Human Tim/Timeless-interacting protein, Tipin, is required for efficient progression of S phase and DNA replication checkpoint

Tipin was originally isolated as a protein interacting with Timeless/Tim1/Tim (Tim), which is known to be involved in both circadian rhythm and cell cycle checkpoint regulation. The endogenous Tim and Tipin proteins in human cells, interacting through the N-terminal segment of each molecule, form a complex throughout the cell cycle. Tipin and Tim are expressed in the interphase nuclei mostly at constant levels during the cell cycle, and small fractions are recovered in the chromatin-enriched fractions during S phase. Depletion of endogenous Tipin results in reduced growth rate, and this may be due in part to inefficient progression of S phase and DNA synthesis. Knockdown of Tipin induces radioresistant DNA synthesis and inhibits phosphorylation of Chk1 kinase caused by replication stress, as was observed with that of Tim. Knockdown of Tipin or Tim results in reduced protein level and relocation to the cytoplasm of the respective binding partner, suggesting that the complex formation may be required for stabilization and nuclear accumulation of both proteins. Furthermore, both Tipin and Tim may facilitate the accumulation of Claspin in the nuclei under replication stress, whereas nuclear localization of Tipin and Tim is unaffected by Claspin. Our results indicate that mammalian Tipin is a checkpoint mediator that cooperates with Tim and may regulate the nuclear relocation of Claspin in response to replication checkpoint.     

Exercise does not alter subcellular localization, but increases phosphorylation of insulin-signaling proteins in human skeletal muscle

The subcellular localization of insulin signaling proteins is altered by various stimuli such as insulin, insulin-like growth factor I, and oxidative stress and is thought to be an important mechanism that can influence intracellular signal transduction and cellular function. This study examined the possibility that exercise may also alter the subcellular localization of insulin signaling proteins in human skeletal muscle. Nine untrained males performed 60 min of cycling exercise (approximately 67% peak pulmonary O2 uptake). Muscle biopsies were sampled at rest, immediately after exercise, and 3 h postexercise. Muscle was fractionated by centrifugation into the following crude fractions: cytosolic, nuclear, and a high-speed pellet containing membrane and cytoskeletal components. Fractions were analyzed for protein content of insulin receptor, insulin receptor substrate (IRS)-1 and -2, p85 subunit of phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3). There was no significant change in the protein content of the insulin signaling proteins in any of the crude fractions after exercise or 3 h postexercise. Exercise had no significant effect on the phosphorylation of IRS-1 Tyr612 in any of the fractions. In contrast, exercise increased (P < 0.05) the phosphorylation of Akt Ser473 and GSK-3alpha/beta Ser9/21 in the cytosolic fraction only. In conclusion, exercise can increase phosphorylation of downstream insulin signaling proteins specifically in the cytosolic fraction but does not result in changes in the subcellular localization of insulin signaling proteins in human skeletal muscle. Change in the subcellular protein localization is therefore an unlikely mechanism to influence signal transduction pathways and cellular function in skeletal muscle after exercise.     

Specific changes of Ras GTPase-activating protein (GAP) and a GAP-associated p62 protein during calcium-induced keratinocyte differentiation.

Induction of tyrosine phosphorylation occurs as an early and specific event in keratinocyte differentiation. A set of tyrosine-phosphorylated substrates which transduce mitogenic signals by tyrosine kinases has previously been identified. We show here that of these substrates, the Ras GTPase-activating protein, GAP, is specifically affected during calcium-induced keratinocyte differentiation. As early as 10 min after calcium addition to cultured primary mouse keratinocytes, GAP associates with tyrosine-phosphorylated proteins and translocates to the membrane. In addition, a GAP-associated protein of approximately 62 kDa (p62) becomes rapidly and heavily tyrosine phosphorylated in both membrane and cytosolic fractions. This protein corresponds to the major tyrosine-phosphorylated protein that is induced in differentiating keratinocytes as early as 5 min after calcium addition. p62 phosphorylation was not observed after exposure of these cells to epidermal growth factor, phorbol ester, or transforming growth factor beta. In contrast, PLC gamma and P13K were tyrosine phosphorylated after epidermal growth factor, but not calcium, stimulation. Thus, changes of Ras GAP and an associated p62 protein occur as early and specific events in keratinocyte differentiation and appear to involve a calcium-induced tyrosine kinase.     

