Protein and Peptide Letters - Volume 29, Issue 7, 2022

Protein tyrosine phosphatase receptor-type Q (PTPRQ), a member of the type III tyrosine phosphatase receptor (R3 PTPR) family, is composed of three domains, including 18 extracellular fibronectin type III (FN3) repeats, a transmembrane helix, and a cytoplasmic phosphotyrosine phosphatase (PTP) domain. PTPRQ was initially identified as a transcript upregulated in glomerular mesangial cells in a rat model of glomerulonephritis. Subsequently, studies found that PTPRQ has phosphotyrosine phosphatase and phosphatidylinositol phosphatase activities and can regulate cell proliferation, apoptosis, differentiation, and survival. Further in vivo studies showed that PTPRQ is necessary for the maturation of cochlear hair bundles and is considered a potential gene for deafness. In the recent two decades, 21 mutations in PTPRQ have been linked to autosomal recessive hearing loss (DFNB84) and autosomal dominant hearing loss (DFNA73). Recent mutations, deletions, and amplifications of PTPRQ have been observed in many types of cancers, which indicate that PTPRQ might play an essential role in the development of many cancers. In this review, we briefly describe PTPRQ structure and enzyme activity and focus on the correlation between PTPRQ and human disease. A profound understanding of PTPRQ could be helpful in the identification of new therapeutic targets to treat associated diseases.

The coronavirus family consists of pathogens that seriously affect human and animal health. They mostly cause respiratory or enteric diseases, which can be severe and life-threatening, such as coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome (SARS), and Middle East Respiratory Syndrome (MERS) in humans. The conserved coronaviral papain-like protease is an attractive antiviral drug target because it is essential for coronaviral replication, and it also inhibits host innate immune responses. This review focuses on the latest research progress relating to the mechanism of coronavirus infection, the structural and functional characteristics of coronavirus papain-like protease, and the potent inhibitors of the protease.

Background: Osteoarthritis (OA) is an inflammatory joint disorder with high incidence rates. Long non-coding RNAs (LncRNAs) influence OA development. Objectives: In this research, we attempt to figure out the functions of lncRNA BLACAT1 in human articular chondrocyte (HAC) apoptosis and extracellular matrix (ECM) degradation in OA. Methods: Interleukin (IL)-1β was employed to induce HAC damage. Cell viability and apoptosis were detected, with expression patterns of lncRNA BLACAT1, miR-149-5p, and HMGCR, and levels of Caspase-3, Caspase-9, BAX, Bcl-2, COL2A1, and SOX9 determined. Then, lncRNA BLACAT1 was silenced in IL-1β-treated HACs to analyze its role in HAC damage. The target relations of lncRNA BLACAT1 and miR-149-5p and miR-149-5p and HMGCR were verified. In addition, combined experiments were performed as a miR-149-5p inhibitor or HMGCR overexpression was injected into cells with lncRNA BLACAT1 silencing. Results: In IL-1β-treated HACs, lncRNA BLACAT1 and HMGCR were overexpressed while miR- 149-5p was poorly expressed, along with reduced cell viability, enhanced apoptosis, elevated Caspase-3 and Caspase-9 activities, increased BAX level, decreased Bcl-2 level, and declined levels of COL2A1 and SOX9, which were reversed by lncRNA BLACAT1 silencing. LncRNA BLACAT1 targeted miR-149-5p, and miR-149-5p targeted HMGCR. miR-149-5p knockout or HMGCR overexpression annulled the inhibitory role of lncRNA BLACAT1 silencing in HAC apoptosis and ECM degradation. Conclusion: LncRNA BLACAT1 was overexpressed in IL-1β-treated HACs, and the lncRNA BLACAT1/miR-149-5p/HMGCR ceRNA network promoted HAC apoptosis and ECM degradation.

Background: The present study was targeted at investigating the effects of hsa_circRNA_0008092 (circ_0008092) on hepatocellular carcinoma (HCC) cell proliferation, migration, invasion and apoptosis, and its related mechanism. Methods: The gene expression profiles of GSE166678 were downloaded from the Gene Expression Omnibus database, and differentially expressed circRNAs in human HCC were screened out. Besides, circ_0008092, microRNA-502-5p (miR-502-5p) and cyclin D1 (CCND1) expressions in HCC tissues and cell lines were detected by quantitative real-time polymerase chain reaction (qRTPCR). Cell countering kit-8 (CCK-8), Transwell and flow cytometry assays were used to detect the proliferation, migration, invasion and apoptosis of HCC cells. Bioinformatics was utilized to predict the targeted relationships between miR-502-5p and circ_0008092, as well as miR-502-5p and CCND1 mRNA 3'-untranslated region (3’UTR). Western blot assay was applied to detect CCND1 protein expression in HCC cells. Results: Circ_0008092 was highly expressed in HCC tissues and cells, which was associated with a shorter survival time in patients with HCC. Circ_0008092 overexpression promoted proliferation, migration and invasion, and inhibited apoptosis of HCC cells; circ_0008092 knockdown worked oppositely. Circ_0008092 directly targeted miR-502-5p and negatively modulated miR-502-5p expression. CCND1 was a target gene of miR-502-5p, and was positively and indirectly modulated by circ_0008092. Conclusion: Our data suggest that circ_0008092 promotes HCC progression by regulating the miR- 502-5p/CCND1 axis.

