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EPM1 is the most common form of Progressive Myoclonus Epilepsy characterized by late-childhood onset, ever-worsening and disabling myoclonus, seizures, ataxia, psychiatric disease, and shortened lifespan. EPM1 is caused by expansions of a dodecamer repeat sequence in the promoter of CSTB (cystatin B), which dramatically reduces, but does not eliminate, gene expression. The relatively late onset and consistent presence of a minimal amount of protein product makes EPM1 a favorable target for gene replacement therapy. If treated early, these children's normally developed brains could be rescued from the neurodegeneration that otherwise follows, and their cross-reactive immunological material (CRIM) positive status greatly reduces transgene related toxicity. We performed a proof-of-concept CSTB gene replacement study in Cstb knockout mice by introducing full-length human CSTB driven by the CBh promoter packaged in AAV9 and administered at postnatal days 21 and 60. Mice were sacrificed at 2 or 9 months of age, respectively. We observed significant improvements in expression levels of neuroinflammatory pathway genes and cerebellar granule cell layer apoptosis, as well as amelioration of motor impairment. The data suggest that gene replacement is a promising therapeutic modality for EPM1 and could spare affected children and families the ravages of this otherwise severe neurodegenerative disease., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: Cervical cancer (CC) is one of the most common gynecologic malignancies worldwide. Although rapid improvements have been made regarding its prevention and treatment, little is known about disease pathogenesis and the clinical relevance of reliable biomarkers. The present study evaluated the expression of cystatin B (CSTB) as a potential biomarker of CC., Methods: Tissue microarray analysis and immunohistochemical staining were performed to detect CSTB expression, while CSTB mRNA and protein expression levels of freshly isolated CC tissue were measured by quantitative real-time PCR and western blot, respectively. Bioinformatics were used to analyze the CSTB co-expression network and functional enrichments., Results: We observed high CSTB mRNA and protein expression levels in CC tissues, which was confirmed by tissue microarray in a comparison with paired adjacent non-cancerous cervical tissue samples. CSTB gene enrichments and associations with co-expressed genes were also observed. Further analysis showed that elevated CSTB expression was associated with pathological progress in CC., Conclusion: Our data demonstrate that CSTB has the potential to be used as a tissue biomarker with clinical value in patients with CC, which may aid the development of intervention strategies., Competing Interests: Declaration of conflicting interestsThe authors declare that they have no competing interests.

Progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessive disorder, also known as Unverricht-Lundborg disease (ULD). EPM1 patients suffer from photo-sensitive seizures, stimulus-sensitive myoclonus, nocturnal myoclonic seizures, ataxia and dysarthria. In addition, cerebral ataxia and impaired GABAergic inhibition are typically present. EPM1 is caused by mutations in the Cystatin B gene ( CSTB ). The CSTB protein functions as an intracellular thiol protease inhibitor and inhibits Cathepsin function. It also plays a crucial role in brain development and regulates various functions in neurons beyond maintaining cellular proteostasis. These include controlling cell proliferation and differentiation, synaptic functions and protection against oxidative stress, likely through regulation of mitochondrial function. Depending on the differentiation stage and status of neurons, the protein localizes either to the cytoplasm, nucleus, lysosomes or mitochondria. Further, CSTB can also be secreted to the extracellular matrix for interneuron rearrangement and migration. In this review, we will review the various functions of CSTB in the brain and discuss the putative pathophysiological mechanism underlying EPM1.

Cathepsin B is a cysteine protease that is implicated in multiple aspects of Alzheimer's disease pathogenesis. The endogenous inhibitor of this enzyme, cystatin B (CSTB) is encoded on chromosome 21. Thus, individuals who have Down syndrome, a genetic condition caused by having an additional copy of chromosome 21, have an extra copy of an endogenous inhibitor of the enzyme. Individuals who have Down syndrome are also at significantly increased risk of developing early-onset Alzheimer's disease (EOAD). The impact of the additional copy of CSTB on Alzheimer's disease development in people who have Down syndrome is not well understood. Here we compared the biology of cathepsin B and CSTB in individuals who had Down syndrome and Alzheimer's disease, with disomic individuals who had Alzheimer's disease or were ageing healthily. We find that the activity of cathepsin B enzyme is decreased in the brain of people who had Down syndrome and Alzheimer's disease compared with disomic individuals who had Alzheimer's disease. This change occurs independently of an alteration in the abundance of the mature enzyme or the number of cathepsin B + cells. We find that the abundance of CSTB is significantly increased in the brains of individuals who have Down syndrome and Alzheimer's disease compared to disomic individuals both with and without Alzheimer's disease. In mouse and human cellular preclinical models of Down syndrome, three-copies of CSTB increases CSTB protein abundance but this is not sufficient to modulate cathepsin B activity. EOAD and Alzheimer's disease-Down syndrome share many overlapping mechanisms but differences in disease occur in individuals who have trisomy 21. Understanding this biology will ensure that people who have Down syndrome access the most appropriate Alzheimer's disease therapeutics and moreover will provide unique insight into disease pathogenesis more broadly., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Objective: Patients with Unverricht-Lundborg disease/EPM1 develop increasing locomotory disability or ataxia in the course of their disease. To test our hypothesis that negative myoclonus is the reason for this increasing ataxia, we investigated a possible correlation over time., Methods: In 15 patients with EPM1who were confirmed to have a mutation in the CSTB gene, polygraphic video-EEG-EMG recordings were performed in freely moving or standing patients. The criterion for the duration of the negative myoclonus was the measured length of the silent periods on the EMG., Results: All 15 patients had documented negative myoclonus when standing and walking. The mean duration of silent periods significantly increased from 100 (SD: 19.1) ms at time point T1 to 128 (SD: 26.6) ms at T2 in seven of eight patients, based on two recordings and a mean interval of 12.8 (SD: 4.9) years. Using a cross-sectional approach, all 15 patients were classified based on whether they were ambulatory, could walk with aid, or needed a wheelchair. Ambulatory patients had a mean duration of 97.3 (SD: 16.5) ms, patients who could walk with aid had a mean duration of 106.7 (SD: 16) ms, and patients who were wheelchair-bound had a mean duration of 138 (SD: 23.6) ms. In addition to the prolongation of the silent periods, there was an observed increase in frequency of the negative myoclonus, becoming more continuous and tremulous., Significance: Using simultaneous EEG/EMG recordings in freely moving or standing patients, we have shown that the locomotor disability or ataxia is due to negative myoclonus in voluntary innervated muscles. The reason for the progression is the prolongation of the silent periods as measured by the duration of the negative myoclonus and their increase in frequency., (© 2023 International League Against Epilepsy.)