Ash/Grb-2, a SH2/SH3-containing protein, couples to signaling for mitogenesis and cytoskeletal reorganization by EGF and PDGF.

The Src homology (SH) region 2 binds to phosphorylated tyrosine residues and SH3 domains may interact with cytoskeletal molecules and GTPase-activating proteins for Rho/Rac proteins (the small GTP-binding proteins related to Ras). The recently cloned Ash/Grb-2 protein, a 25-28 kDa molecule composed entirely of SH2 and SH3 domains, is a mammalian homolog of the Caenorhabditis elegans Sem-5 protein, which communicates between a receptor protein tyrosine kinase and a Ras protein. In the present study the function of Ash/Grb-2 was investigated by microinjecting cells with an anti-Ash antibody. The antibody abolished both S phase entry and the reorganization of actin assembly to ruffle formation upon stimulation with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). On the other hand, anti-Ash antibody had no effect on S phase entry or actin stress fiber formation induced by either serum or lysophosphatidic acid. Since the induction of DNA synthesis, ruffle induction and stress fiber formation involve a function of Ras, Rac activation and Rho activation respectively, the findings strongly suggest that Ash plays a critical role in the signaling of both pathways downstream from growth factor receptors to Ras and Rac. Consistent with this, Ash co-precipitated with EGF receptor from EGF-stimulated cells. Other proteins of approximately 21, 29, 135 and 160 kDa were also detected in the anti-Ash antibody immunoprecipitates, suggesting a role of Ash as a linker molecule in signal transduction downstream of growth factor receptors.     

Regulation of HSP25 expression and phosphorylation in functionally overloaded rat plantaris and soleus muscles.

Functional overload (FO) is a powerful inducer of muscle hypertrophy and both oxidative and mechanical stress in muscle fibers. Heat shock protein 25 (HSP25) may protect against both of these stressors, and its expression can be regulated by changes in muscle loading and activation. The primary purpose of the present study was to test the hypothesis that chronic FO increases HSP25 expression and phosphorylation (pHSP25) in hypertrophying rat hindlimb muscle. HSP25 and pHSP25 levels were quantified in soluble and insoluble fractions of the soleus and plantaris to determine whether 3 or 7 days of FO increase translocation of HSP25 and/or pHSP25 to the insoluble fraction. p38 protein and phosphorylation (p-p38) was measured to determine its association with changes in pHSP25. HSP25 mRNA showed time-dependent increases in both the soleus and plantaris with FO. Three or seven days of FO increased HSP25 and pHSP25 in the soluble fraction in both muscles, with a greater response in the plantaris. In the insoluble fraction, HSP25 was increased after 3 or 7 days in both muscles, whereas pHSP25 was only increased in the 7-day plantaris. p38 and p-p38 increased in the plantaris at both time points. In the soleus, p-p38 only increased after 7 days. These results show that FO is associated with changes in HSP25 expression and phosphorylation and suggest its role in the remodeling that occurs during muscle hypertrophy. Increases in HSP25 in the insoluble fraction suggest that it may help to stabilize actin and/or other cytoskeletal proteins during the stress of muscle remodeling.     

Protein synthesis and degradation in a leucine auxotroph of Escherichia coli.

