Global Proteome and Phosphoproteome Characterization of Sepsis-induced Kidney Injury

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Highlights

• •Cecal Ligation Puncture (CLP) mouse model to study sepsis-induced kidney disease.
• •Quantitative global proteome and phosphoproteome profiling of mouse kidneys.
• •Highly significant candidate markers for onset and progression of AKI to CKD.
• •Mechanistic insights into sepsis-associated kidney injuries.

     

In-Depth Characterization of the Clostridioides difficile Phosphoproteome to Identify Ser/Thr Kinase Substrates

Clostridioides difficile is the leading cause of postantibiotic diarrhea in adults. During infection, the bacterium must rapidly adapt to the host environment by using survival strategies. Protein phosphorylation is a reversible post-translational modification employed ubiquitously for signal transduction and cellular regulation. Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases have emerged as important players in bacterial cell signaling and pathogenicity. C. difficile encodes two STKs (PrkC and CD2148) and one phosphatase. We optimized a titanium dioxide phosphopeptide enrichment approach to determine the phosphoproteome of C. difficile. We identified and quantified 2500 proteins representing 63% of the theoretical proteome. To identify STK and serine/threonine phosphatase targets, we then performed comparative large-scale phosphoproteomics of the WT strain and isogenic ΔprkC, CD2148, Δstp, and prkC CD2148 mutants. We detected 635 proteins containing phosphorylated peptides. We showed that PrkC is phosphorylated on multiple sites in vivo and autophosphorylates in vitro. We were unable to detect a phosphorylation for CD2148 in vivo, whereas this kinase was phosphorylated in vitro only in the presence of PrkC. Forty-one phosphoproteins were identified as phosphorylated under the control of CD2148, whereas 114 proteins were phosphorylated under the control of PrkC including 27 phosphoproteins more phosphorylated in the ?stp mutant. We also observed enrichment for phosphothreonine among the phosphopeptides more phosphorylated in the Δstp mutant. Both kinases targeted pathways required for metabolism, translation, and stress response, whereas cell division and peptidoglycan metabolism were more specifically controlled by PrkC-dependent phosphorylation in agreement with the phenotypes of the ΔprkC mutant. Using a combination of approaches, we confirmed that FtsK was phosphorylated in vivo under the control of PrkC and that Spo0A was a substrate of PrkC in vitro. This study provides a detailed mapping of kinase–substrate relationships in C. difficile, paving the way for the identification of new biomarkers and therapeutic targets.     

Survivin基因在系统性红斑狼疮患者中的异常表达

目的:检测Survivin基因在系统性红斑狼疮(SLE)患者中的表达变化,探讨Survivin基因在SLE发病过程中的作用机制。方法:抽取32例正常健康对照人群和26例SLE患者的外周血,分离单个核细胞,提取总RNA。半定量RT-PCR法检测Survivin基因的表达,以GAPDH基因为内对照,结果以Survivin基因与GAPDH基因的RT-PCR产物的灰度比值表示。同时分析Survivin基因与SLE患者病情活动度的三个实验室指标dsDNA、补体C3和C4的相关性。结果:半定量RT-PCR显示Survivin基因mR- NA表达在SLE患者组为0.83±0.61,正常对照组为0.49±0.59,SLE患者的Survivin基因mRNA的表达明显高于正常对照组(P<0.05)。SLE患者的抗dsDNA抗体水平与Survivin基因表达呈正相关(R=0.62,P<0.01),C3水平则与Survivin基因表达呈负相关(R=-0.41,P<0.05),而C4水平则与Survivin基因表达无显著相关(R=-0.28,P>0.05)。结论:Survivin基因在SLE患者外周血中异常高表达,可能在SLE的发病机制中起着一定的作用,且其高表达与SLE患者的病情活动度有关。     

