Protein and Peptide Letters - Online First

Dysregulation of mevalonate metabolism is a hallmark of tumorigenesis and therapy resistance across malignancies, though its role in bladder cancer remains unclear. This study aimed to elucidate its impact on prognosis and cisplatin chemosensitivity in bladder cancer.

Transcriptomic data and clinical information of bladder cancer patients were obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases. Non-negative matrix factorization (NMF) was used to cluster mevalonate metabolism-related genes into distinct metabolic subtypes (C1 and C2). Associations between mevalonate metabolism, clinical characteristics, immune infiltration, and cisplatin resistance were analyzed using Gene Set Variation Analysis (GSVA), Kaplan-Meier survival analysis, single-sample Gene Set Enrichment Analysis (ssGSEA), and in vitro experiments.

NMF clustering classified bladder cancer patients into two metabolic subtypes (C1/C2). The C1, characterized by higher mevalonate metabolism (MVAscore), was associated with a poorer prognosis, shorter overall survival (OS), and higher T-stage and pathological grades. Immune analysis showed lower immune cell infiltration in C1. Immune infiltration analysis revealed significantly lower immune infiltration levels in the C1. Further analysis revealed a positive correlation between mevalonate metabolism and platinum resistance, with a notable increase in mevalonate metabolism observed in cisplatin-resistant bladder cancer cells. In vitro, simvastatin inhibited the proliferation of bladder cancer cells and enhanced their sensitivity to cisplatin.

Mevalonate metabolism drives BCa heterogeneity and chemoresistance while suppressing anti-tumor immunity. Its dysregulation serves as both a prognostic biomarker and a target for therapeutic intervention.

Mevalonate metabolism contributes to cisplatin resistance in bladder cancer and represents a potential therapeutic target. Simvastatin targeting this pathway enhances the efficacy of cisplatin, providing a novel personalized chemotherapy strategy.

italic>Mycobacterium tuberculosis (Mtb) is a Gram-positive bacterium that causes tuberculosis (TB). It remains viable for extended periods within host macrophages by entering a dormant state. Alpha crystallin 1 (Acr1) is a 16 kDa protein of Mtb and is reported to be highly upregulated in latent TB. Acr1 suppresses the host’s immune system by impairing the differentiation and maturation of dendritic cells and macrophages. We hypothesize that Mtb judiciously utilizes its Acr1 protein to paralyse the immune system of the host by inducing the release of IL-10 and generating an immunosuppressive environment.

We employed in silico tools to identify highly promiscuous, IL-10-inducing and IL-6-non-inducing epitopes of Mtb. Moreover, the selected epitope was synthesized and tested for its suppressive activity and generation of Tregs.

We identified the presence of a specific epitope in Acr1 (F18) that is responsible for bolstering the release of IL-10 and Tregs through in silico tools and verified the activity by in vitro assays. In hPBMCs, the F18 epitope could suppress the proliferation of CD4 T cells stimulated with PHA and expand the pool of Tregs in a dose-dependent manner.

The F18 epitope from Mtb’s Acr1 protein promotes IL-10 and Treg responses without triggering pro-inflammatory IL-6, suggesting its probable immunoregulatory role. While it holds potential for treating autoimmune diseases, its impact on infection in tuberculosis should be further investigated.

Our findings suggest that the F18 epitope induces IL-10 production and Treg differentiation while inhibiting CD4⁺ T cell proliferation and IL-6 secretion, thereby promoting an immunosuppressive environment. Furthermore, this study highlights the possible role of Acr1 and its immunosuppressive epitope F18 as therapeutic agents for inducing suppressive Tregs, which may help in the management of autoimmune diseases.

Keratinases have an established role in degrading highly stable and insoluble fibers of keratin proteins, which are otherwise difficult to be hydrolyzed by conventional proteases. Keratinases find promising application in degrading poultry waste to valuable products. Moreover, their role in cosmetics, detergents, agriculture and the leather industry is well recognized.

In this study, the keratinase gene from locally isolated Brevibacillus agri bacteria was cloned and expressed in Escherichia coli, and some of its potential applications were explored.

1300 bp amplified gene from Brevibacillus agri was cloned into E. coli DH5α competent cells using pTZ57R/T vector. After blue-white screening, the positive clone was confirmed by colony PCR and restriction analysis. Purified keratinase gene KerH from recombinant pTZR/KerH plasmid was ligated into pET-28a (+) and transferred into competent cells of E. coli DH5α. Following confirmation through colony PCR, and restriction analysis, recombinant plasmid (pET-28a/Ker) from the positive clone was transferred into competent E. coli BL21 cells. The transformed cells were then cultured for up to 8 hours after induction with 0.8 mM IPTG and lysed by sonication. The resulting recombinant keratinase (KerH) was purified by heat treatment and Ni-affinity column and characterized.