The synthesis and degradation of the soluble and sodium dodecyl sulfate-(SDS)-solubilized protein fractions of Escherichia coli were studied in both growing and nongrowing cultures. When separated according to molecular weight on SDS-polyacrylamide gels, the proteins of both fractions of growing cells undergo no measureable differential synthesis or degradation during logarithmic growth. However, when a leucine auxotroph is suspended in medium containing 5.3 muM leucine (a level that will not sustain growth), the SDS-solubilized protein of such a nongrowing culture shows a rapid synthesis of two protein components (32,000 and 12,000 daltons) found only in the out membrane.     

Role of cell-wall biogenesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The involvement of cell-wall polymer synthesis in auxin-mediated elongation of coleoptile segments from Zea mays L. was investigated with particular regard to the growth-limiting outer epidermis. There was no effect of indole acetic acid (IAA) on the incorporation of labeled glucose into the major polysaccharide wall fractions (cellulose, hemicellulose) within the first 2 h of IAA-induced growth. 2,6-Dichlorobenzonitrile inhibited cellulose synthesis strongly but had no effect on IAA-induced segment elongation even after a pretreatment period of 24 h, indicating that the growth response is independent of the apposition of new cellulose microfibrils at the epidermal cell wall. The incorporation of labeled leucine into total and cell-wall protein of the epidermis was promoted by IAA during the first 30 min of IAA-induced growth. Inhibition of IAA-induced growth by protein and RNA-synthesis inhibitors (cycloheximide, cordycepin) was accompanied by an inhibition of leucine incorporation into the epidermal cell wall during the first 30 min of induced growth but had no effect on the concomitant incorporation of monosaccharide precursors into the cellulose or hemicellulose fractions of this wall. It is concluded that at least one of the epidermal cell-wall proteins fulfills the criteria for a growth-limiting protein induced by IAA at the onset of the growth response. In contrast, the synthesis of the polysaccharide wall fractions cellulose and hemicellulose, as well as their transport and integration into the growing epidermal wall, appears to be independent of growth-limiting protein and these processes are therefore no part of the mechanism of growth control by IAA.Abbreviations CHI cycloheximide - COR cordycepin - DCB 2,6-dichlorobenzonitrile - GLP growth-limiting protein(s) - IAA indole-3-acetic acid     

Mitogen activation induces the enhanced synthesis of two heat-shock proteins in human lymphocytes

We have used mitogenic lectin (PHA) and a monoclonal antibody (OKT3) to stimulate human peripheral blood (G0) lymphocytes, in the presence of monocytes, and have found two major preferentially synthesized proteins, 73 and 95 kD, which are induced by the mitogens. The elevated synthesis of both proteins begins approximately 4-6 h after mitogen addition (early to mid G0/G1) before entry into first S phase. Maximum synthesis of both proteins is reached by 12 h after mitogen addition when P95 synthesis represents approximately 4%, and P73 approximately 2%, of the total protein synthesis, compared with less than 0.5% for each protein in cells cultured without mitogen. Thus, the proteins appear to be major components of activated cells. We find that both P73 and P95 are induced by heat stress as well as mitogenic stimulation. The induction of the proteins is not affected by either deleting glucose from the culture media or, alternatively, by supplementing it. Using polyclonal antibodies prepared to each of the proteins isolated from mitogen activated cells and monoclonal antibodies that were raised to heat shock proteins, we are able to show that P95 is electrophoretically and immunologically identical to the HSP 90 induced by heat stress. P73 is one of the 70 kD HSPs, (termed HSC 70; Pelham, H. R. B. 1986. Cell. 46: 959-961), but is different from the most strongly heat inducible form of HSP 70 (72 kD). The distribution of both proteins in subcellular fractions of mitogen activated lymphocytes is similar to the reported localization of the respective HSP's in other cell types. The results suggest that HSP 90 and HSC 70 may have functional roles in stress response and growth processes of human lymphocytes.     

Effects of platelet-derived growth factor on degradation, nuclear translocation of nonhistone proteins, and DNA synthesis.