Background: Both autophagy and glycolysis are essential for pancreatic ductal adenocarcinoma (PDAC) survival due to desmoplasia. We investigated whether targeting a hub gene which participates in both processes could be an efficient strategy for PDAC treatment., Methods: The expression pattern of glycolysis signatures (GS) and autophagy signatures (AS) and their correlation with cystatin B (CSTB) in PDAC were analysed. It was discovered how CSTB affected the growth, glycolysis, and autophagy of PDAC cells. We assessed competitive binding to cathepsin B (CTSB) between CSTB and cystatin C (CSTC) via immunoprecipitation (IP) and immunofluorescence (IF). Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) and luciferase reporter gene assays were used to unveil the mechanism underlying CSTB upregulation. The expression pattern of CSTB was examined in clinical samples and KrasG12D/+, Trp53R172H/+, Pdx1-Cre (KPC) mice., Results: GS and AS were enriched and closely associated in PDAC tissues. CSTB increased autophagic flux and provided substrates for glycolysis. CSTB knockdown attenuated the proliferation of PDAC cells and patient-derived xenografts. The liquid chromatography-tandem mass spectrometry assay indicated CSTB interacted with CTSB and contributed to the proteolytic activity of CTSB in lysosomes. IF and IP assays demonstrated that CSTB competed with CSTC to bind to CTSB. Mutation of the key sites of CSTB abolished the interaction between CSTB and CTSB. CSTB was highly expressed in PDAC due to H3K27acetylation and SP1 expression. High expression of CSTB in PDAC was observed in tissue microarray and patients' serum samples., Conclusions: Our work demonstrated the tumorigenic roles of autophagy and glycolysis in PDAC. CSTB is a key role in orchestrating these processes to ensure energy supply of PDAC cells., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Unverricht-Lundborg disease (ULD), also known as progressive myoclonic epilepsy 1 (EPM1), is a rare autosomal recessive neurodegenerative disorder characterized by a complex symptomatology that includes action- and stimulus-sensitive myoclonus and tonic-clonic seizures. The main cause of the onset and development of ULD is a repeat expansion of a dodecamer sequence localized in the promoter region of the gene encoding cystatin B (CSTB), an inhibitor of lysosomal proteases. Although this is the predominant mutation found in most patients, the physio-pathological mechanisms underlying the disease complexity remain largely unknown. In this work, we used patient-specific iPSCs and their neuronal derivatives to gain insight into the molecular and genetic machinery responsible for the disease in two Italian siblings affected by different phenotypes of ULD. Specifically, fragment length analysis on amplified CSTB promoters found homozygous status for dodecamer expansion in both patients and showed that the number of dodecamer repeats is the same in both. Furthermore, the luciferase reporter assay showed that the CSTB promoter activity was similarly reduced in both lines compared to the control. This information allowed us to draw important conclusions: (1) the phenotypic differences of the patients do not seem to be strictly dependent on the genetic mutation around the CSTB gene, and (2) that some other molecular mechanisms, not yet clearly identified, might be taken into account. In line with the inhibitory role of cystatin B on cathepsins, molecular investigations performed on iPSCs-derived neurons showed an increased expression of lysosomal cathepsins (B, D, and L) and a reduced expression of CSTB protein. Intriguingly, the increase in cathepsin expression does not appear to be correlated with the residual amount of CSTB, suggesting that other mechanisms, in addition to the regulation of cathepsins, could be involved in the pathological complexity of the disease.

Cystatins are a diverse group of cysteine protease inhibitors widely present among various organisms. Beyond their protease inhibitor function, cystatins play a crucial role in diverse pathophysiological conditions in animals, including neurodegenerative disorders, tumor progression, inflammatory diseases, and immune response. However, the role of cystatins in immunity against viral and bacterial infections in fish remains to be elucidated. In this study, the cystatin B from big-belly seahorse, Hippocampus abdominalis, designated as HaCSTB, was identified and characterized. HaCSTB shared the highest homology with type 1 cystatin family members of teleosts and had three cystatin catalytic domains with no signal peptides or disulfide bonds. HaCSTB transcripts were mainly expressed in peripheral blood cells (PBCs), followed by the testis and pouch of healthy big-belly seahorses. Immune challenge with lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (Poly I:C), and Streptococcus iniae induced upregulation of relative HaCSTB mRNA expression in PBCs. Subcellular localization analysis revealed the distribution of HaCSTB in the cytosol, mitochondria, and nuclei of fathead minnow cells (FHM). Recombinant HaCSTB (rHaCSTB) exhibited potent in vitro inhibitory activity against papain, a cysteine protease, in a concentration-, pH-, and temperature-dependent manner. Overexpression of HaCSTB in viral hemorrhagic septicemia virus (VHSV)-susceptible FHM cells increased cell viability and reduced VHSV-induced apoptosis. Collectively, these results suggest that HaCSTB might engage in the teleostean immune protection against bacteria and viruses., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background and Purpose: Unverricht-Lundborg disease (ULD) is a common type of progressive myoclonic epilepsy (PME). It is caused mostly by biallelic dodecamer repeat expansions in the promoter region of CSTB gene. Despite highly prevalent in the Mediterranean countries, no studies have been reported from Egypt. This article study the presence of CSTB gene mutations among Egyptian patients clinically suspected with ULD, and describes the clinical and genetic characteristics of those with confirmed gene mutation., Methods: Medical records of patients following up in two specialized epilepsy clinics in Cairo, Egypt were retrospectively reviewed. Twenty patients who belonged to 13 unrelated families were provisionally diagnosed with ULD based on the clinical presentation. Genetic testing was done. Clinical characteristics, demographic data and EEG findings were documented., Results: Genetic studies confirmed the presence of the CSTB dodecamer repeat expansion in 14 patients from 8 families (frequency 70 %). The mean duration of the follow-up was 5 years. Male to female distribution was 1:1 with a mean age of onset 9.7 years. Consanguinity was noted in 4 families. Eight patients had their first seizure between the age of 10 and 20 years. Myoclonic jerks ranged in severity from mild in three unrelated patients to severe in one. Only 3 had cognitive impairment., Conclusion: Our study confirms the presence of CSTB mutation among Egyptian patients suspected with ULD. There was no clear phenotype-genotype correlation among the studied group of patients. In addition, we noticed variable inter and intra familial severity among patients from the same family., (Copyright © 2021 Elsevier B.V. All rights reserved.)

No robust biomarkers have yet been identified for autism spectrum disorder (ASD) or autistic traits. Familial factors likely influence biomarkers such as protein concentrations. Comparing twins with ASD or high autistic traits to the less affected co-twin allows estimating the impact of familial confounding. We measured 203 proteins in cerebrospinal fluid (n = 86) and serum (n = 127) in twins (mean age 14.2 years, 44.9% females) enriched for ASD and other neurodevelopmental conditions. Autistic traits were assessed by using the parent-report version of the Social Responsiveness Scale-2. In cerebrospinal fluid, autistic traits correlated negatively with three proteins and positively with one. In serum, autistic traits correlated positively with 15 and negatively with one. Also in serum, six were positively-and one negatively-associated with ASD. A pathway analysis of these proteins revealed immune system enrichment. In within twin pair analyses, autistic traits were associated with serum B-cell activating factor (BAFF) only, whereas Cystatin B (CSTB) remained significantly associated with ASD. These associations did not remain significant when only considering monozygotic twins. For the remainder, the within-pair analysis indicated familial confounding, including shared environment and genes, influencing both autism and protein levels. Our findings indicate proteins involved in immunity as putative biomarkers of autistic traits and ASD with partial genetic confounding. Although some results are in line with previous studies in general, further studies are needed for replication., (© 2021 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Cystatin B (CSTB) acts as an inhibitor of cysteine proteases of the cathepsin family and loss-of-function mutations result in human brain diseases with a genotype-phenotype correlation. In the most severe case, CSTB-deficiency disrupts brain development, and yet the molecular basis of this mechanism is missing. Here, we establish CSTB as a regulator of chromatin structure during neural stem cell renewal and differentiation. Murine neural precursor cells (NPCs) undergo transient proteolytic cleavage of the N-terminal histone H3 tail by cathepsins B and L upon induction of differentiation into neurons and glia. In contrast, CSTB-deficiency triggers premature H3 tail cleavage in undifferentiated self-renewing NPCs and sustained H3 tail proteolysis in differentiating neural cells. This leads to significant transcriptional changes in NPCs, particularly of nuclear-encoded mitochondrial genes. In turn, these transcriptional alterations impair the enhanced mitochondrial respiration that is induced upon neural stem cell differentiation. Collectively, our findings reveal the basis of epigenetic regulation in the molecular pathogenesis of CSTB deficiency., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

