Glycoproteins as diagnostic and prognostic biomarkers for neurodegenerative diseases: A glycoproteomic approach.

Neurodegenerative diseases (NDs) are incurable and can develop progressively debilitating disorders, including dementia and ataxias. Alzheimer's disease and Parkinson's disease are the most common NDs that mainly affect the elderly people. There is an urgent need to develop new diagnostic tools so that patients can be accurately stratified at an early stage. As a common post-translational modification, protein glycosylation plays a key role in physiological and pathological processes. The abnormal changes in glycosylation are associated with the altered biological pathways in NDs. The pathogenesis-related proteins, like amyloid-ß and microtubule-associated protein tau, have altered glycosylation. Importantly, specific glycosylation changes in cerebrospinal fluid, blood and urine are valuable for revealing neurodegeneration in the early stages. This review describes the emerging biomarkers based on glycoproteomics in NDs, highlighting the potential applications of glycoprotein biomarkers in the early detection of diseases, monitoring of the disease progression, and measurement of the therapeutic responses. The mass spectrometry-based strategies for characterizing glycoprotein biomarkers are also introduced.

Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-coupled Affinity Purification/Mass Spectrometry Analysis Revealed a Novel Role of Neurofibromin in mTOR Signaling.

Neurofibromin (NF1) is a well known tumor suppressor that is commonly mutated in cancer patients. It physically interacts with RAS and negatively regulates RAS GTPase activity. Despite the importance of NF1 in cancer, a high quality endogenous NF1 interactome has yet to be established. In this study, we combined clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene knock-out technology with affinity purification using antibodies against endogenous proteins, followed by mass spectrometry analysis, to sensitively and accurately detect NF1 protein-protein interactions in unaltered in vivo settings. Using this system, we analyzed endogenous NF1-associated protein complexes and identified 49 high-confidence candidate interaction proteins, including RAS and other functionally relevant proteins. Through functional validation, we found that NF1 negatively regulates mechanistic target of rapamycin signaling (mTOR) in a LAMTOR1-dependent manner. In addition, the cell growth and survival of NF1-deficient cells have become dependent on hyperactivation of the mTOR pathway, and the tumorigenic properties of these cells have become dependent on LAMTOR1. Taken together, our findings may provide novel insights into therapeutic approaches targeting NF1-deficient tumors.

Analysis of Salmonella PhoP/PhoQ regulation by dimethyl-SRM-based quantitative proteomics.

SRM (selected reaction monitoring), a tandem mass spectrometry-based method characterized by high repeatability and accuracy, is an effective tool for the quantification of predetermined proteins. In this study, we built a time-scheduled dimethyl-SRM method that can provide the precise relative quantification of 92 proteins in one run. By applying this method to the Salmonella PhoP/PhoQ two-component system, we found that the expression of selected PhoP/PhoQ-activated proteins in response to Mg(2+) concentrations could be divided into two distinct patterns. For the time-course SRM experiment, we found that the dynamics of the selected PhoP/PhoQ-activated proteins could be divided into three distinct patterns, providing a new clue regarding PhoP/PhoQ activation and regulation. Moreover, the results for iron homeostasis proteins in response to Mg(2+) concentrations revealed that the PhoP/PhoQ two-component system may serve as a repressor for iron uptake proteins. And ribosomal protein levels clearly showed a response to different Mg(2+) concentrations and to time.

Quantitative Proteomics Reveals the Roles of Peroxisome-associated Proteins in Antiviral Innate Immune Responses.

Compared with whole-cell proteomic analysis, subcellular proteomic analysis is advantageous not only for the increased coverage of low abundance proteins but also for generating organelle-specific data containing information regarding dynamic protein movement. In the present study, peroxisome-enriched fractions from Sendai virus (SeV)-infected or uninfected HepG2 cells were obtained and subjected to quantitative proteomics analysis. We identified 311 proteins that were significantly changed by SeV infection. Among these altered proteins, 25 are immune response-related proteins. Further bioinformatic analysis indicated that SeV infection inhibits cell cycle-related proteins and membrane attack complex-related proteins, all of which are beneficial for the survival and replication of SeV within host cells. Using Luciferase reporter assays on several innate immune-related reporters, we performed functional analysis on 11 candidate proteins. We identified LGALS3BP and CALU as potential negative regulators of the virus-induced activation of the type I interferons.

RNF26 temporally regulates virus-triggered type I interferon induction by two distinct mechanisms.