Stimulation of resting normal rat kidney fibroblasts, prelabeled with [3H]leucine, by platelet-derived growth factor (PDGF) caused inhibition of cellular protein degradation and a parallel increased nuclear translocation of 3H-labeled nonhistone proteins (3H-NHP) and DNA synthesis. Nuclear translocation of these proteins was independent of protein synthesis. Fractionation of the nuclear 3H-NHP in a pH gradient of 2.5-6.5 showed that the protein fractions with a high degree of proteolysis in resting cells corresponded to the protein fractions with a high extent of translocation in stimulated cells, suggesting that degradation and translocation of these proteins may be related. PDGF inhibited cellular uptake of [3H]chloroquine, suggesting that PDGF inhibits NHP degradation via the lysosomal pathway. These observations support the hypothesis that PDGF induces NHP translocation to the nucleus by inhibiting lysosomal degradation of these proteins.     

Phosphorylation events catalyzed by major cell signaling proteins differ in response to thermal and osmotic stress among native (Mytilus californianus and Mytilus trossulus) and invasive (Mytilus galloprovincialis) species of mussels

Sharp environmental gradients encountered within the intertidal zone have driven the evolution of physiological adaptations that allow its inhabitants to maintain cellular function in the presence of fluctuating abiotic factors. These adaptations are mediated by gene-regulatory networks that, despite their inherent complexity, must remain evolvable and capable of responding to different selection pressures associated with specific ecological niches. Phosphorylation events catalyzed by cell-signaling enzymes represent a parsimonious mechanism to integrate new functional or regulatory properties into these gene-regulatory networks. In this study, proteins phosphorylated on consensus sequences for protein kinases A, B, and C; cyclin-dependent kinases; and mitogen-activated protein kinases, as well as the abundance of phosphorylated stress-activated protein kinase (phospho-SAPK/JNK), were quantified in order to ascertain whether phosphorylation events are divergent among native (Mytilus californianus and Mytilus trossulus) and invasive (Mytilus galloprovincialis) species of mussels that differ in their tolerance toward environmental stress. Abundances of phosphorylated substrate proteins for each of the major signaling proteins that were investigated, as well as the abundance of phospho-SAPK/JNK, differed both within and between species during thermal and osmotic stress. These data suggest that modulating protein function via phosphorylation may be an important mechanism to integrate novel properties into stress-regulatory networks. In turn, differential phosphorylation during environmental stress may contribute to species-specific tolerances toward abiotic stress, interspecies dynamics, and biogeographic patterns in Mytilus congeners.     

Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading

Lack of neurite growth in optic nerve explants in vitro has been suggested to be due to nonpermissive substrate properties of higher vertebrate central nervous system (CNS) white matter. We have searched for surface components in CNS white matter, which would prevent neurite growth. CNS, but not peripheral nervous system (PNS) myelin fractions from rat and chick were highly nonpermissive substrates in vitro. We have used an in vitro spreading assay with 3T3 cells to quantify substrate qualities of membrane fractions and of isolated membrane proteins reconstituted in artificial lipid vesicles. CNS myelin nonpermissiveness was abolished by treatment with proteases and was not associated with myelin lipid. Nonpermissive proteins were found to be membrane bound and yielded highly nonpermissive substrates upon reconstitution into liposomes. Size fractionation of myelin protein by SDS-PAGE revealed two highly nonpermissive minor protein fractions of Mr 35 and 250-kD. Removal of 35- and of 250-kD protein fractions yielded a CNS myelin protein fraction with permissive substrate properties. Supplementation of permissive membrane protein fractions (PNS, liver) with low amounts of 35- or of 250-kD CNS myelin protein was sufficient to generate highly nonpermissive substrates. Inhibitory 35- and 250-kD proteins were found to be enriched in CNS white matter and were found in optic nerve cell cultures which contained highly nonpermissive, differentiated oligodendrocytes. The data presented demonstrate the existence of membrane proteins with potent nonpermissive substrate properties. Distribution and properties suggest that these proteins might play a crucial inhibitory role during development and regeneration in CNS white matter.     

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells.

