Ca2+-Stimulated AMPK-Dependent Phosphorylation of Exo1 Protects Stressed Replication Forks from Aberrant Resection.

Abnormal processing of stressed replication forks by nucleases can cause fork collapse, genomic instability, and cell death. Despite its importance, it is poorly understood how the cell properly controls nucleases to prevent detrimental fork processing. Here, we report a signaling pathway that controls the activity of exonuclease Exo1 to prevent aberrant fork resection during replication stress. Our results indicate that replication stress elevates intracellular Ca2+ concentration ([Ca2+]i), leading to activation of CaMKK2 and the downstream kinase 5' AMP-activated protein kinase (AMPK). Following activation, AMPK directly phosphorylates Exo1 at serine 746 to promote 14-3-3 binding and inhibit Exo1 recruitment to stressed replication forks, thereby avoiding unscheduled fork resection. Disruption of this signaling pathway results in excessive ssDNA, chromosomal instability, and hypersensitivity to replication stress inducers. These findings reveal a link between [Ca2+]i and the replication stress response as well as a function of the Ca2+-CaMKK2-AMPK signaling axis in safeguarding fork structure to maintain genome stability.

Parallel Proteomic Comparison of Mutants With Altered Carbon Metabolism Reveals Hik8 Regulation of PII Phosphorylation and Glycogen Accumulation in a Cyanobacterium.

Carbon metabolism is central to photosynthetic organisms and involves the coordinated operation and regulation of numerous proteins. In cyanobacteria, proteins involved in carbon metabolism are regulated by multiple regulators including the RNA polymerase sigma factor SigE, the histidine kinases Hik8, Hik31 and its plasmid-borne paralog Slr6041, and the response regulator Rre37. To understand the specificity and the cross-talk of such regulations, we simultaneously and quantitatively compared the proteomes of the gene knockout mutants for the regulators. A number of proteins showing differential expression in one or more mutants were identified, including four proteins that are unanimously upregulated or downregulated in all five mutants. These represent the important nodes of the intricate and elegant regulatory network for carbon metabolism. Moreover, serine phosphorylation of PII, a key signaling protein sensing and regulating in vivo carbon/nitrogen (C/N) homeostasis through reversible phosphorylation, is massively increased with a concomitant significant decrease in glycogen content only in the hik8-knockout mutant, which also displays impaired dark viability. An unphosphorylatable PII S49A substitution restored the glycogen content and rescued the dark viability of the mutant. Together, our study not only establishes the quantitative relationship between the targets and the corresponding regulators and elucidated their specificity and cross-talk but also unveils that Hik8 regulates glycogen accumulation through negative regulation of PII phosphorylation, providing the first line of evidence that links the two-component system with PII-mediated signal transduction and implicates them in the regulation of carbon metabolism.

Glycosyltransferase Slr1064 regulates carbon metabolism by modulating the levels of UDP-GlcNAc in Synechocystis sp. PCC 6803.

Glycosyltransferases (GTs) are enzymes that transfer sugars to various targets. They play important roles in diverse biological processes, including photosynthesis, cell motility, exopolysaccharide biosynthesis, and lipid metabolism; however, their involvement in regulating carbon metabolism in Synechocystis sp. PCC 6803 has not been reported. We identified a novel GT protein, Slr1064, involved in carbon metabolism. The effect of slr1064 deletion on the growth of Synechocystis cells and functional mechanisms of Slr1064 on carbon metabolism were thoroughly investigated through physiological, biochemistry, proteomic, and metabolic analyses. We found that this GT, which is mainly distributed in the membrane compartment, is essential for the growth of Synechocystis under heterotrophic and mixotrophic conditions, but not under autotrophic conditions. The deletion of slr1064 hampers the turnover rate of Gap2 under mixotrophic conditions and disrupts the assembly of the PRK/GAPDH/CP12 complex under dark culture conditions. Additionally, UDP-GlcNAc, the pivotal metabolite responsible for the O-GlcNAc modification of GAPDH, is downregulated in the Δslr1064. Our work provides new insights into the role of GTs in carbon metabolism in Synechocystis and elucidate the mechanism by which carbon metabolism is regulated in this important model organism.

