Protein Disulfide Isomerase 2 of Chlamydomonas reinhardtii Is Involved in Circadian Rhythm Regulation

Protein disulfide isomerases （PDIs） are known to play important roles in the folding of nascent proteins and in the formation of disulfide bonds. Recently, we identified a PDI from Chlamydomonas reinhardtii （CrPDI2） by a mass spectrometry approach that is specifically enriched by heparin affinity chromatography in samples taken during the night phase. Here, we show that the recombinant CrPDI2 is a redox-active protein. It is reduced by thioredoxin reductase and catalyzes itself the reduction of insulin chains and the oxidative refolding of scrambled RNase A. By immunoblots, we confirm a high-amplitude change in abundance of the heparin-bound CrPDI2 during subjective night. Interestingly, we find that CrPDI2 is present in protein complexes of different sizes at both day and night. Among three identified interac- tion partners, one （a 2-cys peroxiredoxin） is present only during the night phase. To study a potential function of CrPDI2 within the circadian system, we have overexpressed its gene. Two transgenic lines were used to measure the rhythm of phototaxis~ In the transgenic strains, a change in the acrophase was observed. This indicates that CrPDI2 is involved in the circadian signaling pathway and, together with the night phase-specific interaction of CrPDI2 and a peroxiredoxin, these findings suggest a close coupling of redox processes and the circadian clock in C. reinhardtii.     

Photoconversion of Lysotracker Red to a green fluorescent molecule

Dear Editor： Lysotracker Red DND-99 （Invitrogen-Molecular Probes） is a fluorophore in the form of a conjugated multi-pyrrole ring structure containing a weakly basic amine that selectively accumulates in acidic compartments and exhibits red fluorescence （excitation： 577 nm, emission： 590 nm） （Figure 1A）. It is structurally related to Lysotracker Green （Figure 1B） but has an additional pyrrole ring in conjugation with the primary structure, which produces a longer wavelength emission. Lysotracker Red is commonly used in multicolor imaging studies as a lysosomal marker to determine intracellular localization of a protein of interest by fluorescence and confocal microscopy [1-5] and is recommended by the manufacturer for this application. While using Lysotracker Red to study the localization of a protein fused to green fluorescent protein （GFP）, we observed an additional strong green fluorescent signal that colocalized with Lysotracker Red. After careful examination, however, we noted that the added green signal appeared only after illumination of the cells by a standard 100W mercury epi-fluorescence light source equipped with a 560/40 excitation filter （Leica TX2）. Prior to exposing the field to broadband excitation light, it was possible to visualize by confocal scanning （488nm excitation line） cells that exclusively expressed GFP. Remarkably, after exposure to broadband excitation light, green fluorescence appeared in all cells irrespective of GFP expression, displayed signal intensity similar to that of GFP, and colocalized with Lysotracker Red （Figure S1）.[第一段]     

Autophagy and cancer

Autophagy is a homeostatic and evolutionarily conserved mechanism of self-digestion by which the cells degrade and recycle long-lived proteins and excess or damaged organelles.Autophagy is activated in response to both physiological and pathological stimuli including growth factor depletion,energy deficiency or the upregulation of Bcl-2 protein expression.A novel role of autophagy in various cancers has been proposed.Interestingly,evidence that supports both a positive and negative role of autophagy in the pathogenesis of cancer has been reported.As a tumor suppression mechanism,autophagy maintains genome stability,induces senescence and possibly autophagic cell death.On the other hand,autophagy participates in tumor growth and maintenance by supplying metabolic substrate,limiting oxidative stress,and maintaining cancer stem cell population.It has been proposed that the differential roles of autophagy in cancer are disease type and stage specific.In addition,substrate selectivity might be involved in carrying out the specific effect of autophagy in cancer,and represents one of the potential directions for future studies.     