People with Down syndrome (DS), caused by trisomy of chromosome 21 have a greatly increased risk of developing Alzheimer's disease (AD). This is in part because of triplication of a chromosome 21 gene, APP. This gene encodes amyloid precursor protein, which is cleaved to form amyloid-β that accumulates in the brains of people who have AD. Recent experimental results demonstrate that a gene or genes on chromosome 21, other than APP, when triplicated significantly accelerate amyloid-β pathology in a transgenic mouse model of amyloid-β deposition. Multiple lines of evidence indicate that cysteine cathepsin activity influences APP cleavage and amyloid-β accumulation. Located on human chromosome 21 (Hsa21) is an endogenous inhibitor of cathepsin proteases, CYSTATIN B (CSTB) which is proposed to regulate cysteine cathepsin activity in vivo. Here we determined if three copies of the mouse gene Cstb is sufficient to modulate amyloid-β accumulation and cathepsin activity in a transgenic APP mouse model. Duplication of Cstb resulted in an increase in transcriptional and translational levels of Cstb in the mouse cortex but had no effect on the deposition of insoluble amyloid-β plaques or the levels of soluble or insoluble amyloid-β42, amyloid-β40, or amyloid-β38 in 6-month old mice. In addition, the increased CSTB did not alter the activity of cathepsin B enzyme in the cortex of 3-month or 6-month old mice. These results indicate that the single-gene duplication of Cstb is insufficient to elicit a disease-modifying phenotype in the dupCstb x tgAPP mice, underscoring the complexity of the genetic basis of AD-DS and the importance of multiple gene interactions in disease., Competing Interests: F.K.W. has undertaken consultancy for Elkington and Fife Patent Lawyers unrelated to the work in the manuscript and is also a PLoS One Academic Editor. This does not alter our adherence to PLoS ONE policies on sharing data and materials.

Unverricht-Lundborg disease (ULD) is an inherited form of progressive myoclonus epilepsy caused by mutations in the gene encoding Cystatin B (CSTB), an inhibitor of lysosomal proteases. The most common mutation described in ULD patients is an unstable expansion of a dodecamer sequence located in the CSTB gene promoter. This expansion is causative of the downregulation of CSTB gene expression and, consequently, of its inhibitory activity. Here we report the generation of induced pluripotent stem cell (iPSC) lines from two Italian siblings having a family history of ULD and affected by different clinical and pathological phenotypes of the disease., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Taurochenodeoxycholic acid (TCDCA) is one of the major active components in bile acid. It was proven to have inhibitory activities on inflammation and also participate in host immuno-regulation. TCDCA exerts anti-inflammatory and immuno-regulatory effects through the glucocorticoid receptor (GR) mediated genomic signaling pathway and the G protein-coupled bile acid receptor 5 (TGR5) mediated AC-cAMP-PKA signaling pathway. However, it is unclear whether GR or TGR5 plays an important role in the regulatory effects of TCDCA. In order to further investigate this effects mechanism of TCDCA, the research use the transcriptome to identify the major genes and pathway in the anti-inflammatory and immuno-regulatory effects., Methods: After the Fibroblast-like synoviocytes (FLS) being treated by different concentrations (10 - 5 , 10 - 6 and 10 - 7  M) of TCDCA for 12 h, the resulting mRNA was analyzed by RNA-seq. The differentially expressed genes were screened from sequencing results using bioinformatics techniques. In the next step, other published literature were referred in order to find out whether those genes mentioned above are related to inflammation. The final selected differentially expressed genes associated with inflammation were then validated by q-PCR and western blot assays., Results: Five genes associated with anti-inflammatory and immuno-regulatory effects, include Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Glutathione peroxidase 3 (GPX3), Serine/arginine-rich splicing factor-9 (SRSF9), Connective tissue growth factor (CTGF) and Cystatin B (CSTB) were identified. TCDCA at the concentrations of 10 - 5 , 10 - 6 and 10 - 7  M significantly (p < 0.05) up-regulate the mRNA and protein expression of SRSF9 and GPX3 and also up-regulate the mRNA expression of CSTB, CTGF and GAPDH. RNA-seq results of GPX3 and SRSF9 expression were consistent with q-PCR results, while q-PCR results of CTGF, GAPDH showed inconsistent with their RNA-seq results. Q-PCR result of CSTB expression also showed inconsistent with the RNA-seq result., Conclusions: The anti-inflammatory and immuno-regulatory activities of TCDCA are proven to be related to the up-regulation expression of GPX3, SRSF9 and CSTB.

Aim of the Study: The aim of this study was to explore genetic findings and the phenotype in Polish patients with Unverricht-Lundborg disease (ULD)., Materials and Methods: We retrospectively evaluated mutations in the cystatin B (CSTB) gene and clinical presentation in a cohort of patients with ULD. The study population consisted of 19 (14 males) patients with genetically confirmed disease., Results: Sixteen patients were homozygous for the expanded dodecamer repeat mutation alleles, one subject was compound heterozygous for the dodecamer repeat expansion and other mutation, in two, the type of mutation has not yet been established. The numbers of repeats in the CSTB gene varied from 60 to 81. Clinical information was available for 16 subjects. The disease course was progressive in all patients, leading to severe disability, mainly due to myoclonus, in nine., Conclusions and Clinical Implications: Genetic findings and the clinical picture of our patients with ULD were in accordance with available studies. The most common genetic defect underlying ULD was homozygosity for an unstable expansion of a dodecamer repeat in the CSTB gene. Patients with action or/and stimulus sensitive myoclonus or intractable myoclonus epilepsy, especially with onset in late childhood/adolescence should be screened for ULD., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest related to this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: Microglial phagocytosis of apoptotic cells is an essential component of the brain regenerative response during neurodegeneration. Whereas it is very efficient in physiological conditions, it is impaired in mouse and human mesial temporal lobe epilepsy, and now we extend our studies to a model of progressive myoclonus epilepsy type 1 in mice lacking cystatin B (CSTB)., Methods: We used confocal imaging and stereology-based quantification of apoptosis and phagocytosis of the hippocampus of Cstb knockout (KO) mice, an in vitro model of phagocytosis and siRNAs to acutely reduce Cstb expression, and a virtual three-dimensional (3D) model to analyze the physical relationship between apoptosis, phagocytosis, and active hippocampal neurons., Results: Microglial phagocytosis was impaired in the hippocampus of Cstb KO mice at 1 month of age, when seizures arise and hippocampal atrophy begins. This impairment was not related to the lack of Cstb in microglia alone, as shown by in vitro experiments with microglial Cstb depletion. The phagocytosis impairment was also unrelated to seizures, as it was also present in Cstb KO mice at postnatal day 14, before seizures begin. Importantly, phagocytosis impairment was restricted to the granule cell layer and spared the subgranular zone, where there are no active neurons. Furthermore, apoptotic cells (both phagocytosed and not phagocytosed) in Cstb-deficient mice were at close proximity to active cFos + neurons, and a virtual 3D model demonstrated that the physical relationship between apoptotic cells and cFos + neurons was specific for Cstb KO mice., Significance: These results suggest a complex crosstalk between apoptosis, phagocytosis, and neuronal activity, hinting that local neuronal activity could be related to phagocytosis dysfunction in Cstb KO mice. Overall, these data suggest that phagocytosis impairment is an early feature of hippocampal damage in epilepsy and opens novel therapeutic approaches for epileptic patients based on targeting microglial phagocytosis., (© 2020 The Authors. Epilepsia published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Cystatins B is an endogenous cysteine cathepsin inhibitor. In shrimp, cystatins B-like (CSTB-L) has not been characterized and its role in WSSV infection is largely unknown. In this study, a full-length 699 bp CSTB-L sequence with 291 bp open reading frame encoding a 96 amino acid from L.vannamei (Lv) was first cloned. The tissue distribution assay indicated that LvCSTB-L presented ubiquitous expression in most examined tissues, with the most predominant expression in the hepatopancreas and the weakest expression in the muscles. LvCSTB-L transcripts could be induced in the intestine and hepatopancreas by WSSV challenge. The relative expression level of IE1 and VP28 in the LvCSTB-L knockdown shrimp were increased significantly. In addition, the shrimp cumulative mortality was remarkably (p < 0.01) increased after LvCSTB-L knockdown. Moreover, following the LvCSTB-L silencing, significant decreases in the mRNA levels of p53, p38, caspase3, STAT and ERK were also observed. The results suggested that LvCSTB-L could play positively roles in antiviral immune response by JAK-STAT, MAPK and apoptotic pathway. These findings would further our understanding of shrimp antiviral response, and therefore help for virus control and prevention., (Copyright © 2020. Published by Elsevier Ltd.)