Proximity Labeling Facilitates Defining the Proteome Neighborhood of Photosystem II Oxygen Evolution Complex in a Model Cyanobacterium.

Ascorbate peroxidase (APEX)-based proximity labeling coupled with mass spectrometry has a great potential for spatiotemporal identification of proteins proximal to a protein complex of interest. Using this approach is feasible to define the proteome neighborhood of important protein complexes in a popular photosynthetic model cyanobacterium Synechocystis sp. PCC6803 (hereafter named as Synechocystis). To this end, we developed a robust workflow for APEX2-based proximity labeling in Synechocystis and used the workflow to identify proteins proximal to the photosystem II (PS II) oxygen evolution complex (OEC) through fusion APEX2 with a luminal OEC subunit, PsbO. In total, 38 integral membrane proteins (IMPs) and 93 luminal proteins were identified as proximal to the OEC. A significant portion of these proteins are involved in PS II assembly, maturation, and repair, while the majority of the rest were not previously implicated with PS II. The IMPs include subunits of PS II and cytochrome b6/f, but not of photosystem I (except for PsaL) and ATP synthases, suggesting that the latter two complexes are spatially separated from the OEC with a distance longer than the APEX2 labeling radius. Besides, the topologies of six IMPs were successfully predicted because their lumen-facing regions exclusively contain potential APEX2 labeling sites. The luminal proteins include 66 proteins with a predicted signal peptide and 57 proteins localized also in periplasm, providing important targets to study the regulation and selectivity of protein translocation. Together, we not only developed a robust workflow for the application of APEX2-based proximity labeling in Synechocystis and showcased the feasibility to define the neighborhood proteome of an important protein complex with a short radius but also discovered a set of the proteins that potentially interact with and regulate PS II structure and function.

Spatial Proteome Reorganization of a Photosynthetic Model Cyanobacterium in Response to Abiotic Stresses.

Spatial proteome reorganization in response to a changing environment represents a different layer of adaptation mechanism in addition to differential expression of a subset of stress responsive genes in photosynthetic organisms. Profiling such reorganization events is critically important to extend our understanding how photosynthetic organisms adapt to adverse environments. Thus, we treated a unicellular photosynthetic model cyanobacterium, Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), with five different types of abiotic stresses including nitrogen starvation, iron deficiency, cold, heat, and darkness, and systematically identified proteins showing stress-induced differential expression and/or redistribution between the membrane and the soluble fractions using a quantitative proteomics approach. A number of proteins showing such a redistribution in response to a single or multiple types of abiotic stresses were identified. These include 12 ribosomal proteins displaying unanimous cold-induced redistribution to the membrane and the protein FurA, a master regulator of iron acquisition, displaying iron deficiency- and nitrogen starvation-induced redistribution to the membrane. Such findings shed light on a novel regulatory mechanism underlying the corresponding stress responses, and establish the results in the present study as an important resource for future studies intended to understand how photosynthetic organisms cope with adverse environments.

Evaluation of the Potential Risk of Advanced Peak Determination in Distorting Isobaric Labeling-Based Single-Shot Proteome Quantitation.

The recent development and implementation of the advanced peak determination (APD) algorithm with MS instrument dramatically increased the sampling of low abundance features for MS/MS fragmentation. After in-depth evaluation, it is found that with APD on, many chimeric spectra are acquired through co-fragmentation of high abundance contaminants with low abundance targets, and such co-fragmentations are largely avoided when APD is off. To evaluate whether such a co-fragmentation could significantly distort the accuracy of the isobaric-labeling based quantitation of the low abundance target, a single-shot TMT experiment is performed using a two-proteome model, whereby each TMT channel contains premixed peptides from human and a cyanobacterium with a known ratio. Unexpectedly, it is found that APD does not significantly distort TMT ratios, probably because the majority of the APD-specific chimeric spectra are not identifiable. Nevertheless, a few examples of significant distortion of TMT ratios of low abundance peptides caused by APD is found through manual inspection, and suggests that APD should be off in a single-shot TMT experiment to avoid the laborious and time-costing manual inspection.