Hsp90 inhibition results in autophagy-mediated proteasome-independent degradation of IκB kinase （IKK）

Autophagic and proteasomal proteolysis are two major pathways for degradation of cellular constituents. Current models suggest that autophagy is responsible for the nonselective bulk degradation of long-lived proteins and organelles while the proteasome specifically degrades short-lived proteins including misfolded proteins caused by the absence of Hsp90 function. Here, we show that the IκB kinase （IKK）, an essential activator of NF-κB, is selectively degraded by autophagy when Hsp90 is inhibited by geldanamycin （GA）, a specific Hsp90 inhibitor showing highly effective anti-tumor activity. We find that in this case inactivation of ubiquitination or proteasome fails to block IKK degradation. However, inhibition of autophagy by an autophagy inhibitor or knockout of Atg5, a key component of the autophagy pathway, significantly rescues IKK from GA-induced degradation. These findings provide the first evidence that an Hsp90 client may be degraded by a mechanism different from the proteasome pathway and establish a molecular link among Hsp90, NF-κB and autophagy     

Molecular dynamics reveal a novel kinase-substrate interface that regulates protein translation

A key control point in gene expression is the initiation of protein translation, with a universal stress response being constituted by in- hibitory phosphoryiation of the eukaryotic initiation factor 2α （el F2oL）. In humans, four kinases sense diverse physiological stresses to regulate elF2α to control cell differentiation, adaptation, and survival. Here we develop a computational molecular model of elF2α and one of its kinases, the protein kinase R, to simulate the dynamics of their interaction. Predictions generated by coarse-grained dynamics simulations suggest a novel mode of action. Experimentation substantiates these predictions, identifying a previously unrecognized interface in the protein complex, which is constituted by dynamic residues in both elF2α and its kinases that are crucial to regulate protein translation. These findings call for a reinterpretation of the current mechanism of action of the el F2α kinases and demonstrate the value of conducting computational analysis to evaluate protein function.     

Retromer Subunits VPS35A and VPS29 Mediate Prevacuolar Compartment （PVC） Function in Arabidopsis

Intracellular protein routing is mediated by vesicular transport which is tightly regulated in eukaryotes. The protein and lipid homeostasis depends on coordinated delivery of de novo synthesized or recycled cargoes to the plasma membrane by exocytosis and their subsequent removal by rerouting them for recycling or degradation. Here, we report the characterization of protein affected trafficking 3 （pat3） mutant that we identified by an epifluorescence-based for- ward genetic screen for mutants defective in subcellular distribution of Arabidopsis auxin transporter PIN1-GFR While pat3 displays largely normal plant morphology and development in nutrient-rich conditions, it shows strong ectopic intracellular accumulations of different plasma membrane cargoes in structures that resemble prevacuolar compart- ments （PVC） with an aberrant morphology. Genetic mapping revealed that pat3 is defective in vacuolar protein sorting 35A （VPS35A）, a putative subunit of the retromer complex that mediates retrograde trafficking between the PVC and trans-Golgi network. Similarly, a mutant defective in another retromer subunit, vps29, shows comparable subcellular defects in PVC morphology and protein accumulation. Thus, our data provide evidence that the retromer components VPS35A and VPS29 are essential for normal PVC morphology and normal trafficking of plasma membrane proteins in plants. In addition, we show that, out of the three VPS35 retromer subunits present in Arabidopsis thaliana genome, the VPS35 homolog A plays a prevailing role in trafficking to the lyric vacuole, presenting another level of complexity in the retromer-dependent vacuolar sorting.     

Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development

Pollen germination on the surface of compatible stigmatic tissues is an essential step for plant fertilization. Here we report that the Arabidopsis mutant bcll is male sterile as a result of the failure ofpollen germination. We show that the bcll mutant allele cannot be transmitted by male gametophytes and no homozygous bcll mutants were obtained. Analysis of pollen developmental stages indicates that the bcll mutation affects pollen germination but not pollen maturation. Molecular analysis demonstrates that the failure of pollen germination was caused by the disruption of AtBECLIN 1. AtBECLIN 1 is expressed predominantly in mature pollen and encodes a protein with significant homology to Beclin1/Atg6/Vps30 required for the processes of autophagy and vacuolar protein sorting （VPS） in yeast. We also show that AtBECLIN 1 is required for normal plant development, and that genes related to autophagy, VPS and the glycosylphosphatidylinositol anchor system, were affected by the deficiency of AtBECLIN 1.     