磷蛋白组的研究技术及其进展

真核细胞中蛋白质磷酸化是一个重要事件。真核细胞利用可逆的蛋白磷酸化来控制许多细胞过程包括信号转换、基因表达、细胞周期等。磷蛋白组的研究涉及磷蛋白的分离和鉴定 ,磷酸化残基定位和定量分析。由于蛋白质磷酸化是一个动态过程 ,在细胞中磷蛋白含量低 ,磷酸化位点可变 ,且磷酸肽的质谱信号常常会受到抑制 ,所以磷蛋白的分析存在更多的困难。本文介绍了国内外在磷酸蛋白的分离鉴定及定量分析方面的研究技术以及进展情况。目前 ,质谱仍然是核心的鉴定技术 ,寻找更好富集方法是最大的挑战。定量蛋白组学是对蛋白质的差异表达进行精确的定量分析。目前还不存在一种独立的方法可以完成磷蛋白的分离、鉴定 ,以及磷酸位点的定位和定量分析。随着样品分离技术和相关仪器的发展 ,磷酸蛋白快速、准确、全面分析鉴定将能够实现。     

Tandem Mass Tag Approach Utilizing Pervanadate BOOST Channels Delivers Deeper Quantitative Characterization of the Tyrosine Phosphoproteome

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Highlights

• •Detection of low-abundance phosphotyrosine-containing peptides is challenging.
• •Multiplexed TMT allows inclusion of modification(pTyr)-saturated boost channels.
• •Boost channels facilitate selection of pTyr precursor ions for fragmentation.
• •Quantitation depth is increased while maintaining accuracy and precision.

     

Phosphoproteome analysis of fission yeast

Phosphorylation is a key regulator of many events in eukaryotic cells. The acquisition of large-scale phosphorylation data sets from model organisms can pinpoint conserved regulatory inputs and reveal kinase-substrate relationships. Here, we provide the first large-scale phosphorylation analysis of the fission yeast, Schizosaccharomyces pombe. Protein from thiabendazole-treated cells was separated by preparative SDS-PAGE and digested with trypsin. The resulting peptides were subjected to either IMAC or TiO2 phosphopeptide enrichment methods and then analyzed by LC-MS/MS using an LTQ-Orbitrap mass spectrometer. In total, 2887 distinct phosphorylation sites were identified from 1194 proteins with an estimated false-discovery rate of <0.5% at the peptide level. A comparison of the two different enrichment methods is presented, supporting the finding that they are complementary. Finally, phosphorylation sites were examined for phosphorylation-specific motifs and evolutionary conservation. These analyses revealed both motifs and specific phosphorylation events identified in S. pombe were conserved and predicted novel phosphorylation in mammals.     

Phosphoproteome of resting human platelets

Beside their main physiological function in hemostasis, platelets are also highly involved in pathological processes, such as atherothrombosis and inflammation. During hemostasis, binding of adhesive substrates to tyrosine-kinase-linked adhesion receptors and/or soluble agonists to G-protein coupled receptors leads to a cascade of intracellular signaling processes based on substrate (de)phosphorylation. The same mechanisms are involved in platelet activation at sites of atherosclerotic plaque rupture, contributing to vessel occlusion and consequently to pathologic states, such as myocardial infarction, stroke, or peripheral artery disease. To gain a deeper insight into platelet function, we analyzed the phosphoproteome of resting platelets and identified 564 phosphorylation sites from more than 270 proteins, of which many have not been described in platelets before. Among those were several unknown potential protein kinase A (PKA) and protein kinase G (PKG) substrates. Because platelet inhibition is tightly regulated especially by PKA and PKG activity, these proteins may represent important new targets for cardiovascular research. Thus, our finding that GPIbalpha is phosphorylated at Ser603 in resting platelets may represent a novel mechanism for the regulation of one of the most important platelet receptor (GPIb-IX-V) mediated signaling pathways by PKA/PKG.     