Nitration-induced ubiquitination and degradation control quality of ERK1.

The mitogen-activated protein kinase ERK1/2 (ERKs, extracellular-regulated protein kinases) plays important roles in a wide spectrum of cellular processes and have been implicated in many disease states. The spatiotemporal regulation of ERK activity has been extensively studied. However, scarce information has been available regarding the quality control of the kinases to scavenge malfunctioning ERKs. Using site-specific mutagenesis and mass spectrometry, we found that the disruption of the conserved H-bond between Y210 and E237 of ERK1 through point mutation at or naturally occurring nitration on Y210 initiates a quality control program dependent on chaperon systems and CHIP (C-terminal of Hsp70-interacting protein)-mediated ubiquitination and degradation. The H-bond is also important for the quality control of ERK2, but through a distinct mechanism. These findings clearly demonstrate how malfunctioning ERKs are eliminated when cells are in certain stress conditions or unhealthy states, and could represent a general mechanism for scavenging malfunctioning kinases in stress conditions.

Mitogen-Activated Protein Kinase Cascade MKK7-MPK6 Plays Important Roles in Plant Development and Regulates Shoot Branching by Phosphorylating PIN1 in Arabidopsis.

Emerging evidences exhibit that mitogen-activated protein kinase (MAPK/MPK) signaling pathways are connected with many aspects of plant development. The complexity of MAPK cascades raises challenges not only to identify the MAPK module in planta but also to define the specific role of an individual module. So far, our knowledge of MAPK signaling has been largely restricted to a small subset of MAPK cascades. Our previous study has characterized an Arabidopsis bushy and dwarf1 (bud1) mutant, in which the MAP Kinase Kinase 7 (MKK7) was constitutively activated, resulting in multiple phenotypic alterations. In this study, we found that MPK3 and MPK6 are the substrates for phosphorylation by MKK7 in planta. Genetic analysis showed that MKK7-MPK6 cascade is specifically responsible for the regulation of shoot branching, hypocotyl gravitropism, filament elongation, and lateral root formation, while MKK7-MPK3 cascade is mainly involved in leaf morphology. We further demonstrated that the MKK7-MPK6 cascade controls shoot branching by phosphorylating Ser 337 on PIN1, which affects the basal localization of PIN1 in xylem parenchyma cells and polar auxin transport in the primary stem. Our results not only specify the functions of the MKK7-MPK6 cascade but also reveal a novel mechanism for PIN1 phosphorylation, establishing a molecular link between the MAPK cascade and auxin-regulated plant development.

Translating Divergent Environmental Stresses into a Common Proteome Response through Hik33 in a Model Cyanobacterium.

The histidine kinase Hik33 plays important roles in mediating cyanobacterial response to divergent types of abiotic stresses including cold, salt, high light (HL), and osmotic stresses. However, how these functions are regulated by Hik33 remains to be addressed. Using a hik33-deficient strain (Δhik33) of Synechocystis sp. PCC 6803 (Synechocystis) and quantitative proteomics, we found that Hik33 depletion induces differential protein expression highly similar to that induced by divergent types of stresses. This typically includes downregulation of proteins in photosynthesis and carbon assimilation that are necessary for cell propagation, and upregulation of heat shock proteins, chaperons, and proteases that are important for cell survival. This observation indicates that depletion of Hik33 alone mimics divergent types of abiotic stresses, and that Hik33 could be important for preventing abnormal stress response in the normal condition. Moreover, we found the majority of proteins of plasmid origin were significantly upregulated in Δhik33, though their biological significance remains to be addressed. Together, the systematically characterized Hik33-regulated cyanobacterial proteome, which is largely involved in stress responses, builds the molecular basis for Hik33 as a general regulator of stress responses.

Translating Divergent Environmental Stresses into a Common Proteome Response through the Histidine Kinase 33 (Hik33) in a Model Cyanobacterium.

