Shaohua Zhan

Protein content and functional characteristics of serum‐purified exosomes from patients with colorectal cancer revealed by quantitative proteomics

Tumor cells of colorectal cancer (CRC) release exosomes into the circulation. These exosomes can mediate communication between cells and affect various tumor‐related processes in their target cells. We present a quantitative proteomics analysis of the exosomes purified from serum of patients with CRC and normal volunteers; data are available via ProteomeXchange with identifier PXD003875. We identified 918 proteins with an overlap of 725 Gene IDs in the Exocarta proteins list. Compared with the serum‐purified exosomes (SPEs) of normal volunteers, we found 36 proteins upregulated and 22 proteins downregulated in the SPEs of CRC patients. Bioinformatics analysis revealed that upregulated proteins are involved in processes that modulate the pretumorigenic microenvironment for metastasis. In contrast, differentially expressed proteins (DEPs) that play critical roles in tumor growth and cell survival were principally downregulated. Our study demonstrates that SPEs of CRC patients play a pivotal role in promoting the tumor invasiveness, but have minimal influence on putative alterations in tumor survival or proliferation. According to bioinformatics analysis, we speculate that the protein contents of exosomes might be associated with whether they are involved in premetastatic niche establishment or growth and survival of metastatic tumor cells. This information will be helpful in elucidating the pathophysiological functions of tumor‐derived exosomes, and aid in the development of CRC diagnostics and therapeutics.

Temporal lobe in human aging: A quantitative protein profiling study of samples from Chinese Human Brain Bank.

The temporal lobe is a portion of the cerebral cortex with critical functionality. The age-related protein profile changes in the human temporal lobe have not been previously studied. This 4-plex tandem mass tag labeled proteomic study was performed on samples of temporal lobe from Chinese donors. Tissue samples were assigned to four age groups: Group A (the young, age: 34±13 years); Group B (the elderly, 62±5 years); Group C (the aged, 84±4 years) and Group D (the old, 95±1 years). Pooled samples from the different groups were subjected to proteomics and bioinformatics analysis to identify age-related changes in protein expression and associated pathways. We isolated 5072 proteins, and found that 67 proteins were downregulated and 109 proteins were upregulated in one or more groups during the aging process. Western blotting assays were performed to verify the proteomic results. Bioinformatic analysis identified proteins involved in neuronal degeneration, including proteins involved in neuronal firing, myelin sheath damage, and cell structure stability. We also observed the accumulation of extracellular matrix and lysosomal proteins which imply the occurrence of fibrosis and autophagy. Our results suggest a series of changes across a wide range of proteins in the human temporal lobe that may relate to aging and age-related neurodegenerative disorders.

Comprehensive proteome analysis of lysosomes reveals the diverse function of macrophages in immune responses

Phagocytosis and autophagy in macrophages have been shown to be essential to both innate and adaptive immunity. Lysosomes are the main catabolic subcellular organelles responsible for degradation and recycling of both extracellular and intracellular material, which are the final steps in phagocytosis and autophagy. However, the molecular mechanisms underlying lysosomal functions after infection remain obscure. In this study, we conducted a quantitative proteomics analysis of the changes in constitution and glycosylation of proteins in lysosomes derived from murine RAW 264.7 macrophage cells treated with different types of pathogens comprising examples of bacteria (Listeria monocytogenes, L. m), DNA viruses (herpes simplex virus type-1, HSV-1) and RNA viruses (vesicular stomatitis virus, VSV). In total, 3,704 lysosome-related proteins and 300 potential glycosylation sites on 193 proteins were identified. Comparative analysis showed that the aforementioned pathogens induced distinct alterations in the proteome of the lysosome, which is closely associated with the immune functions of macrophages, such as toll-like receptor activation, inflammation and antigen-presentation. The most significant changes in proteins and fluctuations in glycosylation were also determined. Furthermore, Western blot analysis showed that the changes in expression of these proteins were undetectable at the whole cell level. Thus, our study provides unique insights into the function of lysosomes in macrophage activation and immune responses.

Protein profile changes in the frontotemporal lobes in human severe traumatic brain injury.