The transmembrane domain of TACE regulates protein ectodomain shedding

Numerous membrane proteins are cleaved by tumor necrosis factor-α converting enzyme （TACE）, which causes the release of their ectodomains. An ADAM （a disintegrin and metalloprotease domain） family member, TACE contains several noncatalytic domains whose roles in ectodomain shedding have yet to be fully resolved. Here, we have explored the function of the transmembrane domain （TM） of TACE by coupling molecular engineering and functional analysis. A TM-free TACE construct that is anchored to the plasma membrane by a glycosylphosphatidylinositol （GPI）-binding polypeptide failed to restore shedding of transforming growth factor-or （TGF-α）, tumor necrosis factor-α （TNF-α） and L-selectin in cells lacking endogenous TACE activity. Substitution of the TACE TM with that of the prolactin receptor or platelet-derived growth factor receptor （PDGFR） also resulted in severe loss of TGF-α shedding, but had no effects on the cleavage of TNF-α and L-selectin. Replacement of the TM in TGF-α with that of L-selectin enabled TGF-α shedding by the TACE mutants carrying the TM of prolactin receptor and PDGFR. Taken together, our observations suggest that anchorage of TACE to the lipid bilayer through a TM is required for efficient cleavage of a broad spectrum of substrates, and that the amino-acid sequence of TACE TM may play a role in regulatory specificity among TACE substrates.     

Regulation of proteasomes in prion disease

The hallmark of prion disease is the accumulation of mis folded protein PrPsc, which is toxic to neuronal cells. The proteasome system is responsible for the rapid, precise, and timely degradation of proteins and plays an important role in cellular protein quality control. Increasing evidence indi cates impaired activity of proteasomes in prion diseases. Accumulated PrPsc can directly or indirectly affect prote asome activity. Misfolded protein may influence the assem bly and activity of 19S regulatory particle, or post translational modification of 20S proteasome, which may adversely affect the protein degradation activity of protea somes. In this review, we summarized the recent findings concerning the possible regulation of proteasomes in prion and other neurodegenerative diseases. The proteasome system may enhance its degradation activity by changing its structure, and this activity can also be increased by related chaperones when neuronal cells are subject to stress. When the proteasome system is inhibited, degradation of protein aggregates via autophagy may increase as a compensatory system. It is possible that a balance exists between the prote asome and autophagy in vivo; when one is impaired, the ac tivity of the other may increase to maintain homeostasis. However, more studies are needed to elucidate the relation ship between the proteasome system and autophagy.     

Manipulation of autophagy by HCMV infection is involved in mTOR and influences the replication of virus

Autophagy is an evolutionarily conserved cellular degrad- ation process, in which intracellular components are digested by the lysosomal pathway. While this process maintains cel- lular homeostasis by constitutively recycling cytoplasmic organelles and proteins, it can also be stimulated by environ- mental stress conditions, such as starvation, oxidative stress, and accumulation of misfolded proteins. During viral infec- tion, autophagy can act as a host surveillance mechanism, where viral antigens are delivered to endosomal and lyso- somal compartments enriched in immune sensors that can activate autophagy upon stimulation. Viruses, in turn, have adapted several ways in which to evade this antiviral mech- anism. While many viruses block various points along the autophagic pathway, some actively subvert autophagy for their own benefit. It has been proposed that viral manipula- tion of autophagy might directly and/or indirectly facilitate most stages of the viral lifecycle. In some circumstances, autophagy can act as a pro-viral pathway promoting viral replication or exiting from cells [1]. For example, hepatitis C virus （HCV） induces autophagosome accumulation, but then inhibits their ability to fuse with lysosomes [2]. HCV may also require autophagy during early infection [3]. For human cytomegalovirus （HCMV）, however, there is controversy surrounding whether it stimulates autophagy during early in- fection [4] or inhibits autophagy [5,6]. In the present study, we investigated whether and how HCMV modulates autophagy in vitro over time. Human erythroleukemia （HEL） cells were infected with HCMV at 1 plaque forming unit （PFU）/cell for 0, 12, 24, 36, 48, and 60 h and stained with acridine orange （Sigma, St Louis, USA） to evaluate autophagosome formation by flow cyto- metry. HCMV first triggered, then later inhibited, acidic vesicle and autolysosome accumulation,     