The histidine kinase Hik33 plays important roles in mediating cyanobacterial response to divergent types of abiotic stresses including cold, salt, high light (HL), and osmotic stresses. However, how these functions are regulated by Hik33 remains to be addressed. Using a hik33-deficient strain (Δhik33) of Synechocystis sp. PCC 6803 (Synechocystis) and quantitative proteomics, we found that Hik33 depletion induces differential protein expression highly like that induced by divergent types of stresses. This typically includes downregulation of proteins in photosynthesis and carbon assimilation that are necessary for cell propagation, and upregulation of heat shock proteins, chaperons, and proteases that are important for cell survival. This observation indicates that depletion of Hik33 alone mimics divergent types of abiotic stresses, and that Hik33 could be important for preventing abnormal stress response in the normal condition. Moreover, we found most proteins of plasmid origin were significantly upregulated in Δhik33, though their biological significance remains to be addressed. Together, the systematically characterized Hik33-regulated cyanobacterial proteome, which is largely involved in stress responses, builds the molecular basis for Hik33 as a general regulator of stress responses.

[Advances of the technologies in large-scale membrane proteome identification].

Membrane proteins play important functions not only as receptors and transporters, but also in many other important intracellular functions such as photosynthetic and respiratory electron transport. Identification of membrane proteins is a necessary step to understand their functions. Membrane proteins are generally highly hydrophobic and difficult to be resolved by aqueous solutions, and large-scale proteomic identification of membrane proteins has been a great technical challenge. Significant efforts have been invested in the field to improve the solubility of membrane proteins in aqueous solutions that are compatible for mass spectrometry analysis. This review summarizes the main technological achievements in the field of membrane proteomics particularly for the improvement of membrane protein identification, and uses the photosynthetic model cyanobacterium Synechocystis sp. PCC6803 as an example to illustrate how technology advances push forward the field in terms of the increased coverage of membrane proteome identification.

Activation of the Oxidative Pentose Phosphate Pathway is Critical for Photomixotrophic Growth of a hik33-Deletion Mutant of Synechocystis sp. PCC 6803.

The histidine kinase Hik33 plays a central role in acclimation to changing environments in cyanobacteria. Deletion of hik33 induces a strong stress-like response in Synechocystis sp. PCC 6803 (Synechocystis) as represented by repressed photoautotrophic growth and photosynthesis, and differential expression of stress-responsive proteins. In contrast, the photomixotrophic growth of the hik33-deletion mutant (Δhik33) with glucose as the exogenous carbon source is only marginally repressed. To investigate how glucose rescues the growth of Δhik33, the proteomes of the photomixotrophically growing wild-type (WT) and the mutant strains of Synechocystis are quantitatively analyzed. It is found that glucose induces upregulation of the oxidative pentose phosphate (OPP) pathway. Depletion of glucose-6-phosphate dehydrogenase (G6PDH), which catalyzes the first and the rate-limiting step of the OPP pathway, significantly inhibits the photomixotrophic growth of Δhik33 but not of the WT. The result suggests that the OPP pathway, which is usually nonfunctional in the photomixotrophically growing WT, plays a major role in the photomixotrophic growth of Δhik33.

Systematically ranking the tightness of membrane association for peripheral membrane proteins (PMPs).

Large-scale quantitative evaluation of the tightness of membrane association for nontransmembrane proteins is important for identifying true peripheral membrane proteins with functional significance. Herein, we simultaneously ranked more than 1000 proteins of the photosynthetic model organism Synechocystis sp. PCC 6803 for their relative tightness of membrane association using a proteomic approach. Using multiple precisely ranked and experimentally verified peripheral subunits of photosynthetic protein complexes as the landmarks, we found that proteins involved in two-component signal transduction systems and transporters are overall tightly associated with the membranes, whereas the associations of ribosomal proteins are much weaker. Moreover, we found that hypothetical proteins containing the same domains generally have similar tightness. This work provided a global view of the structural organization of the membrane proteome with respect to divergent functions, and built the foundation for future investigation of the dynamic membrane proteome reorganization in response to different environmental or internal stimuli.