Phosphoproteome analysis of Drosophila melanogaster embryos

Protein phosphorylation is a key regulatory event in most cellular processes and development. Mass spectrometry-based proteomics provides a framework for the large-scale identification and characterization of phosphorylation sites. Here, we used a well-established phosphopeptide enrichment and identification strategy including the combination of strong cation exchange chromatography, immobilized metal affinity chromatography, and high-accuracy mass spectrometry instrumentation to study phosphorylation in developing Drosophila embryos. In total, 13,720 different phosphorylation sites were discovered from 2702 proteins with an estimated false-discovery rate (FDR) of 0.63% at the peptide level. Because of the large size of the data set, both novel and known phosphorylation motifs were extracted using the Motif-X algorithm, including those representative of potential ordered phosphorylation events.     

Anti-C1q Antibodies as a Follow-Up Marker in SLE Patients

In cross-sectional studies autoantibodies against complement C1q (anti-C1q) were found to be highly associated with active lupus nephritis. The aim of this retrospective study was to determine the value of anti-C1q as follow-up marker of disease activity and renal involvement in patients with systemic lupus erythematosus (SLE). Fifty-two patients with SLE and a minimum of three anti-C1q measurements during follow-up were analyzed. Anti-C1q levels correlated with global disease activity scores. In subgroup analyses, patients without renal involvement did not show a significant correlation between anti-C1q levels and disease activity. In contrast, in patients with renal involvement, anti-C1q levels correlated well with global disease activity. In addition, a positive correlation with the urine protein-to-creatinine ratio and anti-dsDNA antibody levels as well as a negative correlation with complement levels was observed. Anti-C1q antibodies were found to strongly correlate with parameters of SLE disease activity during follow-up, in particular with regard to renal involvement.     

SLE血清中抗-DNA抗体分离和性质的研究

本文用国产高分子树脂(T)接枝小牛胸腺DNA,通过亲合层析从系统性红斑狼疮SLE患者血清中纯化出抗-ds DNA抗体和抗-ss DNA抗体。酶联免疫吸附分析(ELISA)的研究表明:SLE抗-DNA抗体和DNA结合的差异性很大,是高度非均一性的。抗-ss DNA抗体不仅组成成分比抗-ds DNA抗体复杂,ss DNA/抗-ssDNA亲合能力也明显高于ds DNA/抗-ds DNA。纯化的抗-DNA抗体以IgG类抗体占主导,同时也有其它类型抗体存在(例如IgM等)。抗-ds DNA抗体有较抗-ss DNA抗体高的IgG含量(两者的IgG/IgM分别是7.0和4.0),说明IgG抗-DNA抗体更倾向于同dsDNA结合。     

Phosphoproteome analysis of Lotus japonicus seeds

In this study, we report the first dataset of phosphoproteins of the seeds of a model plant, Lotus japonicus. This dataset might be useful in studying the regulatory mechanisms of seed germination in legume plants. By proteomic analysis of seeds following water absorption, we identified a total of 721 phosphopeptides derived from 343 phosphoproteins in cotyledons, and 931 phosphopeptides from 473 phosphoproteins in hypocotyls. Kinase‐specific prediction analyses revealed that different kinases were activated in cotyledons and hypocotyls. In particular, many peptides containing ATM‐kinase target motifs, X‐X‐pS/pT‐Q‐X‐X, were detected in cotyledons. Moreover, by real‐time RT‐PCR analysis, we found that expression of a homolog of ATM kinase is upregulated specifically in cotyledons, suggesting that this ATM‐kinase homolog plays a significant role in cell proliferation in the cotyledons of L. japonicus seeds. The data have been deposited to the ProteomeXchange with identifier PXD000053 ( http://proteomecentral.proteomexchange.org/dataset/PXD000053 ).     

High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination

Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.     

