Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits.

The proteasome is a multi-subunit proteolytic complex that plays a central role in protein degradation in all eukaryotic cells. It regulates many vital cellular processes therefore its dysfunction can lead to various pathologies including cancer and neurodegeneration. Isolation of enzymatically active proteasomes is a key step to the successful study of the proteasome regulation and functions. Here we describe a simple and efficient protocol for immunoaffinity purification of the functional 20S proteasomes from human HEK 293T cells after transient overexpression of specific proteasome subunits tagged with 3xFLAG. To construct 3xFLAG-fusion proteins, DNA sequences encoding the 20S proteasome subunits PSMB5, PSMA5, and PSMA3 were cloned into mammalian expression vector pIRES-hrGFP-1a. The corresponding recombinant proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA3-3xFLAG were transiently overexpressed in human HEK 293T cells and were shown to be partially incorporated into the intact proteasome complexes. 20S proteasomes were immunoprecipitated from HEK 293T cell extracts under mild conditions using antibodies against FLAG peptide. Isolation of highly purified 20S proteasomes were confirmed by SDS-PAGE and Western blotting using antibodies against different proteasome subunits. Affinity purified 20S proteasomes were shown to possess chymotrypsin- and trypsin-like peptidase activities confirming their functionality. This simple single-step affinity method of the 20S proteasome purification can be instrumental to subsequent functional studies of proteasomes in human cells.

Unveiling the role of PSMA5 in glioma progression and prognosis.

Glioma is the most aggressive intracranial malignancy and is associated with poor survival rates and limited quality of life, impairing neuropsychological function and cognitive competence in survivors. The Proteasome Subunit Alpha Type-5 (PSMA5) is a multicatalytic proteinase complex that has been linked with tumor progression but is rarely reported in glioma. This study investigates the expression pattern, prognostic characteristics, and potential biological functions of PSMA5 in glioma. PSMA5 was significantly overexpressed in 28 types of cancer when compared to normal tissue. Furthermore, elevated levels of PSMA5 were observed in patients with wild-type isocitrate dehydrogenase 1 and exhibited a positive correlation with tumor grade. It was also found to be a standalone predictor of outcomes in glioma patients. Additionally, inhibiting PSMA5-induced cell cycle arrest may provide a therapeutic option for glioma.

Role and mechanism of IRF9 in promoting the progression of rheumatoid arthritis by regulating macrophage polarization via PSMA5.

Aim: To explore the mechanisms of IRF9 in the progression of rheumatoid arthritis(RA), and the effects of IRF9 on M1/M2 polarization. Methods: RA dataset (GSE55457) was downloaded from GEO. Correlation analysis between IRF9 and its downstream target protein PSMA5 was performed using bioinformatics analysis. The M1/M2 cell ratio of peripheral blood mononuclear cells which from 20 healthy specimen and 40 RA patients was determined. The expression of IRF9 and PSMA5 was detected using qPCR and Western blot. Then, knockdown IRF9 in RAW264.7 cell line (sh-IRF9 RAW264.7) was constructed. The effect of sh-IRF9 RAW264.7 on RA was explored by constructing a CIA mouse model. Results: IRF9 is upregulated in RA and is of good early screening effect. The results of pathway analysis showed that IRF9 targets and regulates the PSMA5 signaling pathway. IRF9 and PSMA5 were significantly elevated in RA patients, M1/M2 ratio was also increased. The effects of IRF9 on RAW264.7 macrophages were deeply explored in vitro, revealing that knockdown of IRF9 suppressed PSMA5, M1/M2 ratio and the secretion of pro-inflammatory factor in RAW264.7. In mouse in vivo experiments, sh-IRF9 RAW264.7 cells were found to modulate RA by downregulating PSMA5, modulating the M1/M2 ratio through enhancing the anti-inflammatory factor, and suppressing the pro-inflammatory factor. Conclusion: IRF9 promoted the progression of RA via regulating macrophage polarization through PSMA5.

Knockdown of liver cancer cell-secreted exosomal PSMA5 controls macrophage polarization to restrain cancer progression by blocking JAK2/STAT3 signaling.

INTRODUCTION: Tumor-associated macrophages, a major component of the tumor microenvironment, undergo polarization into M2 macrophages (M2), and thereby exert an immunosuppressive effect to induce cancer metastasis. This study strives to uncover a molecular mechanism underlying this event in hepatocellular carcinoma (HCC). METHODS: Proteasome subunit alpha 5 (PSMA5) expression in liver hepatocellular carcinoma (LIHC) tissues and its association with LIHC patients were predicted using StarBase. PSMA5 level in human HCC cells was manipulated via transfection. Exosomes were isolated from HCC cells, and internalized into macrophages which were cocultured with HCC cells. Exosome internalization was observed after fluorescence labeling. HCC cell migration and invasion were evaluated by wound healing and Transwell assays. Xenograft assay was performed to investigate the role of PSMA5 in in vitro tumorigenesis. M2 polarization was assessed by enzyme-linked immunosorbent assay, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. PSMA5 expression in exosomes and Janus Kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) activation in macrophages and tumors were detected by Western blot analysis. RESULTS: High PSMA5 expression was observed in LIHC tissues and associated with compromised survival of LIHC patients. PSMA5 knockdown inhibited HCC cell migration and invasion. PSMA5 knockdown in HCC cells downregulated PSMA5 level in exosomes from these HCC cells. HCC cell-isolated exosomes were successfully internalized into macrophages, and facilitated M2 polarization and JAK2/STAT3 pathway activation. HCC cell-secreted exosomal PSMA5 knockdown inhibited the exosome-induced effect on macrophages, and attenuated the promotion induced by exosome-treated macrophages on HCC cell migration/invasion and tumorigenesis along with in vivo M2 polarization and JAK2/STAT3 pathway activation. CONCLUSION: HCC cell-secreted exosomal PSMA5 knockdown hinders M2 polarization to suppress cancer progression by restraining JAK2/STAT3 signaling.