Hepatitis B virus surface protein-induced hPIAS1 transcription requires TAL1, E47, MYOG, NFI, and MAPK signal pathways.

The protein inhibitor of activated STAT1 (PIAS1) plays important roles in regulating virus-induced chronic hepatitis, but the interaction between hepatitis B virus (HBV) and hPIAS1 is not clear. Our aim was to verify if HBV encoding proteins enhance the transcription of hPIAS1 and which cis-elements and transcription factors were involved in the mechanism. In order to do, so a series of molecular biological methods, along with functional and histological studies, were performed. We found that the HBV surface protein (HBs) enhanced hPIAS1 transcription through the activities of TAL1, E47, myogenin (MYOG), and NFI, dependent on the activation of p38MAPK and ERK signaling pathways in vitro, which might contribute to the ineffectiveness of treatment in CHB patients. Furthermore, liver samples from patients with high HBsAg levels and HBV DNA displayed increased hPIAS1 expression and high levels of TAL1, E47, MYOG, and NFI, compared to those patients with low HBsAg levels and HBV DNA, and healthy controls. These findings suggest that the HBs protein-induced hPIAS1 transcription requires TAL1, E47, MYOG, NFI, and MAPK signal pathways. It provides new potential targets for antiviral therapeutic strategies for controlling HBV-associated diseases.

Clathrin and AP2 are required for phagocytic receptor-mediated apoptotic cell clearance in Caenorhabditis elegans.

Clathrin and the multi-subunit adaptor protein complex AP2 are central players in clathrin-mediated endocytosis by which the cell selectively internalizes surface materials. Here, we report the essential role of clathrin and AP2 in phagocytosis of apoptotic cells. In Caenorhabditis elegans, depletion of the clathrin heavy chain CHC-1 and individual components of AP2 led to a significant accumulation of germ cell corpses, which resulted from defects in both cell corpse engulfment and phagosome maturation required for corpse removal. CHC-1 and AP2 components associate with phagosomes in an inter-dependent manner. Importantly, we found that the phagocytic receptor CED-1 interacts with the α subunit of AP2, while the CED-6/Gulp adaptor forms a complex with both CHC-1 and the AP2 complex, which likely mediates the rearrangement of the actin cytoskeleton required for cell corpse engulfment triggered by the CED-1 signaling pathway. In addition, CHC-1 and AP2 promote the phagosomal association of LST-4/Snx9/18/33 and DYN-1/dynamin by forming a complex with them, thereby facilitating the maturation of phagosomes necessary for corpse degradation. These findings reveal a non-classical role of clathrin and AP2 and establish them as indispensable regulators in phagocytic receptor-mediated apoptotic cell clearance.

Toward the complete proteome of Synechocystis sp. PCC 6803.

The proteome of the photosynthetic model organism Synechocystis sp. PCC 6803 has been extensively analyzed in the last 15 years for the purpose of identifying proteins specifically expressed in subcellular compartments or differentially expressed in different environmental or internal conditions. This review summarizes the progress achieved so far with the emphasis on the impact of different techniques, both in sample preparation and protein identification, on the increasing coverage of proteome identification. In addition, this review evaluates the current completeness of proteome identification, and provides insights on the potential factors that could affect the complete identification of the Synechocystis proteome.

SOCS3 expression correlates with severity of inflammation in mouse hepatitis virus strain 3-induced acute liver failure and HBV-ACLF.