Viral infection triggers induction of type I interferons (IFNs), which are critical mediators of innate antiviral immune response. Mediator of IRF3 activation (MITA, also called STING) is an adapter essential for virus-triggered IFN induction pathways. How post-translational modifications regulate the activity of MITA is not fully elucidated. In expression screens, we identified RING finger protein 26 (RNF26), an E3 ubiquitin ligase, could mediate polyubiquitination of MITA. Interestingly, RNF26 promoted K11-linked polyubiquitination of MITA at lysine 150, a residue also targeted by RNF5 for K48-linked polyubiquitination. Further experiments indicated that RNF26 protected MITA from RNF5-mediated K48-linked polyubiquitination and degradation that was required for quick and efficient type I IFN and proinflammatory cytokine induction after viral infection. On the other hand, RNF26 was required to limit excessive type I IFN response but not proinflammatory cytokine induction by promoting autophagic degradation of IRF3. Consistently, knockdown of RNF26 inhibited the expression of IFNB1 gene in various cells at the early phase and promoted it at the late phase of viral infection, respectively. Furthermore, knockdown of RNF26 inhibited viral replication, indicating that RNF26 antagonizes cellular antiviral response. Our findings thus suggest that RNF26 temporally regulates innate antiviral response by two distinct mechanisms.

Protein turnover analysis in Salmonella Typhimurium during infection by dynamic SILAC, Topograph, and quantitative proteomics.

Protein turnover affects protein abundance and phenotypes. Comprehensive investigation of protein turnover dynamics has the potential to provide substantial information about gene expression. Here we report a large-scale protein turnover study in Salmonella Typhimurium during infection by quantitative proteomics. Murine macrophage-like RAW 264.7 cells were infected with SILAC labeled Salmonella. Bacterial cells were extracted after 0, 30, 60, 120, and 240 min. Mass spectrometry analyses yielded information about Salmonella protein turnover dynamics and a software program named Topograph was used for the calculation of protein half lives. The half lives of 311 proteins from intracellular Salmonella were obtained. For bacteria cultured in control medium (DMEM), the half lives for 870 proteins were obtained. The calculated median of protein half lives was 69.13 and 99.30 min for the infection group and the DMEM group, respectively, indicating an elevated protein turnover at the initial stage of infection. Gene ontology analyses revealed that a number of protein functional groups were significantly regulated by infection, including proteins involved in ribosome, periplasmic space, cellular amino acid metabolic process, ion binding, and catalytic activity. The half lives of proteins involved in purine metabolism pathway were found to be significantly shortened during infection.

C-terminal Src kinase (Csk)-mediated phosphorylation of eukaryotic elongation factor 2 (eEF2) promotes proteolytic cleavage and nuclear translocation of eEF2.

Protein-tyrosine kinase C-terminal Src kinase (Csk) was originally purified as a kinase for phosphorylating Src and other Src family kinases. The phosphorylation of a C-terminal tyrosine residue of Src family kinases suppresses their kinase activity. Therefore, most physiological studies regarding Csk function have been focused on Csk as a negative regulator of Src family tyrosine kinases and as a potential tumor suppressor. Paradoxically, the protein levels of Csk were elevated in some human carcinomas. In this report, we show that eukaryotic elongation factor 2 (eEF2) is a new protein substrate of Csk and could locate in the nucleus. We demonstrate that Csk-mediated phosphorylation of eEF2 has no effect on its cytoplasmic function in regulating protein translation. However, phosphorylation of eEF2 enhances its proteolytic cleavage and the nuclear translocation of the cleaved eEF2 through a SUMOylation-regulated process. Furthermore, we show that cleaved fragments of eEF2 can induce nuclear morphological changes and aneuploidy similar to those in cancer cells, suggesting that there is an additional mechanism for Csk in tumorigenesis through regulation of eEF2 subcellular localization.

Proteomic Analysis of the Human Tankyrase Protein Interaction Network Reveals Its Role in Pexophagy.

Tankyrase 1 (TNKS) and tankyrase 2 (TNKS2) belong to the poly(ADP-ribose) polymerase family of proteins, which use nicotinamide adenine dinucleotide to modify substrate proteins with ADP-ribose modifications. Emerging evidence has revealed the pathological relevance of TNKS and TNKS2, and identified these two enzymes as potential drug targets. However, the cellular functions and regulatory mechanisms of TNKS/2 are still largely unknown. Through a proteomic analysis, we defined the protein-protein interaction network for human TNKS/2 and revealed more than 100 high-confidence interacting proteins with numerous biological functions in this network. Finally, through functional validation, we uncovered a role for TNKS/2 in peroxisome homeostasis and determined that this function is independent of TNKS enzyme activities. Our proteomic study of the TNKS/2 protein interaction network provides a rich resource for further exploration of tankyrase functions in numerous cellular processes.