The Genetic Diversity of Proteasome Genes in the T1DM Polish Population.

Autoimmune metabolic diseases generate numerous healthy and social problems. The possible association of SNPs in the ubiquitin-proteasome system (UPS) with human pathology is under intensive study. OBJECTIVE: In the present study, the genetic variations in PSMB5 (rs11543947), PSMA6 (rs2277460, rs1048990), PSMC6 (rs2295826, rs2295827) and PSMA3 (rs2348071) UPS gene cluster was investigated in type 1 diabetes and healthy donors in the Polish population. METHODS: The study comprised 105 patients with type 1 diabetes mellitus (T1DM) and 214 controls. All were genotyped by PCR and restriction digestion analysis or Sanger sequencing. RESULTS: Rs1048990 and rs2348071 were found to be neutral to T1DM (p-value: 0.499 and 0.656, respectively). According to the multiple loci genotype (MLG) analysis, the major homozygote of the tested polymorphisms had a protective effect. The most common MLG in the T1DM group was characterised by simultaneous risk factors at rs11543947, rs2277460, rs2295826 and rs2295827 (pvalue: <0.0001 vs. MGL1). Multiple locus haplotype analysis revealed a similar dependence, with common alleles at all tested loci demonstrating a protective effect, and the rare alleles increasing T1DM risk (p-value: <0.0001 vs. MLH1). CONCLUSION: Our study suggests that the proteasome gene polymorphisms rs11543947, rs2277460, rs2295826, and rs2295827 could be potential markers for T1DM susceptibility in the Polish population.

Identification of eight novel proteasome variants in five unrelated cases of proteasome-associated autoinflammatory syndromes (PRAAS).

Mutations in genes coding for proteasome subunits and/or proteasome assembly helpers typically cause recurring autoinflammation referred to as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperatures (CANDLE) or proteasome-associated autoinflammatory syndrome (PRAAS). Patients with CANDLE/PRAAS present with mostly chronically elevated type I interferon scores that emerge as a consequence of increased proteotoxic stress by mechanisms that are not fully understood. Here, we report on five unrelated patients with CANDLE/PRAAS carrying novel inherited proteasome missense and/or nonsense variants. Four patients were compound heterozygous for novel pathogenic variants in the known CANDLE/PRAAS associated genes, PSMB8 and PSMB10, whereas one patient showed additive loss-of-function mutations in PSMB8. Variants in two previously not associated proteasome genes, PSMA5 and PSMC5, were found in a patient who also carried the PSMB8 founder mutation, p.T75M. All newly identified mutations substantially impact the steady-state expression of the affected proteasome subunits and/or their incorporation into mature 26S proteasomes. Our observations expand the spectrum of PRAAS-associated genetic variants and improve a molecular diagnosis and genetic counseling of patients with sterile autoinflammation.

Conserved Mitotic Phosphorylation of a Proteasome Subunit Regulates Cell Proliferation.

Reversible phosphorylation has emerged as an important mechanism for regulating proteasome function in various physiological processes. Essentially all proteasome phosphorylations characterized thus far occur on proteasome holoenzyme or subcomplexes to regulate substrate degradation. Here, we report a highly conserved phosphorylation that only exists on the unassembled α5 subunit of the proteasome. The modified residue, α5-Ser16, is within a SP motif typically recognized by cyclin-dependent kinases (CDKs). Using a phospho-specific antibody generated against this site, we found that α5-S16 phosphorylation is mitosis-specific in both yeast and mammalian cells. Blocking this site with a S16A mutation caused growth defect and G2/M arrest of the cell cycle. α5-S16 phosphorylation depends on CDK1 activity and is highly abundant in some but not all mitotic cells. Immunoprecipitation and mass spectrometry (IP-MS) studies identified numerous proteins that could interact with phosphorylated α5, including PLK1, a key regulator of mitosis. α5-PLK1 interaction increased upon mitosis and could be facilitated by S16 phosphorylation. CDK1 activation downstream of PLK1 activity was delayed in S16A mutant cells, suggesting an important role of α5-S16 phosphorylation in regulating PLK1 and mitosis. These data have revealed an unappreciated function of "exo-proteasome" phosphorylation of a proteasome subunit and may bring new insights to our understanding of cell cycle control.