CSTA plays a role in osteoclast formation and bone resorption by mediating the DAP12/TREM2 pathway.

Cystatin A (CSTA) is a cysteine protease inhibitor that is expressed highly during osteoporosis. However, the exact role of CSTA in osteoporosis remains unknown. In this study, we examined the role of CSTA in the formation, differentiation, and bone resorption of osteoclasts. We extracted bone marrow cells from 8-week-old wildtype mice to obtain RANKL and M-CSF-induced osteoclasts. We performed CSTA overexpression and knockdown experiments in the cells. We analyzed the role of CSTA in the process of osteoclasts by trap staining. In addition, we studied the contribution of CSTA to osteogenesis through the DAP12/TREM2 (DNAX-activating protein of 12 kDa/Triggering receptor expressed on myeloid cells-2) complex. We analyzed the role of CSTA in postmenopausal osteoporosis using OVX mouse models. We found that the silencing of CSTA inhibited the differentiation and formation of osteoclasts. The loss of CSTA weakened the expression of osteoclast marker genes. In contrast, overexpression of CSTA significantly increased differentiation and formation of osteoclasts and enhanced bone resorption. Immunofluorescence staining indicated that CSTA and DAP12 are co-expressed in osteoclasts, and the loss of either DAP12 or TREM2 inhibited osteoclast differentiation and bone resorption. Suppression of CSTA decreased DAP12 and TREM2 expression, whereas overexpression of CSTA rescued the loss of TREM2 expression caused by DAP12 knockdown. Co-immunoprecipitation and co-localization experiments indicated that CSTA interacted with DAP12. In addition, we found that injection of si-CSTA into OVX mice significantly improved bone parameters. Our research indicates that CSTA interacts with the DAP12/TREM2 complex and could be a potential targeted therapy for osteoporosis management.

Acral peeling skin syndrome resulting from a novel homozygous mutation in the CSTA gene-A report of two cases.

Acral peeling skin syndrome is a rare genodermatosis characterized by asymptomatic peeling of the acral skin. It is usually caused by biallelic mutations in the gene TGM5. However, biallelic mutations in the CSTA gene have also been described to cause APSS with exfoliative ichthyosis, so far in only five pedigrees. Here, we report two new pedigrees, each with one patient having APSS, due to a novel CSTA mutation.

BtsT, a Novel and Specific Pyruvate/H+ Symporter in Escherichia coli.

The peptide transporter carbon starvation (CstA) family (transporter classification [TC] 2.A.114) belongs to the second largest superfamily of secondary transporters, the amino acid/polyamine/organocation (APC) superfamily. No representative of the CstA family has previously been characterized either biochemically or structurally, but we have now identified the function of one of its members, the transport protein YjiY of Escherichia coli Expression of the yjiY gene is regulated by the LytS-like histidine kinase BtsS, a sensor of extracellular pyruvate, together with the LytTR-like response regulator BtsR. YjiY consists of 716 amino acids, which form 18 putative transmembrane helices. Transport studies with intact cells provided evidence that YjiY is a specific and high-affinity transporter for pyruvate (Km , 16 µM). Furthermore, reconstitution of the purified YjiY into proteoliposomes revealed that YjiY is a pyruvate/H+ symporter. It has long been assumed that E. coli possesses a transporter(s) for pyruvate, but the present study is the first to definitively identify such a protein. Based on its function, we propose to change the name of the uncharacterized gene yjiY to btsT for Brenztraubensäure (the German word for pyruvate) transporter.IMPORTANCE BtsT (formerly known as YjiY) is found in many commensal and pathogenic representatives of the Enterobacteriaceae This study for the first time characterizes a pyruvate transporter in E. coli, BtsT, as a specific pyruvate/H+ symporter. When nutrients are limiting, BtsT takes up pyruvate from the medium, thus enabling it to be used as a carbon source for the growth and survival of E. coli.

Identification and validation of Cystatin A as a novel promising therapeutic target for gastric cancer.

Background: The effect of pharmacological treatment of gastric cancer (GC) is limited, thus, it holds significant scientific importance to thoroughly investigate the molecular mechanisms underlying GC development and identify novel molecules capable of substantially extending patients' survival. This study utilized bioinformatics techniques to identify 11 genes associated with recurrence-free survival (RFS) in GC patients and investigated the potential biological functions of these genes through single-cell transcriptomic analysis. Subsequently, a single gene Cystatin A (CSTA) was selected for further analysis to explore its impact on signaling pathways and treatment. Methods: Differentially expressed genes (DEGs) were identified and overlapped in the analysis of RFS to identify potential prognostic genes for GC patients, based on data from the Cancer Genome Atlas-stomach adenocarcinoma (TCGA-STAD) and GSE54129. Subsequently, a prognostic model based on RFS in GC patients was established. Single-cell sequencing data were employed to explore the potential functions of these model genes. CSTA, one of the RFS-related genes, was further investigated using immunohistochemistry (IHC), Cell Counting Kit 8 (CCK-8), transwell, scratch, colony formation assays, flow cytometry, and Western blotting methods. Results: Through bioinformatics analysis, we identified 23 RFS-related genes in GC. Using the least absolute shrinkage and selection operator (LASSO)-Cox method, an RFS prognostic model was developed which pinpointed 11 GC prognosis-related (GPR) genes as significant factors influencing RFS in GC patients. The single-cell analysis revealed their potential role in affecting differentiation and maturation of pre-fibroblasts thereby impacting RFS in GC patients. CSTA exhibited low expression levels in GC tissues. Overexpression of CSTA promoted apoptosis in GC cells through the caspase-dependent apoptotic pathway and enhanced their response to cisplatin via this same pathway. Conclusions: The 11 GPR genes are primarily enriched within a specific type of stromal cell exhibiting heightened communication, metabolism, and differentiation levels. The gene signature of these stromal cells has implications for patient prognosis. Additionally, CSTA, a gene related to prognosis, has been shown to influence apoptosis levels in GC cells.