Recently, suppressor of cytokine signaling-3 (SOCS3) has been shown to be an inducible endogenous negative regulator of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway which is relevant in inflammatory response, while its functions in acute liver failure and HBV-induced acute-on-chronic liver failure (HBV-ACLF) have not been fully elucidated. In this study, we explored the role of SOCS3 in the development of mouse hepatitis virus strain 3 (MHV-3)-induced acute liver failure and its expression in liver and peripheral blood mononuclear cells (PBMCs) of patients with HBV-ACLF. Inflammation-related gene expression was detected by real-time PCR, immunohistochemistry and Western blotting. The correlation between SOCS3 level and liver injury was studied. Our results showed that the SOCS3 expression was significantly elevated in both the liver tissue and PBMCs from patients with HBV-ACLF compared to mild chronic hepatitis B (CHB). Moreover, a time course study showed that SOCS3 level was increased remarkably in the liver of BALB/cJ mice at 72 h post-infection. Pro-inflammatory cytokines, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α, were also increased significantly at 72 h post-infection. There was a close correlation between hepatic SOCS3 level and IL-6, and the severity of liver injury defined by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, respectively. These data suggested that SOCS3 may play a pivotal role in the pathogenesis of MHV-3-induced acute liver failure and HBV-ACLF.

GreenPhos, a universal method for in-depth measurement of plant phosphoproteomes with high quantitative reproducibility.

Protein phosphorylation regulates a variety of important cellular and physiological processes in plants. In-depth profiling of plant phosphoproteomes has been more technically challenging than that of animal phosphoproteomes. This is largely due to the need to improve protein extraction efficiency from plant cells, which have a dense cell wall, and to minimize sample loss resulting from the stringent sample clean-up steps required for the removal of a large amount of biomolecules interfering with phosphopeptide purification and mass spectrometry analysis. To this end, we developed a method with a streamlined workflow for highly efficient purification of phosphopeptides from tissues of various green organisms including Arabidopsis, rice, tomato, and Chlamydomonas reinhardtii, enabling in-depth identification with high quantitative reproducibility of about 11 000 phosphosites, the greatest depth achieved so far with single liquid chromatography-mass spectrometry (LC-MS) runs operated in a data-dependent acquisition (DDA) mode. The mainstay features of the method are the minimal sample loss achieved through elimination of sample clean-up before protease digestion and of desalting before phosphopeptide enrichment and hence the dramatic increases of time- and cost-effectiveness. The method, named GreenPhos, combined with single-shot LC-MS, enabled in-depth quantitative identification of Arabidopsis phosphoproteins, including differentially phosphorylated spliceosomal proteins, at multiple time points during salt stress and a number of kinase substrate motifs. GreenPhos is expected to serve as a universal method for purification of plant phosphopeptides, which, if samples are further fractionated and analyzed by multiple LC-MS runs, could enable measurement of plant phosphoproteomes with an unprecedented depth using a given mass spectrometry technology.

Evaluation of the Potential Risk of Analyzing Phosphopeptides with Easy-nLC 1200-Coupled Mass Spectrometers.

Liquid chromatography-mass spectrometry (LC-MS) is a major tool for the large-scale qualitative and/or quantitative analysis of protein phosphorylation in cells or tissues. The performance of LC is pivotal for the success of phosphoproteomics in both sensitivity and reproducibility. Here, we report that the widely used Easy-nLC 1200 has poor performance in analyzing phosphopeptides, particularly in terms of sensitivity and reproducibility, whereas its predecessor, Easy-nLC 1000, has a much better performance. Therefore, we suggest that Easy-nLC 1200 is not appropriate for LC-MS-based proteomics analysis for samples with a limited amount, particularly phosphopeptides from plants.

The quantitative proteome atlas of a model cyanobacterium.

Cyanobacteria are a group of oxygenic photosynthetic bacteria with great potentials in biotechnological applications and advantages as models for photosynthesis research. The subcellular localizations of the majority of proteins in any cyanobacteria remain undetermined, representing a major challenge in using cyanobacteria for both basic and industrial researches. Here, using label-free quantitative proteomics, we map 2027 proteins of Synechocystis sp. PCC6803, a model cyanobacterium, to different subcellular compartments and generate a proteome atlas with such information. The atlas leads to numerous unexpected but important findings, including the predominant localization of the histidine kinases Hik33 and Hik27 on the thylakoid but not the plasma membrane. Such information completely changes the concept regarding how the two kinases are activated. Together, the atlas provides subcellular localization information for nearly 60% proteome of a model cyanobacterium, and will serve as an important resource for the cyanobacterial research community.