Severe traumatic brain injury (sTBI) is a serious public health issue with high morbidity and mortality rates. Previous proteomic studies on sTBI have mainly focused on human cerebrospinal fluid and serum, as well as on brain protein changes in murine models. However, human proteomic data in sTBI brain is still scarce. We used proteomic and bioinformatic strategies to investigate variations in protein expression levels in human brains after sTBI, using samples from the Department of Neurosurgery, Affiliated Hospital of Hebei University (Hebei, China). Our proteomic data identified 4031 proteins, of which 160 proteins were overexpressed and 5 proteins were downregulated. Bioinformatics analysis showed significant changes in biological pathways including glial cell differentiation, complement activation and apolipoprotein catalysis in the statin pathway. Western blot verification of protein changes in a subset of the available tissue samples showed results that were consistent with the proteomic data. This study is one of the first to investigate the whole proteome of human sTBI brains, and provide a characteristic signature and overall landscape of the sTBI brain proteome.

Quantitative proteomic profiling for clarification of the crucial roles of lysosomes in microbial infections

HighlightsWe analyzed changes in the lysosomal proteome after microbial infection.We used a combination of bioinformatics and proteomics strategies.Changes in the lysosomal proteome varied with infection by diverse microbes.This novel approach is suitable for investigations of the enriched organelles. Abstract Lysosomes play vital roles in both innate and adaptive immunity. It is widely accepted that lysosomes do not function exclusively as a digestive organelle. It is also involved in the process of immune cells against pathogens. However, the changes in the lysosomal proteome caused by infection with various microbes are still largely unknown, and our understanding of the proteome of the purified lysosome is another obstacle that needs to be resolved. Here, we performed a proteomic study on lysosomes enriched from THP1 cells after infection with Listeria monocytogenes (L.m), Herpes Simplex Virus 1 (HSV‐1) and Vesicular Stomatitis Virus (VSV). In combination with the gene ontology (GO) analysis, we identified 284 lysosomal‐related proteins from a total of 4560 proteins. We also constructed the protein‐protein interaction networks for the differentially expressed proteins and revealed the core lysosomal proteins, including SRC in the L. m treated group, SRC, GLB1, HEXA and HEXB in the HSV‐1 treated group and GLB1, CTSA, CTSB, HEXA and HEXB in the VSV treated group, which are involved in responding to diverse microbial infections. This study not only reveals variable lysosome responses depending on the bacterial or virus infection, but also provides the evidence based on which we propose a novel approach to proteome research for investigation of the function of the enriched organelles.

Proteomic Profiling of Brain and Testis Reveals the Diverse Changes in Ribosomal Proteins in fmr1 Knockout Mice

Fragile X syndrome (FXS), the leading cause of inherited forms of mental retardation and autism, is caused by the transcriptional silencing of fmr1 encoding the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that is a widely expressed, but primarily in the brain and testis, and associated approximately 4% of transcripts. Macro-orchidism is a common symptom associated with FXS both in humans and mice. Thus, we analyze the pooled samples of cerebral cortex, hippocampus and testis from both the fmr1-KO and wild-type mice by a LC-MS/MS proteomic study. Among the identified proteins, most of those showing significant changes in expression were up- or downregulated in the absence of FMRP. Proteins (FMRP, RPS8, RPL23a and ATPIF1, RPL6, GAP43, MTCH2 and MPZ in brain, and FMRP, CAH3, AKR1B7 and C9 in testis) identified by MS/MS were also verified by Western blotting. The Gene Ontology and WikiPathways analysis revealed that the differentially expressed proteins were clustered in the polyribosome and RNA-binding protein categories in both cerebral cortex and hippocampus, but not in testis. Although this study was limited by the little number of samples, our results provide detailed insights into the ribosomal protein profiles of cerebral cortex, hippocampus and testis in the absence of FMRP. Our studies also provide a better understanding of protein profile changes and the underlying dysregulated pathways arising from fmr1 silencing in FXS.

