Objective:To explore the biomarkers associated with multiple myeloma in remission phase (MM-RP) in order to provide potential indicators for disease monitoring and prognostic evaluation.Methods:Bone marrow blood samples were prospectively collected from 9 newly diagnosed multiple myeloma (NDMM) patients and 9 MM-RP patients in Beijing Chaoyang Hospital of Capital Medical University from January to October 2020. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed for proteomic analysis in 3 independent experiments, each containing 3 paired NDMM and MM-RP samples. Differentially expressed proteins (DEP) consistently identified across all 3 experiments were considered potential MM-RP biomarkers. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database were used for enrichment analyses of the biological functions of these potential biomarkers. Protein-protein interaction (PPI) networks were constructed using the STRING 11.5 database, and α-2-macroglobulin (α2M) was identified as a hub protein. The UCSC Xena database was utilized, and the overall survival (OS) of MM patients with high or low α2M expression [stratified by the average level of α2M transcriptome sequencing (RNA-seq) data] was analyzed by using Kaplan-Meier. A multivariate Cox proportional hazards model adjusted for age, International Staging System (ISS) stage and treatment regimen was employed to analyze the impact of α2M expression on OS of MM patients. The Human Protein Atlas (HPA) database was used to examine α2M mRNA expression patterns in 33 cancer types. The correlation of drug sensitivity [50% inhibiting concentration ( IC50)] with α2M expression was assessed using pharmacogenomic data from the GSCALite platform. Results:Among 104 proteins consistently identified in 3 proteomic experiments, 34 DEP were found between NDMM and MM-RP (∣fold change∣>1.0 and P < 0.05), including 25 upregulated DEP and 9 downregulated DEP in MM-RP. GO analysis showed that the identified MM-RP potential markers were mainly involved in biological processes such as complement activation and humoral immune response, and the molecular functions mainly involved serine hydrolase activity, serine peptidase activity, etc., and were mainly distributed in secretory granules, blood particles, and other parts; KEGG enrichment analysis showed that biomarkers were mainly enriched in the complement and coagulation cascade pathways. In the human α2M PPI network constructed using data from the STRING database, there were 10 proteins that interacted with α2M, with a connectivity of 7.82, and had direct interactions with 71% of the proteins in the network, the betweenness centrality value was 0.06, and the closeness centrality value was 1, indicating significant network centrality feature of α2M. In the constructed PPI network of α2M protein and DEP screened by proteomics, there were interactions between α2M protein and 11 MM-RP markers screened by proteomics, and the betweenness centrality value of α2M reached 0.50, the closeness centrality value was 0.67, indicating that α2M was at the core position of the PPI network. UCSC Xena analysis revealed that MM patients with low α2M expression (523 cases) had worse OS than those with high expression (336 cases) ( P =0.024). Multivariate Cox regression analysis confirmed that low α2M expression was an independent risk factor for poor OS (compared with high α2M expression: HR = 0.726, 95% CI: 0.550-0.960, P = 0.024). HPA database analysis demonstrated that the α2M expression levels were variable in different types of cancer, its level in glioblastoma multiforme, clear cell renal cell carcinoma, and stomach adenocarcinoma was higher than that in normal tissues (all P < 0.05), and its level in urothelial carcinoma, breast cancer and cervical squamous cell carcinoma was lower than that in normal tissues (all P < 0.05). GSCALite analysis revealed negative correlations between α2M expression level and IC50 values of B-Raf kinase inhibitors, B-Raf V600E inhibitors and dabrafenib mesylate. Conclusions:α2M expression level in MM-RP patients is lower than that in NDMM patients, and its expression level may be related to the prognosis of patients, which is expected to become a novel biomarker reflecting the disease activity of MM.

Clear cell renal cell carcinoma(ccRCC)is a frequently occurring renal cancer.The Von Hippel-Lindau disease tumor suppressor VHL,a known tumor suppressor gene,is frequently mutated in about 50％of patients with ccRCC.However,it is unclear whether VHL influences the progression of ccRCC tumors expressing wild-type VHL.In the present study,we found that higher expression of VHL was correlated with the better disease-free survival(DFS)in ccRCC patients using The Cancer Genome Atlas(TCGA)datasets.We revealed that VHL overexpression in ccRCC cells inhibited epithelial-mesenchymal transition(EMT),sterol regulatory element-binding protein 1(SREBP1)-regulated triglyceride synthesis,and cell proliferation.Proteomic anal-ysis provided us a global view that VHL regulated four biological processes,including metabolism,immune regulation,apoptosis,and cell movement.Importantly,we found that VHL overexpression led to up-regulated expression of proteins associated with antigen processing and interferon-responsive proteins,thus rendering ccRCC cells more sensitive to interferon treatment.We defined an interferon-responsive signature(IRS)composed of ten interferon-responsive proteins,whose mRNA expression levels were positively correlated with DFS in ccRCC patients.Taken together,our results propose that the subset of ccRCC patients with high VHL expression benefit from immunotherapy.

