetic mutation leads to APP overexpression. While short term overexpression may protect against excitotoxicity, chronically elevated APP would permanently retard neuronal activity and serve as a substrate for dementia. This study argues for a reinterpretation of APP mutations as support for the role of the Aβ peptide in AD.