Quantitative Proteomics Analysis of Sporadic Medullary Thyroid Cancer Reveals FN1 as a Potential Novel Candidate Prognostic Biomarker

BACKGROUND Sporadic medullary thyroid cancer (MTC) is a rare neuroendocrine tumor. Currently, although the diagnosis of sporadic MTC is relatively simple, the need to discover novel candidate prognostic biomarkers for sporadic MTC and investigate the underlying mechanism involved in this rare disease is urgent. MATERIALS AND METHODS We employed tandem mass tag-based liquid chromatography-mass spectrometry to identify and analyze differentially expressed proteins (DEPs) in sporadic MTC. Western blotting was used to validate the DEPs. Immunohistochemistry was performed to investigate FN1 and RPS6KA3 in an independent set of sporadic MTC tissues. Immunohistochemical data were analyzed by different statistical methods. RESULTS Three hundred eighty-eight DEPs were identified in mass spectrometry, mainly involved in the extracellular matrix, cytoskeletal remodeling, or oxidoreductase activity. Among them, THBS1, MMP9, FN1, RPS6KA3, SYT1, and carcinoembryonic antigen were successfully validated by Western blot. In addition, FN1 and RPS6KA3, enriched in extracellular matrix (ECM) remodeling and the mitogen-activated protein kinase (MAPK) signaling pathway, respectively, were investigated in an independent set of sporadic MTC tissues. Receiver-operator characteristic curve analysis showed that FN1 and RPS6KA3 can be used for discriminating sporadic MTC tumorous tissues from paired normal thyroid tissues, and the clinical biomarker calcitonin was positively correlated with FN1 and RPS6KA3 in tumorous tissues. Furthermore, the immunohistochemical scores of FN1 in tumorous tissue showed an inverse relationship with tumor classification, lymph node classification, and American Joint Committee on Cancer stage. Through univariate and multivariate analysis for progression-free survival, we also found that low FN1 expression in tumorous tissues was an independent worse prognostic factor for progression-free survival. CONCLUSION We identified that the pathophysiology of sporadic MTC involve numerous pathways, including the synaptic vesicle pathway, the MAPK signaling pathway, and the ECM remodeling pathway. Furthermore, our study also identified FN1 as novel prognostic biomarkers related to the pathophysiologic changes in sporadic MTC. IMPLICATIONS FOR PRACTICE Proteomic dissection and prognostic biomarkers are scarce in sporadic medullary thyroid cancer (MTC). This article reports the use of proteomics technology to comprehensively investigate the molecular mechanisms of sporadic MTC, which resulted in the identification of FN1 as a novel candidate prognostic biomarker.

Multiple roles of Ring 1 and YY1 binding protein in physiology and disease

Ring 1 and YY1 binding protein (RYBP) was first identified in 1999, and its structure includes a conserved Npl4 Zinc finger motif at the N‐terminus, a central region that is characteristically enriched with arginine and lysine residues and a C‐terminal region enriched with serine and threonine amino acids. Over nearly 20 years, multiple studies have found that RYBP functions as an organ developmental adaptor. There is also evidence that RYBP regulates the expression of different genes involved in various aspects of biological processes, via a mechanism that is dependent on interactions with components of PcG complexes and/or through binding to different transcriptional factors. In addition, RYBP interacts directly or indirectly with apoptosis‐associated proteins to mediate anti‐apoptotic or pro‐apoptotic activity in both the cytoplasm and nucleus of various cell types. Furthermore, RYBP has also been shown to act as tumour suppressor gene in different solid tumours, but as an oncogene in lymphoma and melanoma. In this review, we summarize our current understanding of the functions of this multifaceted RYBP in physiological and pathological conditions, including embryonic development, apoptosis and cancer, as well as its role as a component of polycomb repressive complex 1.

Multiple functions of the E3 ubiquitin ligase CHIP in immunity

ABSTRACT The carboxyl terminal of Hsp70-interacting protein (CHIP) is an E3 ubiquitin ligase that plays a pivotal role in the protein quality control system by shifting the balance of the folding-refolding machinery toward the degradative pathway. However, the precise mechanisms by which nonnative proteins are selected for degradation by CHIP either directly or indirectly via chaperone Hsp70 or Hsp90 are still not clear. In this review, we aim to provide a comprehensive model of the mechanism by which CHIP degrades its substrate in a chaperone-dependent or direct manner. In addition, through tight regulation of the protein level of its substrates, CHIP plays important roles in many physiological and pathological conditions, including cancers, neurological disorders, cardiac diseases, bone metabolism, immunity, and so on. Nonetheless, the precise mechanisms underlying the regulation of the immune system by CHIP are still poorly understood despite accumulating developments in our understanding of the regulatory roles of CHIP in both innate and adaptive immune responses. In this review, we also aim to provide a view of CHIP-mediated regulation of immune responses and the signaling pathways involved in the model described. Finally, we discuss the roles of CHIP in immune-related diseases.