Blotting, Western ; Cell Cycle Checkpoints ; Cell Line, Tumor ; Chaperonin 60 ; metabolism ; HEK293 Cells ; Humans ; Mitochondria ; metabolism ; Reactive Oxygen Species ; metabolism ; Real-Time Polymerase Chain Reaction ; Sirtuin 3 ; genetics ; metabolism

Chromosomal Proteins, Non-Histone ; chemistry ; genetics ; metabolism ; HeLa Cells ; Histones ; chemistry ; genetics ; metabolism ; Humans ; Protein Domains

Mutations or inactivation of parkin, an E3 ubiquitin ligase, are associated with familial form or sporadic Parkinson's disease (PD), respectively, which manifested with the selective vulnerability of neuronal cells in substantia nigra (SN) and striatum (STR) regions. However, the underlying molecular mechanism linking parkin with the etiology of PD remains elusive. Here we report that p62, a critical regulator for protein quality control, inclusion body formation, selective autophagy and diverse signaling pathways, is a new substrate of parkin. P62 levels were increased in the SN and STR regions, but not in other brain regions in parkin knockout mice. Parkin directly interacts with and ubiquitinates p62 at the K13 to promote proteasomal degradation of p62 even in the absence of ATG5. Pathogenic mutations, knockdown of parkin or mutation of p62 at K13 prevented the degradation of p62. We further showed that parkin deficiency mice have pronounced loss of tyrosine hydroxylase positive neurons and have worse performance in motor test when treated with 6-hydroxydopamine hydrochloride in aged mice. These results suggest that, in addition to their critical role in regulating autophagy, p62 are subjected to parkin mediated proteasomal degradation and implicate that the dysregulation of parkin/p62 axis may involve in the selective vulnerability of neuronal cells during the onset of PD pathogenesis.
Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; HEK293 Cells ; Heat-Shock Proteins ; chemistry ; metabolism ; Humans ; Lysine ; metabolism ; Mice ; Neurons ; metabolism ; pathology ; Oxidopamine ; pharmacology ; Parkinson Disease ; metabolism ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Protein Stability ; Proteolysis ; drug effects ; Sequestosome-1 Protein ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination ; drug effects

Animals ; Eukaryotic Initiation Factors ; genetics ; metabolism ; Humans ; Phosphorylation ; physiology ; Proteomics ; Ribosomal Proteins ; genetics ; metabolism ; TOR Serine-Threonine Kinases ; genetics ; metabolism

Amino Acid Sequence ; Autophagy ; Binding Sites ; Genomics ; Molecular Sequence Data ; Phosphorylation ; Saccharomyces cerevisiae Proteins ; chemistry ; genetics ; metabolism

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.
Alanine ; chemistry ; metabolism ; Amino Acid Motifs ; Aspartic Acid ; chemistry ; metabolism ; Autophagy ; genetics ; Autophagy-Related Proteins ; Carrier Proteins ; chemistry ; metabolism ; Gene Expression Regulation, Fungal ; Membrane Proteins ; chemistry ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Nitrogen ; deficiency ; Phagosomes ; chemistry ; drug effects ; metabolism ; Phosphorylation ; Protein Transport ; Saccharomyces cerevisiae ; drug effects ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; chemistry ; genetics ; metabolism ; Serine ; chemistry ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology

Adaptor protein ClpS is an essential regulator of prokaryotic ATP-dependent protease ClpAP, which delivers certain protein substrates with specific amino acid sequences to ClpAP for degradation. However, ClpS also functions as the inhibitor of the ClpAP-mediated protein degradation for other proteins. Here, we constructed the clpS-overexpression Mycobacterium smegmatis strain, and showed for the first time that overexpression of ClpS increased the resistance of M. smegmatis to rifampicin that is one of most widely used antibiotic drugs in treatment of tuberculosis. Using quantitative proteomic technology, we systematically analyzed effects of ClpS overexpression on changes in M. smegmatis proteome, and proposed that the increased rifampicin resistance was caused by ClpS-regulated drug sedimentation and drug metabolism. Our results indicate that the changes in degradation related proteins enhanced drug resistance and quantitative proteomic analysis is an important tool for understanding molecular mechanisms responsible for bacteria drug resistance.
ATP-Dependent Proteases ; metabolism ; Drug Resistance, Bacterial ; Endopeptidase Clp ; metabolism ; Mycobacterium smegmatis ; drug effects ; metabolism ; Proteolysis ; Proteomics ; Rifampin ; pharmacology

The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.
Cryoelectron Microscopy ; Escherichia coli ; metabolism ; Escherichia coli Proteins ; genetics ; metabolism ; GTP Phosphohydrolases ; genetics ; metabolism ; Mass Spectrometry ; Protein Structure, Secondary ; Protein Structure, Tertiary ; RNA, Ribosomal ; analysis ; metabolism ; Ribosomal Proteins ; chemistry ; genetics ; metabolism ; Ribosome Subunits, Small, Bacterial ; chemistry ; metabolism ; ultrastructure ; Salts ; chemistry