Cystatins represent a large superfamily of proteins involved in the competitive reversible inhibition of C1 class cysteine proteases. Plant-derived papain proteases and cysteine cathepsins are the major cysteine proteases that interact with cystatins. The cystatin superfamily can be further classified into three groups: stefins, cystatins, and kininogens. Among these, cystatin B is categorized under stefins. Cystatin B lacks a signal sequence, disulfide bonds, and carbohydrate groups. However, it contains the conserved cystatin family signature, including a single cystatin-like domain, cysteine protease inhibitory signature concealing pentapeptide (QXVXG) consensus sequence, and two conserved neighboring glycine ( 8 GG 9 ) residues at the N-terminal. In the current study, a member of cystatin B was identified from Korean black rockfish (Sebastes schlegeli) using a cDNA database and designated as RfCytB. The full-length cDNA of RfCytB was 573 bp long, with a coding region of 294 bp. The 5'-untranslated region (UTR) comprised 55 bp, and the 263-bp-long 3'-UTR included a polyadenylation signal sequence and a poly-A tail. The coding sequence encodes a polypeptide comprising 97 amino acids, with a predicted molecular weight of 11 kDa and theoretical isoelectric point of 6.3. RfCytB shared homology features with similar molecules from other teleost and vertebrate species, and was clustered with Cystatin family 1 in our phylogenetic reconstruction. RfCytB was ubiquitously expressed in all tissue types of healthy animals, with the highest levels of expression observed in gill and spleen. Temporal expression of RfCytB displayed significant up-regulation upon infection with Aeromonas salmonicida. Recombinantly expressed RfCytB showed a concentration-dependent inhibitory activity towards papain, with a high thermal stability. Transient expression of RfCytB in LPS activated murine macrophages, thereby inducing the expression of genes related to pro-inflammatory conditions, such as iNOS and TNF α. These results provide evidence for its protease inhibitory and immunity relevant roles in hosts., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Progressive myoclonus epilepsy (PME) of Unverricht-Lundborg type (EPM1) is an autosomal recessive neurodegenerative disorder with the highest incidence of PME worldwide. Mutations in the gene encoding cystatin B (CSTB) are the primary genetic cause of EPM1. Here, we investigate the role of CSTB during neurogenesis in vivo in the developing mouse brain and in vitro in human cerebral organoids (hCOs) derived from EPM1 patients. We find that CSTB (but not one of its pathological variants) is secreted into the mouse cerebral spinal fluid and the conditioned media from hCOs. In embryonic mouse brain, we find that functional CSTB influences progenitors' proliferation and modulates neuronal distribution by attracting interneurons to the site of secretion via cell-non-autonomous mechanisms. Similarly, in patient-derived hCOs, low levels of functional CSTB result in an alteration of progenitor's proliferation, premature differentiation, and changes in interneurons migration. Secretion and extracellular matrix organization are the biological processes particularly affected as suggested by a proteomic analysis in patients' hCOs. Overall, our study sheds new light on the cellular mechanisms underlying the development of EPM1., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The prognosis for pancreatic ductal adenocarcinoma (PDAC) remains poor despite decades of effort. The abundant extracellular matrix (ECM) in PDAC comprises a major fraction of the tumor mass and plays various roles in promoting resistance to therapies. However, nonselective depletion of ECM has led to poor patient outcomes. Consistent with that observation, we previously showed that individual matrisome proteins derived from stromal cells correlate with either long or short patient survival. In marked contrast, those derived from cancer cells correlate strongly with poor survival. Here, we studied three cancer cell-derived matrisome proteins that are significantly overrepresented during PDAC progression, AGRN (agrin), SERPINB5 (serine protease inhibitor B5), and CSTB (cystatin B). Using both overexpression and knockdown experiments, we demonstrate that all three are promoters of PDAC metastasis. Furthermore, these proteins operate at different metastatic steps. AGRN promoted epithelial-to-mesenchymal transition in primary tumors, whereas SERPINB5 and CSTB enhanced late steps in the metastatic cascade by elevating invadopodia formation and in vivo extravasation. All three genes were associated with a poor prognosis in human patients and high levels of SERPINB5, secreted by cancer cells and deposited in the ECM, correlated with poor patient prognosis. This study provides strong evidence that cancer cell-derived matrisome proteins can be causal in promoting tumorigenesis and metastasis and lead to poor patient survival. Therefore, compared with the bulk matrix, mostly made by stromal cells, precise interventions targeting cancer cell-derived matrisome proteins, such as AGRN, SERPINB5, and CSTB, may represent preferred potential therapeutic targets. SIGNIFICANCE: This study provides insights into the biological roles of cancer cell-derived matrisome proteins in PDAC and supports the notion that these proteins are protumorigenic and better therapeutic targets., (©2020 American Association for Cancer Research.)

Endometrial cancer is one of the most common cancers in women worldwide, affecting more than 300,000 women annually. Dysregulated gene expression, especially those mediated by microRNAs, play important role in the development and progression of cancer. This study aimed to investigate differentially expressed genes in endometrial adenocarcinoma using next generation sequencing (NGS) and bioinformatics. The gene expression profiles and microRNA profiles of endometrial adenocarcinoma (cancer part) and normal endometrial tissue (non-cancer part) were assessed with NGS. We identified 56 significantly dysregulated genes, including 47 upregulated and 9 downregulated genes, in endometrial adenocarcinoma. Most of these genes were associated with defense response, response to stimulus, and immune system process, and further pathway analysis showed that human papillomavirus infection was the most significant pathway in endometrial adenocarcinoma. In addition, these genes were also associated with decreased cell death and survival as well as increased cellular movement. The analyses using Human Protein Atlas, identified 6 genes ( PEG10 , CLDN1 , ASS1 , WNT7A , GLDC , and RSAD2 ) significantly associated with poorer prognosis and 3 genes ( SFN , PIGR , and CDKN1A ) significantly associated with better prognosis. Combining with the data of microRNA profiles using microRNA target predicting tools, two significantly dysregulated microRNA-mediated gene expression changes in endometrial adenocarcinoma were identified: downregulated hsa-miR-127-5p with upregulated CSTB and upregulated hsa-miR-218-5p with downregulated HPGD . These findings may contribute important new insights into possible novel diagnostic or therapeutic strategies for endometrial adenocarcinoma., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Lung injury is one of the pathological hallmarks of most respiratory tract diseases including asthma, acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). It involves progressive pulmonary tissue damages which are usually irreversible and incurable. Therefore, strategies to facilitate drug development against lung injury are needed. Here, we characterized the zebrafish folate-deficiency (FD) transgenic line that lacks a fully-developed swim bladder. Whole-mount in-situ hybridization revealed comparable distribution patterns of swim bladder tissue markers between wild-type and FD larvae, suggesting a proper development of swim bladder in early embryonic stages. Unexpectedly, neutrophils infiltration was not observed in the defective swim bladder. Microarray analysis revealed a significant increase and decrease of the transcripts for cathepsin L and a cystatin B (CSTB)-like (zCSTB-like) proteins, respectively, in FD larvae. The distribution of cathepsin L and the zCSTB-like transcripts was spatio-temporally specific in developing wild-type embryos and, in appropriate measure, correlated with their potential roles in maintaining swim bladder integrity. Supplementing with 5-formyltetrahydrofolate successfully prevented the swim bladder anomaly and the imbalanced expression of cathepsin L and the zCSTB-like protein induced by folate deficiency. Injecting the purified recombinant zebrafish zCSTB-like protein alleviated FD-induced swim bladder anomaly. We concluded that the imbalanced expression of cathepsin L and the zCSTB-like protein contributed to the swim bladder malformation induced by FD and suggested the potential application of this transgenic line to model the lung injury and ECM remodeling associated with protease/protease inhibitor imbalance.