Identification of the Phosphorylated Residues in TveIF5A by Mass Spectrometry

The initiation factor elF5A in Trichomonas vaginalis （TvelF5A） is previously shown to undergo hypusination, phosphorylation and glycosylation. Three different pI isoforms of TvelF5A have been reported. The most acidic isoform （pI 5.2） corresponds to the precursor TvelF5A, whereas the mature TvelF5A appears to be the most basic isoform （pI 5.5）. In addition, the intermediary isoform （pI 5.3） is found only under polyamine-depleted conditions and restored with exogenous putrescine. We propose that differences in PI are due to phosphorylation of the TvelF5A isoforms. Here, we have identified phosphorylation sites using mass spectrometry. The mature TvelF5A contains four phosphorylated residues （$3, T55, T78 and T82）. Phosphorylation at $3 and T82 is also identified in the intermediary TvelF5A, while no phosphorylated residues are found in the precursor TvelF5A. It has been demonstrated that elF5A proteins from plants and yeast are phosphorylated by a casein kinase 2 （CK2）. Interestingly, a gene encoding a protein highly similar to CK2 （TvCK2） is found in T. vaginalis, which might be involved in the phosphorylation of TvelF5A in T. vaginalis.     

Salt Stress in Arabidopsis： Lipid Transfer Protein AZI1 and Its Control by Mitogen-Activated Protein Kinase MPK3

A plant＇s capability to cope with environmental challenges largely relies on signal transmission through mitogen-activated protein kinase （MAPK） cascades. In Arabidopsis thaliana, MPK3 is particularly strongly associated with numerous abiotic and biotic stress responses. Identification of MPK3 substrates is a milestone towards improving stress resistance in plants. Here, we characterize AZI1, a lipid transfer protein （LTP）-related hybrid proline-rich protein （HyPRP）, as a novel target of MPK3. AZI1 is phosphorylated by MPK3 in vitro. As documented by co-immunoprecipitation and bimolecular fluorescence complementation experiments, AZI1 interacts with MPK3 to form protein complexes in planta. Furthermore, null mutants of azil are hypersensitive to salt stress, while AZIl-overexpressing lines are markedly more tolerant. AZI1 overexpression in the mpk3 genetic background partially alleviates the salt-hypersensitive phenotype of this mutant, but functional MPK3 appears to be required for the full extent of AZIl-conferred robustness. Notably, this robustness does not come at the expense of normal development. Immunoblot and RT-PCR data point to a role of MPK3 as positive regulator of AZI1 abundance.     

A novel Physarum polycephalum SR protein kinase specifically phosphorylates the RS domain of the human SR protein,ASF/SF2

A 1591-bp cDNA of a serine-rich protein kinase （SRPK）-Iike protein has been identified in Physarum polycephalum （GenBank accession No. DQ140379）. The cDNA contains two repeat sequences at bp 1-153 and bp 395-547. The encoding sequence is 56% homologous to human SRPK1 and is named Physarum SRPK （PSRPK）. Consistent with other SRPKs, the consensus motifs of PSRPK are within the two conserved domains （CDs）. However, divergent motifs between the N-terminal and CDs are much shorter than the corresponding sequences of other SRPKs. To study the structure and function of this protein, we performed co-expression experiment in Escherichia coli and in vitro phosphorylation assay to investigate the phosphorylation effect of recombinant PSRPK on the human SR protein, ASF/SF2. Western blot analysis showed that PSRPK could phosphorylate ASF/SF2 in E. coil cells. Autoradiographic examination showed that both recombinant PSRPK and a truncated form of PSRPK with a 28-aa deletion at the N-terminus could phosphorylate ASF/SF2 and a truncated form of ASF/SF2 that contains the RS domain. However, these two forms of PSRPK could not phosphorylate a truncated form ASF/SF2 that lacks the RS domain. A truncated form of PSRPK that lacks either of CDs does not have any phosphorylation activity. These results indicated that, like other SRPKs, the phosphorylation site in PSRPK is located within the RS domain of the SR protein and that its phosphorylation activity is closely associated with the two CDs. This study on the structure and function of PSRPK demonstrates that it is a new member of the SRPK family.     