Phosphoproteome dynamics during mitotic exit in budding yeast

The cell division cycle culminates in mitosis when two daughter cells are born. As cyclin‐dependent kinase (Cdk) activity reaches its peak, the anaphase‐promoting complex/cyclosome (APC/C) is activated to trigger sister chromatid separation and mitotic spindle elongation, followed by spindle disassembly and cytokinesis. Degradation of mitotic cyclins and activation of Cdk‐counteracting phosphatases are thought to cause protein dephosphorylation to control these sequential events. Here, we use budding yeast to analyze phosphorylation dynamics of 3,456 phosphosites on 1,101 proteins with high temporal resolution as cells progress synchronously through mitosis. This reveals that successive inactivation of S and M phase Cdks and of the mitotic kinase Polo contributes to order these dephosphorylation events. Unexpectedly, we detect as many new phosphorylation events as there are dephosphorylation events. These correlate with late mitotic kinase activation and identify numerous candidate targets of these kinases. These findings revise our view of mitotic exit and portray it as a dynamic process in which a range of mitotic kinases contribute to order both protein dephosphorylation and phosphorylation.     

Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase

Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr³ of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg² and Lys⁴ adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys⁴ of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser¹³⁷ of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser¹³⁷ resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser¹³⁷ might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing.Eukaryotic protein kinases (ePK)¹ constitute a large family of enzymes that coordinate virtually any cellular processes by the phosphorylation of their target proteins at specific sites (1, 2). Active kinases often modulate the activity of other enzymes, including other kinases, thus amplifying and extending an initial signal that affect sometimes thousands of proteins (3). This creates a highly complex network of feedback and forward loops where multiple kinases can mutually influence each other''s activity. Kinases adopt three molecular strategies to select and specifically phosphorylate their substrates in the crowded environment of a cell (2). First, tight control of cellular kinase localization assures that only proteins present in the close proximity of the kinase can be phosphorylated; second, the kinase specific activity can be regulated via post-translational modifications or the recruitment of cofactor molecules; and third, the recognition of specific consensus motifs on substrates ensures that phosphorylation only occurs at the intended site or sites (2).The Haspin kinase is a member of the ePK family that structurally diverges from most ePKs (1, 4). The Haspin kinase domain displays structural features that have never been observed in other ePK family members (5, 6). Specifically, the possibility of activation loop phosphorylation, a frequent regulatory mechanisms to control kinase activity, is absent in Haspin (5). Haspin is characterized by an active conformation that is stabilized by a hydrophobic lock of the helix αC inducing a stable S conformation of the structurally unique activation segment. These specific structural features also create a structurally diverse substrate binding site comprising a highly electronegative cleft for the histone H3 basic tails (5). Interestingly, the recognition of H3 has been shown to be modulated by methylation at H3 residue Lys⁴, thus coupling Haspin activity with epigenetic mechanisms of chromatin regulation (5). Histone H3 that is phosphorylated at Thr³ is so far the only well-characterized Haspin substrate (7). H3Thr³ phosphorylation (H3Thr^3ph) is required for the localization of Aurora B at the centromere (8–10). Inactivation of Haspin catalytic activity by ATP mimetic inhibitors induces Aurora B centromeric delocalization, leading to a loss of phosphorylation in chromatin associated Aurora B substrates (11, 12). To date, apart from this well-characterized centromeric function of Haspin activity, the broader cellular functions of the kinase and the phosphorylation events that control these remain essentially unknown.In this study, we used an integrated biochemical, proteomic, pharmacologic, and structural biology approach to study the Haspin kinase, its substrates and the cellular consequences of its activity. Specifically, we determined a new mode of kinase substrate binding and identified a Haspin kinase substrate recognition motif. We identified 3964 phosphorylation sites in chromatin-associated proteins, quantified their response to Haspin inhibition, and verified the mitotic phosphorylation of MacroH2A Ser¹³⁷ (13) as directly dependent by Haspin activity. Altogether, our data suggest that Haspin regulates the phosphorylation of proteins involved in mechanisms that control gene expression, including the modifications of histones, and provide evidence for novel molecular effects of Haspin activity on mitotic chromatin.     