Correlated with better prognosis, CSTA inhibits metastasis of nasopharyngeal carcinoma cells via suppressing AKT signaling through promoting METTL3 degradation.

BACKGROUND: Metastasis is one of the main obstacles impeding the survival of nasopharyngeal carcinoma (NPC) patients, with the molecular mechanism underlying NPC metastasis still unclear. RESULTS: In this study, Cystatin A (CSTA) was found downregulated in NPC tissues with metastasis compared with those without metastasis. Shorter overall survival and distant metastasis-free survival were found in NPC patients with lower CSTA expression. Using functional assays, we found that CSTA prevented both the in vitro motility of NPC cells and their ability to metastasize in vivo. Transcriptome sequencing and western blot analysis revealed that CSTA inhibited the phosphorylation of AKT. Moreover, activating AKT using AKT agonist SG79 rescued the motility of CSTA-overexpressing NPC cells, whereas, treatment with AKT inhibitor MK2206 inhibited the motility of CSTA-knockdown NPC cells. Mechanically, immunoprecipitation coupled mass spectrometry found that CSTA interacted with the N6-adenosine-methyltransferase subunit METTL3 and promoted its ubiquitin-proteasome-mediated degradation following the upregulation of NKX3-1 and LHPP, which are negative regulators of AKT. Furthermore, knock-down of NKX3-1 and LHPP enhanced the motility of CSTA-overexpressing NPC cells. CONCLUSIONS: The inhibitory effect of CSTA upon NPC metastasis mainly depended on suppressing AKT signaling by the upregulation of NKX3-1 and LHPP expression resulting from the binding between CSTA and METLL3. Our study suggests that the CSTA-METLL3-NKX3-1/LHPP-AKT axis could be of therapeutic value for inhibiting NPC metastasis.

Quantitative analysis of gene expression changes in response to genotoxic compounds.

Techniques that quantify molecular endpoints sufficiently sensitive to identify and classify potentially toxic compounds have wide potential for high-throughput in vitro screening. Expression of three genes, RAD51C, TP53 and cystatin A (CSTA), in HEPG2 cells was measured by Q-PCR amplification. In parallel, we developed alternative assays for the same 3 gene signature based on an acridinium-ester chemiluminescent reporter molecule. HEPG2 cells were challenged with eighteen different compounds (n=18) chosen to represent compounds that are genotoxic (n=8), non-genotoxic non-carcinogenic (n=2) or have a less well defined mechanism of action with respect to genotoxicity (n=8). At least one of the three genes displayed dysregulated expression in the majority of compounds tested by Q-PCR and ten compounds changed the CSTA expression significantly. Acridinium-ester labelled probes for the three genes were synthesised and tested. Analytical sensitivity was characterised and suggested a limit of detection generally better than 0.1fmol but often 10-50 attomol. A linear amplification step was optimised and this quantitative method detected statistically significant increases in RAD51C and CSTA expression in agreement with the Q-PCR results, demonstrating the potential of this technology. The broad agreement of the amplified chemiluminescent method and Q-PCR in measuring gene expression suggests wider potential application for this chemiluminescent technology.

Insights into desmosome biology from inherited human skin disease and cardiocutaneous syndromes.

The importance of desmosomes in tissue homeostasis is highlighted by natural and engineered mutations in desmosomal genes, which compromise the skin or heart and in some instances both. Desmosomal gene mutations account for 45-50% of cases of arrhythmogenic right ventricular cardiomyopathy, and are mutated in an array of other disorders such as striate palmoplantar keratoderma, hypotrichosis with or without skin vesicles and lethal acantholytic epidermolysis bullosa. Recently, we reported loss-of-function mutations in the human ADAM17 gene, encoding for the 'sheddase' ADAM17, a transmembrane protein which cleaves extracellular domains of substrate proteins including TNF-α, growth factors and desmoglein (DSG) 2. Patients present with cardiomyopathy and an inflammatory skin and bowel syndrome with defective DSG processing. In contrast, the dominantly inherited tylosis with oesophageal cancer appears to result from gain-of-function in ADAM17 due to increased processing via iRHOM2. This review discusses the heterogeneity of mutations in desmosomes and their regulatory proteins.

Acral peeling skin syndrome resembling epidermolysis bullosa simplex in a 10-month-old boy.

The acral peeling skin syndrome (APSS) is a rare autosomal recessive disorder clinically characterized by asymptomatic desquamation of the skin limited to the hands and feet and histologically by cleavage at the stratum granulosum and stratum corneum level [Kiritsi et al.: J Invest Dermatol 2010;130:1741-1746]. We report on a 10-month-old boy with a history of skin peeling limited to the hands and feet since 2 months of age. Clinical examination revealed erythematous erosions with peripheral desquamation and flaccid blisters. DNA mutation analysis detected two heterozygous TGM5 mutations: c.2T>C, p.M1T in exon 1 and c.337G>T, p.G113C in exon 3 in keeping with the diagnosis of APSS. The clinical presentation of APSS alone might be confusing and strongly resemble epidermolysis bullosa simplex making the differential diagnosis difficult.