We previously reported that cystatin B (CSTB) is a progression marker of human ovarian cancer (OC); however, the regulatory mechanism of CSTB and its function in OC remain unclear. The present study aimed to explore the mechanism underlying transforming growth factor-β (TGF‑β) 1‑mediated CSTB regulation, and to examine the function of CSTB on OC cell proliferation and apoptosis. Using the online program, miRWalk, a microRNA (miR)‑143‑3p was detected, which contains a homologous sequence of the potential binding site to the 3'‑untranslated region (3'‑UTR) of CSTB. A dual‑luciferase reporter assay confirmed the interaction between miR‑143‑3p and CSTB 3'‑UTR. Treating OC cells with miR‑143‑3p mimics or inhibitors resulted in a decrease or an increase of CSTB expression at mRNA and protein levels, respectively. Additionally, CSTB was significantly overexpressed, whereas miR‑143‑3p was downregulated in human OC tissues compared with normal ovarian tissues. A negative correlation between miR‑143‑3p and CSTB mRNA expression was observed in ovarian malignant tumors. The levels of primary and mature miR‑143‑3p expression were upregulated in OC cells after TGF‑β1 treatment; the action of TGF‑β1 was abolished in the presence of an inhibitor of TGF‑β type I receptor. These results indicated an axis between TGF‑β, miR‑143‑3p and CSTB in OC cells. Furthermore, high levels of CSTB expression were associated with the poor overall survival of patients with OC. Knockdown of CSTB resulted in a decrease in OC cell proliferation and arrested cells in G2/M phase. In addition, suppression of CSTB induced cell apoptosis. In conclusion, CSTB was overexpressed and miR‑143‑3p was downregulated in ovarian malignant tumors. Mature miR‑143‑3p directly bound CSTB 3'‑UTR, leading to a decrease in CSTB expression in OC cells, which was regulated by TGF‑β1. Our findings suggest the potential therapeutic application of targeting the TGF‑β/miR‑143‑3p/CSTB axis for treating patients with OC.

Proline residues play a prominent role in protein folding and aggregation. We investigated the influence of single prolines and their combination on oligomerization and the amyloid fibrillation reaction of human stefin B (stB). The proline mutants influenced the distribution of oligomers between monomers, dimers, and tetramers as shown by the size-exclusion chromatography. Only P74S showed higher oligomers, reminiscent of the molten globule reported previously for the P74S of stB-Y31 variant. The proline mutants also inhibited to various degree the amyloid fibrillation reaction. At 30 and 37 °C, inhibition was complete for the P74S single mutant, two double mutants (P6L P74S and P74S P79S), and for the triple mutant P6L P11S P74S. At 30 °C the single mutant P6L completely inhibited the reaction, while P11S and P79S formed amyloid fibrils with a prolonged lag phase. P36D did not show a lag phase, reminiscent of a downhill polymerization model. At 37 °C in addition to P36D, P11S, and P79S, P6L and P11S P74S also started to fibrillate; however, the yield of the fibrils was much lower than that of the wild-type protein as judged by transmission electron microscopy. Thus, Pro 74 cis/trans isomerization proves to be the key event, acting as a switch toward an amyloid transition. Using our previous model of nucleation and growth, we simulated the kinetics of all the mutants that exhibited sigmoidal fibrillation curves. To our surprise, the nucleation phase was most affected by Pro cis/trans isomerism, rather than the fibril elongation phase.

Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin. These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation., (© 2019 Wiley Periodicals, Inc.)

Domain swapping is the process by which identical monomeric proteins exchange structural elements to generate dimers/oligomers. Although engineered domain swapping is a compelling strategy for protein assembly, its application has been limited due to the lack of simple and reliable design approaches. Here, we demonstrate that the hydrophobic five-residue 'cystatin motif' (QVVAG) from the domain-swapping protein Stefin B, when engineered into a solvent-exposed, tight surface loop between two β-strands prevents the loop from folding back upon itself, and drives domain swapping in non-domain-swapping proteins. High-resolution structural studies demonstrate that engineering the QVVAG stretch independently into various surface loops of four structurally distinct non-domain-swapping proteins enabled the design of different modes of domain swapping in these proteins, including single, double and open-ended domain swapping. These results suggest that the introduction of the QVVAG motif can be used as a mutational approach for engineering domain swapping in diverse β-hairpin proteins.

Human stefin B is a protease inhibitor from the family of cystatins. It was reported that it forms oligomers in cells. We have shown that it has a role in cell's response to misfolded proteins. We also have shown that its oligomers bind amyloid-beta (Aβ). Here, we discuss ways, how stefin B could reduce build-up of protein aggregates by other proteins and consequently reduces ROS and, how this might be connected to autophagy. When overexpressed, stefin B forms protein aggregates itself and these protein aggregates induce autophagy. Similarly, cystatin C was shown to bind Aβ and to induce autophagy. It is also suggested how more knowledge about the role of stefin B in a cell's response to misfolded proteins could be used to modulate progressive myoclonus epilepsy of type 1 EPM1 disease.