Background: SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as a receptor for entering the host cells. Production of the ACE2 molecule is important because of its potency to use as a blocker and therapeutic agent against SARS-CoV-2 for the prophylaxis and treatment of COVID-19. Objective: The recombinant human ACE2 (rhACE2) is prone to form an inclusion body when expressed in the bacterial cells. Methods: We used the SUMO tag fused to the rhACE2 molecule to increase the expression level and solubility of the fusion protein. Afterward, the freeze-thawing method plus 2 M urea solubilized aggregated proteins. Subsequently, the affinity of solubilized rhACE2 to the receptor binding domain (RBD) of the SARS-CoV-2 spike was assayed by ELISA and SPR methods. Results: SUMO protein succeeded in increasing the expression level but not solubilization of the fusion protein. The freeze-thawing method could solubilize and recover the aggregated fusion proteins significantly. Also, ELISA and SPR assays confirmed the interaction between solubilized rhACE2 and RBD with high affinity. Conclusion: The SUMO tag and freeze-thawing method would be utilized for high-level expression and solubilization of recombinant rhACE2 protein.

Background: Circular RNAs (circRNAs) are pivotal in cancer biology. Nevertheless, the biological functions of circular RNA hsa_circ_0000437 (circ_0000437) have not yet been elucidated. In the present study, we studied the expression characteristics of circ_0000437 in endometrial carcinoma (EC) and explored the roles and potential mechanisms of circ_0000437 in EC progression. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was adopted to detect the expressions of circ_0000437, microRNA-626 (miR-626) and cyclin-dependent kinase inhibitor 1B (CDKN1B) in EC tissues and cells. 5-Ethynyl-2'-deoxyuridine (EdU), cell counting kit-8 (CCK-8) and Transwell assays were performed to evaluate EC cell proliferation and invasion. The expressions of CDKN1B and epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin and N-cadherin) were detected by Western blot. Moreover, the targeted relationship between miR- 626 and circ_0000437 or CDKN1B was determined by a dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Results: Circ_0000437 expression was reduced in EC tissues, and the low expression of circ_0000437 was positively correlated with the lymph node metastasis and high TNM stage of EC patients. Knocking down circ_0000437 promoted the proliferation, invasion and EMT of EC cells. Circ_0000437 directly targeted miR-626 and negatively modulated miR-626 expression in EC cells. CDKN1B was identified as the downstream target of miR-626 in EC cells. Besides, CDKN1B overexpression of miR-626 knockdown reversed the effects of knocking down circ_0000437 on EC cells. Conclusion: Circ_0000437 regulates the miR-626/CDKN1B pathway to suppress the proliferation, invasion and EMT of EC cells. This indicates that circ_0000437 may be a promising biomarker and therapy target for EC.

Objective: Nasopharyngeal carcinoma (NPC) is a squamous cell carcinoma. LncRNA CTBP1-AS2 (CTBP1-AS2) has effects on tumor cell growth. This study explored the mechanism of CTBP1-AS2 on NPC cells. Methods: CTBP1-AS2 expressions in immortalized nasopharyngeal epithelial (NP69) and 6 human NPC cells were detected by RT-qPCR and SUNE-1/CNE-1 cells with relative high/low expressions were selected. Cell proliferation and apoptosis were detected by CCK-8, colony formation assays, and flow cytometry. The binding sites between CTBP1-AS2 and miR-140-5p and miR-140-5p and BMP2 were predicted, and the binding relationships were verified by dual-luciferase assay. BMP2 level was detected by Western blot. miR-140-5p was silenced, or BMP2 was overexpressed in SUNE-1 cells with si-CTBP1-AS2 to study the effects of miR-140-5p and BMP2 on CTBP1-AS2 silencing-inhibited malignant behaviors. Results: CTBP1-AS2 was upregulated in NPC cells. CTBP1-AS2 silencing suppressed NPC cell proliferation and promoted apoptosis. CTBP1-AS2 silencing in SUNE-1 cells raised miR-140-5p expression and repressed BMP2 level. CTBP1-AS2 overexpression in CNE-1 cells suppressed miR- 140-5p expression and elevated BMP2 levels. Discussion: In mechanism, miR-140-5p overexpression decreased BMP2 levels, reduced NPC cell proliferation, and promoted apoptosis. miR-140-5p knockdown or BMP2 overexpression enhanced NPC cell proliferation and inhibited apoptosis, thus restoring NPC cell malignant behaviors inhibited by silencing CTBP1-AS2. Conclusion: CTBP1-AS2 decreased miR-140-5p-induced BMP2 inhibition via functioning as a ceRNA of miR-140-5p and promoted BMP2 expression, thereby promoting NPC cell proliferation and suppressing apoptosis.

Background: Granzyme B can induce apoptosis in target cells by direct and indirect activation of caspases and cleavage of central caspase substrates. Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein and its expression increases following prostate cancer progression. Objective: In this study, we designed a fusion protein including mutant granzyme B, the influenza virus hemagglutinin HA-2 N-terminal, and PSMA ligand to construct GrB-HA-PSMA ligand fusion protein as a molecular agent for selective targeting of PSMA-positive (LNCaP) cells. Methods: The DNA sequence of our designed structure was synthesized and cloned into a pET28a expression vector. The recombinant protein was expressed in E. coli origami bacteria and then purified. The expression of the recombinant protein was verified by SDS PAGE and ELISA method. Furthermore, ELISA and flow cytometry assays were utilized to investigate the efficiency of binding and permeability of the recombinant protein into the LNCaP cells. Finally, cell proliferation and apoptosis rate were evaluated by MTT assay and flow cytometry assay, respectively. HeLa and PC3 cell lines were used as controls. Results: The results showed that GrB-HA-PSMA ligand fusion protein could specifically bind and internalize into the PSMA-positive cells. Furthermore, treatment of the cells with GrB-HA-PSMA ligand fusion protein resulted in increased apoptotic cell death and decreased proliferation of LNCaP cells. Conclusion: Our findings indicate the specificity of GrB-HA-PSMA ligand fusion protein for PSMA-positive cells and suggest that this fusion protein is a potential candidate for prostate cancer targeted therapy.