OsVPS9A Functions Cooperatively with OsRAB5A to Regulate Post-Golgi Dense Vesicle-Mediated Storage Protein Trafficking to the Protein Storage Vacuole in Rice Endosperm Cells

In the rice endosperm cells, glutelins are synthesized on rough endoplasmic reticulum as proglutelins and are sorted to the protein storage vacuoles （PSVs） called protein body IIs （PBIIs）, where they are converted to the mature forms. Dense vesicle （DV）-mediated trafficking of proglutelins in rice seeds has been proposed, but the post-Golgi control of this process is largely unknown. Whether DV can fuse directly with PSV is another matter of debate. In this study, we propose a regulatory mechanism underlying DV-mediated, post-Golgi proglutelin trafficking to PBII （PSV）. gpa2, a loss- of-function mutant of OsVPS9A, which encodes a GEF of OsRAB5A, accumulated uncleaved proglutelins. Proglutelins were mis-targeted to the paramural bodies and to the apoplast along the cell wall in the form of DVs, which led to a con- comitant reduction in PBII size. Previously reported gpal, mutated in OsRab5a, has a similar phenotype, while gpalgpa2 double mutant exacerbated the conditions. In addition, OsVPS9A interacted with OsRAB5A in vitro and in vivo. We con- cluded that OsVPS9A and OsRAB5A may work together and play a regulatory role in DV-mediated post-Golgi proglutelin trafficking to PBII （PSV）. The evidence that DVs might fuse directly to PBII （PSV） to deliver cargos is also presented.     

New GATEWAY vectors for High Throughput Analyses of Protein-Protein Interactions by Bimolecular Fluorescence Complementation

Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation （BiFC） is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N-or C-terminal sub-fragment of S（CFP）3A or Venus, respectively, and enable the performance of multicolor BiFC （mcBiFC）. We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.     

Interaction between Mnk2 and CBCVHL ubiquitin ligase E3 complex

MAP kinase-interacting kinase-2 (Mnk2) is one of the downstream kinases activated by MAP kinases. It phosphorylates the eukaryotic initiation factor 4E (elF4E), although the role of elF4E phosphorylation and the role of Mnk2 in the process of protein translation are not well understood. Except for elF4E, other physiological substrates of Mnk2 are still unidentified. To look for these unidentified substrates and to reveal the physiological function of Mnk2, we performed a yeast two-hybrid screening with Mnk2 as the bait. The results demonstrated Mnk2 could interact with VHL (von Hip-pel-Lindau tumor suppressor), Rbx1 (ring-box 1) and Cul2 (Cullin2) proteins in yeast cells. Furthermore, we validated the interaction between Mnk2 and VHL proteins in mammalian cells by co-immunoprecipitation analysis. Because the three proteins VHL, Rbx1 and Cul2 are all components of the CBCVHL ubiquitin ligase E3 complex, it has been shown that Mnk2 can interact with CBCVHL complex, and is probably one of the new substrates of the CBCVHL complex. Furthermore, during the interaction of Mnk2 with von Hippel-Lindau (VHL) tumor suppressor- binding protein 1 (VBP1), it appears that Mnk2 also joins to modulate cell shape as VBP1 plays an important role in the process of the maturation of the cytoskeleton and in the process of morphogenesis.     

An Autophosphorylation Site of the Protein Kinase SOS2 Is Important for Salt Tolerance in Arabidopsis

The protein kinase SOS2 （Salt Overly Sensitive 2） is essential for salt-stress signaling and tolerance in Arabidopsis. SOS2 is known to be activated by calcium-SOS3 and by phosphorylation at its activation loop. SOS2 is autophosphorylated in vitro, but the autophosphorylation site and its role in salt tolerance are not known. In this study, we identified an autophosphorylation site in SOS2 and analyzed its role in the responses of Arabidopsis to salt stress. Mass spectrometry analysis showed that Ser 228 of SOS2 is autophosphorylated. When this site was mutated to Ala, the autophosphorylation rate of SOS2 decreased. The substrate phosphorylation by the mutated SOS2 was also less than that by the wild-type SOS2. In contrast, changing Ser228 to Asp to mimic the autophosphorylation enhanced substrate phosphorylation by SOS2. Complementation tests in a sos2 mutant showed that the S228A but not the $228D mutation partially disrupted the function of SOS2 in salt tolerance. We also show that activation loop phosphorylation at Thr168 and autophosphorylation at Ser228 cannot substitute for each other, suggesting that both are required for salt tolerance. Our results indicate that Ser 228 of SOS2 is autophosphorylated and that this autophosphorylation is important for SOS2 function under salt stress.