Cystatin B is an intracellular inhibitor that regulates the activities of cysteine proteases. In this study, cystatin B in Japanese flounder (Paralichthys olivaceus) was characterized and its immune function was analyzed. This gene had a high similarity with the sequence of cystatin B in other fish species, and the derived peptide shared typical features of cystatin proteins including the QXVXG motif. The results of quantitative real-time PCR showed that cystatin B mRNA was constitutively expressed in all examined tissues, with the highest level in gill. The stimulations of lipopolysaccharide, peptidoglycan and polyinosinic-polycytidylic acid effectively increased the expression level of cystatin B mRNA. Functional analysis implied that the recombinant P. olivaceus cystatin B purified from Escherichia coli had cysteine protease inhibitory activity and could inhibit bacterial growth by binding to bacteria. Furthermore, we found that P. olivaceus cystatin B had no effects on the expression of inflammatory factors cytokines tumor necrosis factor α, interleukin 10, interleukin 1β and interferon γ. These results indicate that cystatin B of P. olivaceus is potentially involved in immune responses against invading microbial pathogens, and provide a better understanding of the immune mechanisms of cystatins in teleosts., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aims: Dysregulated expression of cystatin B (CSTB) has been implicated in various cancers. The aims of this study were to analyze the CSTB expression and investigate the epigenetic regulation of CSTB in lung and colon cancer cell lines, and also evaluate the clinical outcome of CSTB in primary lung and colorectal tumors., Methods: CSTB expression in lung and colon cancer cell lines was analyzed by real-time RT-PCR and western blotting. Epigenetic regulation of CSTB was examined by demethylation, deacetylation tests and bisulfite sequencing (BS). In primary lung and colorectal tumors, the protein expression of CSTB was evaluated by immunohistochemistry on tissue microarray., Results: CSTB was downregulated in lung cancer cell lines on mRNA and protein levels compared to human bronchial epithelial cells (HBEC). In colon cancer cell lines, CSTB was weakly expressed in Caco2, CX2 and HCT-16 and highly expressed in HT-29, WiDr, SW480 and HRT-18 on mRNA level compared to normal colonic fibroblast cells CCD33Co. After treatment with demethylation agent 5-aza-2'-deoxycytidine, increased CSTB mRNA expression was found in 7 out of 11 lung cancer cell lines including H226, H157, H2170, H1299, COLO677, A549 and H1975, while no obvious alteration was found in colon cancer cell lines. No DNA methylation could be found in the selected CpG islands in two types of cancer cell lines by bisulfite sequencing. In primary tumors, CSTB expression was significantly and inversely correlated with lung tumor stage (pN) and tumor grade (p=0.022 and 0.047, respectively). Kaplan-Meier survival curve revealed a tendency that lung tumors with high CSTB expression had a more favourable prognosis (p=0.062). In colorectal tumors, CSTB was not linked to any clinicopathological parameters including age, size of tumor, lymph node metastasis and tumor grading., Conclusions: CSTB might be a potential prognostic marker for patients with primary lung cancer., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Mutations of the cystatin B gene (CSTB; OMIM 601145) are known to cause Unverricht-Lundborg disease or progressive myoclonic epilepsy-1A (EPM1A, MIM #254800). Most patients are homozygous for an expanded (>30) dodecamer repeat in the promoter region of CSTB, or are compound heterozygotes for the dodecamer repeat and a point mutation. We report two adolescent sisters born to consanguineous parents of Sri Lankan descent who presented with profound global developmental delay, microcephaly, cortical blindness and axial hypotonia with appendicular hypertonia. Neither sibling ever developed head control, independent sitting or ambulation, and never developed speech. The elder sister had a seizure disorder. Both sisters had profound microcephaly and distinct facial features. On serial brain imaging, they had progressive atrophy of the corpus callosum and supratentorial brain, and diffuse hypomyelination with progressive loss of myelin signal. Exome sequencing revealed both siblings to be homozygous for a c.218dupT (p.His75Serfs*2) mutation in exon 3 of CSTB. The neuroimaging features of our patients are consistent with those observed in Cstb-knockout mice, which supports the hypothesis that disease severity is inversely correlated with the amount of residual functional cystatin B protein.

Unverricht-Lundborg disease or progressive myoclonic epilepsy type 1 (EPM1) is an autosomal recessive disease caused by mutation of the cystatin B gene (CSTB), located on chromosome 21q22.3. The most common mutation is an expansion of unstable dodecamer repetition (CCCCGCCCCGCG), whereas other types of mutations are rare. Among these, heterozygous compound mutations are described to induce a more severe phenotype than that of homozygous dodecameric repetition. We report two siblings affected by heterozygous compound mutations carrying a novel mutation of the deletion of three nucleotides in exon 2 of the gene in position 132-134 of the coding sequence (c.132-134del) in the allele not including the dodecamer repetition. This mutation results in the loss of two amino acid residues and insertion of an asparagine in position 44 (p.Lys44&#95;Ser45delinsAsn). Our patients presented a very different clinical picture. The male patient had a severe myoclonus, drug-resistant epilepsy and psychiatric comorbidity, while his affected sister had only very rare seizures and sporadic myoclonic jerks at awakening. The revision of literature about heterozygous compound EPM1 patients confirms this gender phenotypic expressivity, with female patients carrying less severe symptoms than male patients. These data lead to the hypothesis of complex gender-specific factors interacting with CSTB expressivity in EPM1 patients., (Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.)

We describe studies performed thus far on stefin B from the family of cystatins as a model protein for folding and amyloid fibril formation studies. We also briefly mention our studies on aggregation of some of the missense EPM1 mutants of stefin B in cells, which mimic additional pathological traits (gain in toxic function) in selected patients with EPM1 disease. We collected data on the reported interactors of stefin B and discuss several hypotheses of possible cytosolic alternative functions., (Copyright © 2016 John Wiley & Sons, Ltd.)

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited childhood-onset neurodegenerative disorder, characterized by myoclonus, seizures, and ataxia. Mutations in the cystatin B gene (CSTB) underlie EPM1. The CSTB-deficient (Cstb -/- ) mouse model recapitulates key features of EPM1, including myoclonic seizures. The mice show early microglial activation that precedes seizure onset and neuronal loss and leads to neuroinflammation. We here characterized the inflammatory phenotype of Cstb -/- mice in more detail. We found higher concentrations of chemokines and pro-inflammatory cytokines in the serum of Cstb -/- mice and higher CXCL13 expression in activated microglia in Cstb -/- compared to control mouse brains. The elevated chemokine levels were not accompanied by blood-brain barrier disruption, despite increased brain vascularization. Macrophages in the spleen and brain of Cstb -/- mice were predominantly pro-inflammatory. Taken together, these data show that CXCL13 expression is a hallmark of microglial activation in Cstb -/- mice and that the brain inflammation is linked to peripheral inflammatory changes, which might contribute to the disease pathology of EPM1.

This study aimed to investigate the pivotal role of cystatin B (CSTB) in the development of gastric cancer and to explore its possible regulatory mechanism. Human gastric cancer SGC-7901 cells as a model in vitro were transfected with plasmid PCDNA3.1-CSTB and siRNA-CSTB using Lipofectamine 2000. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to determine the relative expression of CSTB and PI3K/Akt/mTOR pathway-related protein. Moreover, MTT assay, Transwell assay, and flow cytometry were used to assess cell proliferation, migration, and apoptosis, respectively. The results showed that CSTB was significantly downregulated in SGC-7901 cells compared with gastric epithelial cells. CSTB was successfully overexpressed and suppressed after cells were transfected with pc-CSTB and si-CSTB, respectively. Moreover, cell viability and migration were significantly decreased after being transfected with pc-CSTB when compared with the control group, while being obviously increased after transfection with si-CSTB. However, cell apoptosis was significantly induced after being transfected with pc-CSTB, while being obviously suppressed after transfection with si-CSTB. Besides, the expression levels of p-PI3K, p-Akt, and p-mTOR proteins were all significantly decreased in the pc-CSTB transfection group when compared with the control group, while being increased in the si-CSTB transfection group. Our findings suggest that CSTB downregulation may promote the development of gastric cancer by affecting cell proliferation and migration, and the PI3K/Akt/mTOR signaling pathway was activated in this process. CSTB may serve as a potential therapeutic target for gastric cancer.

Cystatin B is a cysteine protease inhibitor that induces HIV replication in monocyte-derived macrophages (MDM). This protein interacts with signal transducer and activator of transcription (STAT-1) factor and inhibits the interferon (IFN-β) response in Vero cells by preventing STAT-1 translocation to the nucleus. Cystatin B also decreases the levels of tyrosine-phosphorylated STAT-1 (STAT-1PY). However, the mechanisms of cystatin B regulation on STAT-1 phosphorylation in MDM are unknown. We hypothesized that cystatin B inhibits IFN-β antiviral responses and induces HIV replication in macrophage reservoirs through the inhibition of STAT-1 phosphorylation. Macrophages were transfected with cystatin B siRNA prior to interferon-β treatment or infected with HIV-ADA to determine the effect of cystatin B modulation in STAT-1 localization and activation using immunofluorescence and proximity ligation assays. Cystatin B decreased STAT-1PY and its transportation to the nucleus, while HIV infection retained unphosphorylated STAT (USTAT-1) in the nucleus avoiding its exit to the cytoplasm for eventual phosphorylation. In IFN-β-treated MDM, cystatin B inhibited the nuclear translocation of both, USTAT-1 and STAT-1PY. These results demonstrate that cystatin B interferes with the STAT-1 signaling and IFN-β-antiviral responses perpetuating HIV in macrophage reservoirs., Competing Interests: LMM has a patent application approved for cystatin B, related to this manuscript. Pat No. 8,143,231.