The Plk1-dependent Phosphoproteome of the Early Mitotic Spindle

Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle.During mitosis, multiple processes, such as mitotic entry, spindle assembly, chromosome segregation, and cytokinesis, must be carefully coordinated to ensure the error-free distribution of chromosomes into the newly forming daughter cells. The physical separation of the chromosomes to opposite poles of the cell is driven by the mitotic spindle, a proteinaceous and highly dynamic microtubule (MT)1-based macromolecular machine. Spindle assembly begins early in mitosis and is completed when the bipolar attachment of microtubules to kinetochore (KT) pairs is achieved (1, 2). Polo-like kinase 1 (Plk1), a serine/threonine-specific kinase first identified in Drosophila (3), is one of the key regulators of this essential mitotic process and has therefore attracted much attention (4–6). In agreement with its diverse functions, the localization of Plk1 during mitosis is dynamic. Plk1 first associates with centrosomes in prophase before it localizes to spindle poles and KTs in prometaphase and metaphase. During anaphase, Plk1 is recruited to the central spindle and finally accumulates at the midbody during telophase. Proteomics studies using oriented peptide libraries have shown that two so-called polo boxes at the C-terminal end of Plk1, the polo box domain (PBD), are crucial for the localization of this kinase to cellular structures (7, 8). This domain binds to specific phosphorylated sequence motifs that are created by other priming kinases or are self-primed by Plk1 itself, thus providing an efficient mechanism to regulate localization and substrate selectivity in time and space (9–11).Despite the pleiotropic and critical functions of Plk1 during mitosis, only a limited number of target proteins and phosphorylation sites on substrates have so far been identified or studied in detail (4–6, 12). The difficulties in identification of bona fide Plk1 substrates stem from the low abundance of some substrates, technical limitations for determining in vivo phosphorylation sites, the requirement for Plk1 localization for recognition of some substrates, and the possibility that Plk1 may phosphorylate a broader consensus motif than determined previously (13). Recent developments in mass spectrometry (MS)-based proteomics have allowed the identification of a large number of in vivo phosphorylation sites from complex samples (14). However, the nature of the kinase(s) responsible for most of these phosphorylation events is still unclear, and the assignment of phosphorylation sites to individual kinases remains a challenging task. Previously, we explored the human mitotic spindle by MS and successfully identified a large number of novel spindle proteins and phosphorylation sites (15, 16). Now, the development of quantitative methods to monitor in vivo phosphorylation changes in complex samples (17–19) represents a unique opportunity to address the role of individual kinases in spindle function.To study Plk1 function at the mitotic spindle, we combined quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC) (20) with the isolation of human mitotic spindles and phosphopeptide enrichment. To expand the experimental coverage of Plk1 substrates and gain further insight into direct and indirect functions of Plk1, we compared the phosphoproteomes of mitotic spindles isolated from cells lacking Plk1 activity with spindles from cells with fully active kinase. Two independent approaches were used to interfere with Plk1 activity: protein depletion using an inducible small hairpin (shRNA) cell line and selective inhibition of the kinase by the small molecule inhibitor ZK-thiazolidinone (TAL) (21). Phosphorylation sites found to be down-regulated after Plk1 inhibition/depletion were subsequently validated using in vitro phosphorylation of synthetic peptide arrays. This approach identified many candidate Plk1 substrates, allowed confirmation of direct phosphorylation by Plk1 of more than 100 sites identified in vivo, and suggested a broader phosphorylation consensus motif for this kinase. Collectively, our data set provides a rich resource for in-depth studies on the spindle-associated Plk1-dependent phosphoproteome. This is illustrated by selective follow-up studies in which we validated the Plk1-dependent localization of substrates to centrosomes and kinetochores. In particular, using a phosphospecific antibody, we confirmed Plk1-dependent CENP-F phosphorylation in vivo and demonstrated that CENP-F localization to kinetochores depends on Plk1 kinase activity. Furthermore, we identified several Aurora A-dependent phosphorylation events that are regulated by Plk1, supporting the emerging view of an intimate functional relationship between Plk1 and Aurora A kinase (22, 23).