The blast analysis and homologous sequences in the NCBI database established a close link to Brevibacillus agri. The highest expression from transformed E. coli BL21 was achieved with 0.8 mM IPTG following 6 hours of induction. The resulting recombinant keratinase (KerH), purified by Ni-affinity chromatography, possessed 283 U/mg specific activity and displayed ~45 kDa band on SDS-PAGE and zymogram. Secondary structure analysis and active site prediction was performed computationally. Considering the extensive applications of keratinase, KerH was found to be useful in dehairing animal skin surfaces without any damage. The encapsulated KerH possessed improved stability and better compatibility with commercial detergents. It efficiently removed blood, turmeric, strawberry, and egg yolk stains from the fabric. Furthermore, KerH significantly degraded the poultry feathers and provided a protein hydrolysate that helped in converting damaged, dull and curly hair into healthier, shiny and straightened hair.

These key findings highlight that KerH is a robust keratinase with significant potential as an environmental-friendly alternative to the prevailing harsh chemical treatments in various industries. Encapsulation enhanced its suitability by improving its stability and shelf-life. Its broader substrate specificity, stability and application in detergents and cosmetics underline its commercial importance.

The recombinant KerH from Brevibacillus agri can be considered as a valuable microbial keratinase that can be used as an alternative to the eco hazardous chemicals used in commercial applications of feather degradation, hair protein treatment, feather keratin hydrolysate production and hide dehairing.

This study aimed to investigate the anti-carcinogenic effects of recombinant L-methioninase (rBlmet) on the pancreatic cancer cell line MiaPaCa-2.

In this study, rBlmet was initially cloned, expressed, and purified. To increase enzyme activity, the His-tags on the enzyme were removed using thrombin. rBlmet was then applied to MiaPaCa-2 cells, and the cell viability of MiaPaCa-2 cells was evaluated by neutral red assay after rBlmet treatment. The combined effect of etoposide with rBlmet against MiaPaCa-2 cells was also evaluated for 12 and 24 hours using a neutral red assay. Furthermore, cell morphology was evaluated by Giemsa and DAPI/F-actin staining methods. Survivin and caspase-3 gene expression levels were measured by RT-qPCR.

The specific activity of the enzyme increased after His-tag elimination to 5.62 µmol/mg per minute. rBlmet showed a significant cytotoxic effect on the MiaPaCa-2 cell line. The IC50 value (24 h) of rBlmet for MiaPaCa-2 cells was 3.02 U/mL. In addition, rBlmet increased the cytotoxic effect of etoposide on the MiaPaCa-2 cell line, while it showed less effect on HaCat, which is a normal human cell line. Furthermore, rBlmet increased caspase-3 expression and downregulated survivin gene expression in MiaPaCa-2 cell lines.It successfully inhibited the growth of Mia-PaCa-2 cells by exploiting exogenous methionine amino acid in the growth medium. This study revealed promising results. However, further studies are needed on additional pancreatic cancer cell lines and in vivo models.

Based on these findings, it can be concluded that rBlmet not only has great potential to treat pancreatic cancer in the future but can also be used as an adjuvant to enhance the effectiveness ofchemotherapeutic agents like etoposide.

Tissue Factor (TF) is a crucial transmembrane glycoprotein that triggers blood coagulation upon vascular or tissue injury by binding to plasma factors VII and VIIa. In recent years, the demand for TF has rapidly increased due to its pivotal role in preoperative coagulation tests. However, large-scale production of TF remains challenging despite successful recombinant expression, as incorrect post-translational modifications adversely affect TF activity.

This study aims to investigate the role of post-translational modifications, specifically N-glycosylation, in TF activity and stability. Additionally, it explores strategies to enhance TF production by reducing its degradation through genetic modulation.

We compared TF activity derived from human cells and E. coli to assess the impact of post-translational modifications. Furthermore, we examined the effect of N-glycosylation on TF function. To address TF degradation, we knocked out the HRD1 gene, a key component of the endoplasmic-reticulum-associated degradation (ERAD) pathway, and evaluated its impact on TF stability and activity.

TF produced in human cells exhibited higher activity than TF expressed in E. coli, emphasizing the importance of post-translational modifications. Specifically, N-glycosylation was found to influence TF activity and stability. Additionally, we observed that knocking out the HRD1 gene effectively reduced TF degradation without compromising its activity.

Our findings underscore the crucial role of N-glycosylation in TF function and stability. Moreover, the modulation of the ERAD pathway through knocking out HRD1 presents a promising approach for enhancing TF production. These insights could contribute to the large-scale manufacturing of functionally active TF for clinical and research applications.