We first review the clinical presentation and current therapeutic approaches available for treating Unverricht-Lundborg disease (ULD), a progressive myoclonus epilepsy. Next, we describe the identification of disease causing mutations in the gene encoding cystatin B (CSTB). A Cstb-deficient mouse model, which recapitulates the key features of ULD including myoclonic seizures, ataxia, and neuronal loss, was generated to shed light on the mechanisms contributing to disease pathophysiology. Studies with this model have elucidated the diverse biological roles for Cstb from functioning as a protease inhibitor, to regulating glial activation, oxidative stress, serotonergic neurotransmission, and hyperexcitability. These findings set the stage for future studies that may open avenues to improved therapeutic approaches.

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1, OMIM254800) is an autosomal recessive neurodegenerative disorder characterized by stimulus-sensitive and action-activated myoclonus, tonic-clonic epileptic seizures, and ataxia. Loss-of-function mutations in the gene encoding the cysteine protease inhibitor cystatin B (CSTB) underlie EPM1. The deficiency of CSTB in mice (Cstb-/- mice) generates a phenotype resembling the symptoms of EPM1 patients and is accompanied by microglial activation at two weeks of age and an upregulation of immune system-associated genes in the cerebellum at one month of age. To shed light on molecular pathways and processes linked to CSTB deficiency in microglia we characterized the transcriptome of cultured Cstb-/- mouse microglia using microarray hybridization and RNA sequencing (RNA-seq). The gene expression profiles obtained with these two techniques were in good accordance and not polarized to either pro- or anti-inflammatory status. In Cstb-/- microglia, altogether 184 genes were differentially expressed. Of these, 33 genes were identified by both methods. Several interferon-regulated genes were weaker expressed in Cstb-/- microglia compared to control. This was confirmed by quantitative real-time PCR of the transcripts Irf7 and Stat1. Subsequently, we explored the biological context of CSTB deficiency in microglia more deeply by functional enrichment and canonical pathway analysis. This uncovered a potential role for CSTB in chemotaxis, antigen-presentation, and in immune- and defense response-associated processes by altering JAK-STAT pathway signaling. These data support and expand the previously suggested involvement of inflammatory processes to the disease pathogenesis of EPM1 and connect CSTB deficiency in microglia to altered expression of interferon-regulated genes.

Niemann-Pick C1 (NPC) disease, an autosomal recessive lipid trafficking disorder caused by loss-of-function mutations in the NPC1 gene, is characterized by progressive neurodegeneration resulting in cognitive impairment, ataxia and early death. Little is known about the cellular pathways leading to neuron loss. Here, we studied the effects of diminishing expression of cystatin B, an endogenous inhibitor of cathepsins B, H and L, on the development of NPC neuropathology. We show that decreased expression of cystatin B in patient fibroblasts enhances cathepsin activity. Deletion of the encoding Cstb gene in Npc1-deficient mice resulted in striking deleterious effects, particularly within the cerebellum where diffuse loss of Purkinje cells was observed in young mice. This severe pathology occurred through cell autonomous mechanisms that triggered Purkinje cell death. Moreover, our analyses demonstrated the mislocalization of lysosomal cathepsins within the cytosol of Npc1-deficient Purkinje cells. We provide evidence that this may be a consequence of damage to lysosomal membranes by reactive oxygen species (ROS), leading to the leakage of lysosomal contents that culminates in apoptotic cell death. Consistent with this notion, toxicity from ROS was attenuated in an NPC cell model by cystatin B over-expression or pharmacological inhibition of cathepsin B. The observation that Npc1 and Cstb deletion genetically interact to potently enhance the degenerative phenotype of the NPC cerebellum provides strong support for the notion that lysosomal membrane permeabilization contributes to cerebellar degeneration in NPC disease., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Stefins have been reported to be associated with the progression and metastasis of various malignant tumors. However, the expressions of stefins in hepatocellular carcinoma (HCC) have not been well-defined. In this study, the protein levels of stefin A and stefin B were assessed by immunohistochemical staining, and the mRNA levels were quantified by real-time polymerase chain reaction in 85 primary HCC tissues, 85 surrounding non-cancerous tissues, and 9 normal hepatic tissues. The immunohistochemical staining of cathepsin B and cathepsin D, and the ratio of cathepsins to stefins were assessed. The mRNA expressions of stefin A and stefin B in HCC tissues were significantly higher than surrounding noncancerous tissues and normal hepatic tissues, respectively. A significant positive relationship of stefin A and stefin B was found with node metastasis, tumor size, and Edmondson grade for HCC. Univariate and multivariate analyses revealed that Edmondson grade and stefin B expression were independent factors associated with the risk of lymph node metastasis in HCC. The ratios of cathepsin B to stefin A, cathepsin D to stefin A, cathepsin B to stefin B and cathepsin D to stefin B of the HCC group were significantly higher than that of the surrounding noncancerous group. A significant positive correlation between the ratio of cathepsins to stefins (cathepsin B/stefin A, cathepsin B/stefin B and cathepsin D/stefin B) and node metastasis was demonstrated. We concluded that high expressions of stefin A and stefin B may be an important factor contributing to the development and metastasis of HCC., (© 2016 Wiley Periodicals, Inc.)

Novel biomarkers and non-invasive diagnostic methods are urgently needed for the screening of gastric cancer to reduce its high mortality. We employed quantitative proteomics approach to develop discriminatory biomarker signatures from human saliva for the detection of gastric cancer. Salivary proteins were analyzed and compared between gastric cancer patients and matched control subjects by using tandem mass tags (TMT) technology. More than 500 proteins were identified with quantification, and 48 of them showed significant difference expression (p < 0.05) between normal controls and gastric cancer patients, including 7 up-regulated proteins and 41 down-regulated proteins. Five proteins were selected for initial verification by ELISA and three were successfully verified, namely cystatin B (CSTB), triosephosphate isomerase (TPI1), and deleted in malignant brain tumors 1 protein (DMBT1). All three proteins could differentiate gastric cancer patients from normal control subjects, dramatically (p < 0.05). The combination of these three biomarkers could reach 85% sensitivity and 80% specificity for the detection of gastric cancer with accuracy of 0.93. This study provides the proof of concept of salivary biomarkers for the non-invasive detection of gastric cancer. It is highly encouraging to turn these biomarkers into an applicable clinical test after large scale validation.

Cystatins are reversible cysteine protease inhibitor proteins. They are known to play important roles in controlling cathepsins, neurodegenerative disease, and in immune system regulation. Production of recombinant cystatin proteins is important for biochemical and function characterization. In this study, we cloned and expressed human stefin A, stefin B and cystatin C in Escherichia coli. Human stefin A, stefin B and cystatin C were purified from soluble fraction. For cystatin C, we used various chaperone plasmids to make cystatin C soluble, as it is reported to localize in inclusion bodies. Trigger factor, GroES-GroEL, DnaK-DnaJ-GrpE chaperones lead to the presence of cystatin C in the soluble fraction. Immobilized metal affinity chromatography, glutathione sepharose and anion exchange chromatography techniques were employed for efficient purification of these proteins. Their biological activities were tested by inhibition assays against cathepsin L and H3 protease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The tau protein is central to the etiology of several neurodegenerative diseases, including Alzheimer's disease, a subset of frontotemporal dementias, progressive supranuclear palsy and dementia following traumatic brain injury, yet the proteins it interacts with have not been studied using a systematic discovery approach. Here we employed mild in vivo crosslinking, isobaric labeling, and tandem mass spectrometry to characterize molecular interactions of human tau in a neuroblastoma cell model. The study revealed a robust association of tau with the ribonucleoproteome, including major protein complexes involved in RNA processing and translation, and documented binding of tau to several heat shock proteins, the proteasome and microtubule-associated proteins. Follow-up experiments determined the relative contribution of cellular RNA to the tau interactome and mapped interactions to N- or C-terminal tau domains. We further document that expression of P301L mutant tau disrupts interactions of the C-terminal half of tau with heat shock proteins and the proteasome. The data are consistent with a model whereby a higher propensity of P301L mutant tau to aggregate may reflect a perturbation of its chaperone-assisted stabilization and proteasome-dependent degradation. Finally, using a global proteomics approach, we show that heterologous expression of a tau construct that lacks the C-terminal domain, including the microtubule binding domain, does not cause a discernible shift of the proteome except for a significant direct correlation of steady-state levels of tau and cystatin B., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: This Finnish nationwide study aimed to refine the clinical phenotype variability and to identify factors that could explain the extensive variability in the clinical severity of the symptoms observed among patients with Unverricht-Lundborg disease (progressive myoclonus epilepsy type 1 [EPM1]) homozygous for the dodecamer expansion mutation in the cystatin B (CSTB) gene., Methods: The study population consisted of 66 (33 men and 33 women) patients with genetically confirmed EPM1 homozygous for the CSTB expansion mutation for whom the sizes of the expanded alleles were determined. The clinical evaluation included videorecorded Unified Myoclonus Rating Scale and retrospectively collected medical history. The navigated transcranial magnetic stimulation test was used to determine motor threshold (MT) and silent period (SP) of the motor cortex., Results: An earlier age at onset for EPM1 and longer disease duration were associated with more severe action myoclonus, lower performance IQ, increased MT, and prolonged SP. The number of dodecamer repeats in CSTB alleles varied between 38 and 77. On average, the size of the longer expanded alleles of patients was independently associated with MT, but exerted only a modulating effect on age at onset, myoclonus severity, and SP., Conclusions: As a group, earlier disease onset and longer duration are associated with more severe phenotype. Even though the vast majority of patients with EPM1 have a uniform genetic mutation, the actual size of the longer CSTB expansion mutation allele is likely to have a modulating effect on the age at disease onset, myoclonus severity, and cortical neurophysiology., (© 2015 American Academy of Neurology.)

Epithelial ovarian cancer (EOC) consists of four major subtypes: clear cell carcinoma (CCC), endometrioid adenocarcinoma (EA), mucinous adenocarcinoma (MA) and serous adenocarcinoma (SA). Relative to the other subtypes, the prognosis of CCC is poor due to a high recurrence rate and chemotherapy resistance, but CCC-specific biomarkers have yet to be identified. With the aim of identifying diagnostic and treatment biomarkers for CCC, we analyzed 96 cases of EOC (32 CCC, 13 EA, 19 MA, 32 SA) using liquid chromatography/mass spectrometry (LC/MS) followed by immunohistochemistry (IHC) and quantitative reverse transcription PCR (RT-qPCR). Semi-quantification of protein differences between subtypes showed upregulation of 150 proteins and downregulation of 30 proteins in CCC relative to the other subtypes. Based on hierarchical clustering that revealed a marked distinction in the expression levels of cystatin B (CYTB) and Annexin A4 (ANXA4) in CCC relative to the other subtypes, we focused the study on CYTB and ANXA4 expression in EOCs by IHC, RT-qPCR and western blot analyses using tissue specimens and cultured cells. As a result, compared to the other subtypes, CCC showed significantly high expression levels of CYTB and ANXA4 in the analyses. To examine the possibility of CYTB and ANXA4 as serum diagnostic biomarkers of CCC, we checked the protein levels in conditioned media and cell lysates using culture cells. Compared with the other subtypes, CCC cell lines showed a significantly higher level of expression of CYTB in both conditioned media and cell lysates, while ANXA4 showed a higher level of expression in cell lysates only. Our results demonstrate that CYTB and ANXA4 overexpression may be related to carcinogenesis and histopathological differentiation of CCC. CYTB may be a secreted protein, and may serve as a potential serum diagnostic biomarker of CCC, while ANXA4 may be useful as an intracellular marker.

Genetic factors play a major role in the etiology of juvenile myoclonic epilepsy (JME), a common form of idiopathic generalized epilepsy, but so far, genes related to JME remain largely unknown. JME shares electroclinical features with Unverricht-Lundborg disease (progressive myoclonic epilepsy type 1; EPM1), a form of progressive myoclonus epilepsy characterized by myoclonus, epilepsy, and gradual neurologic deterioration. EPM1 is caused by mutations in the gene that codes for cystatin B (CSTB), an inhibitor of cysteine protease. In the present study, we wished to investigate the role of the CSTB gene in patients with JME. Fifty-seven unrelated patients (35 women; mean age ± standard deviation [SD], 24.1 ± 7.7; mean age ± SD at onset, 15.3 ± 2.4) with JME were enrolled. Twenty-three of 57 patients were the probands of families with JME. The molecular diagnosis was carried out to identify the common dodecamer repeat expansion mutation or other disease-causing mutations in the CSTB gene. The molecular analysis did not depict mutations in any of the 57 patients with JME. Our study did not support a role for the CSTB gene in patients with familial or sporadic JME., (Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.)

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal-recessively inherited neurodegenerative disorder characterized by severely incapacitating myoclonus, seizures, and ataxia, and caused by loss-of-function mutations in the cystatin B gene (CSTB). A central neuropathological finding in the Cstb(-/-) mouse, an animal model for EPM1, is early microglial activation, which precedes astroglial activation, neuronal loss, and onset of myoclonus, thus implying a critical role for microglia in EPM1 pathogenesis. Here, we characterized phenotypic and functional properties of microglia from Cstb(-/-) mice utilizing brain tissue, microglia directly isolated from the brain, and primary microglial cultures. Our results show significantly higher Cstb mRNA expression in microglia than in neurons and astrocytes. In Cstb(-/-) mouse brain, expression of the inflammatory marker p-p38 MAPK and the proportion of both pro-inflammatory M1 and anti-inflammatory M2 microglia is higher than in control mice. Moreover, M1/M2 polarization of microglia in presymptomatic Cstb(-/-) mice is, compared to control mice, skewed towards M2 type at postnatal day 14 (P14), but towards M1 type at P30, a time point associated with onset of myoclonus. At this age, the high expression of both pro-inflammatory inducible nitric oxide synthase (iNOS) and anti-inflammatory arginase 1 (ARG1) in Cstb(-/-) mouse cortex is accompanied by the presence of peripheral immune cells. Consistently, activated Cstb(-/-) microglia show elevated chemokine release and chemotaxis. However, their MHCII surface expression is suppressed. Taken together, our results link CSTB deficiency to neuroinflammation with early activation and dysfunction of microglia and will open new avenues for therapeutic interventions for EPM1., (© 2014 Wiley Periodicals, Inc.)

EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death. We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed., (Copyright © 2014. Published by Elsevier B.V.)

Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO) mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt) and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL) gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.