The effects of exogenous proteins on the incorporation of [3H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A2, 5123C, and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10-20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100-400 microgram/ml. In experiments with lower cell concentrations, a 60% or greater increase in [3H]-thymidine incorporation could be obtained with total calf thymus histone and with F1 and arginine-rich histones from rat liver. At concentrations of 1-2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80-90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10 microgram/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis.     

Two-dimensional electrophoresis database of Listeria monocytogenes EGDe proteome and proteomic analysis of mid-log and stationary growth phase cells

Listeria monocytogenes is the causative agent of listeriosis, one of the most significant foodborne diseases in industrialized countries. The complete genome of the L. monocytogenes EGDe strain, belonging to the serogroup 1/2a, has been sequenced and is comprised of 2853 open reading frames. The objective of the current study was to construct a two-dimensional (2-D) database of the proteome of this strain. The soluble protein fractions of the microorganism were recovered either in the mid-log or in the stationary phase of growth at 37 degrees C. These fractions were analyzed by 2-D electrophoresis (2-DE), using immobilized pH gradient strips of various pH values (3-10, 3-6, and 5-8) for the first-dimensional separations and 12.5% acrylamide gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 201 protein spots corresponding to 126 different proteins were identified by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). The 2-DE maps presented here provide a first basis for further investigations of protein expression in L. monocytogenes. In this way, the comparison of proteome between cells in the exponential or stationary phase of growth at 37 degrees C allowed us to characterize 161 variations in protein spot intensity, of which 38 were identified. Among the differentially expressed proteins were ribosomal proteins (RpsF, RplJ, and RpmE), proteins involved in cellular metabolism (GlpD, PdhD, Pgm, Lmo1372, Lmo2696, and Lmo2743) or in stress adaptation (GroES and ferritin), a fructose-specific phosphotransferase enzyme IIB (Lmo0399) and different post-translational modified forms of listeriolysin (LLO).     

Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks

In response to replicative stress, cells relocate and activate DNA repair and cell cycle arrest proteins such as replication protein A (RPA, a three subunit protein complex required for DNA replication and DNA repair) and the MRN complex (consisting of Mre11, Rad50, and Nbs1; involved in DNA double-strand break repair). There is increasing evidence that both of these complexes play a central role in DNA damage recognition, activation of cell cycle checkpoints, and DNA repair pathways. Here we demonstrate that RPA and the MRN complex co-localize to discrete foci and interact in response to DNA replication fork blockage induced by hydroxyurea (HU) or ultraviolet light (UV). Members of both RPA and the MRN complexes become phosphorylated during S-phase and in response to replication fork blockage. Analysis of RPA and Mre11 in fractionated lysates (cytoplasmic/nucleoplasmic, chromatin-bound, and nuclear matrix fractions) showed increased hyperphosphorylated-RPA and phosphorylated-Mre11 in the chromatin-bound fractions. HU and UV treatment also led to co-localization of hyperphosphorylated RPA and Mre11 to discrete detergent-resistant nuclear foci. An interaction between RPA and Mre11 was demonstrated by co-immunoprecipitation of both protein complexes with anti-Mre11, anti-Rad50, anti-NBS1, or anti-RPA antibodies. Phosphatase treatment with calf intestinal phosphatase or lambda-phosphatase not only de-phosphorylated RPA and Mre11 but also abrogated the ability of RPA and the MRN complex to co-immunoprecipitate. Together, these data demonstrate that RPA and the MRN complex co-localize and interact after HU- or UV-induced replication stress and suggest that protein phosphorylation may play a role in this interaction.     

Inner Nuclear Envelope Proteins SUN1 and SUN2 Play a Prominent Role in the DNA Damage Response

The DNA damage response (DDR) and DNA repair are critical for maintaining genomic stability and evading many human diseases [1, 2]. Recent findings indicate that accumulation of?SUN1, a nuclear envelope (NE) protein, is a significant pathogenic event in Emery-Dreifuss muscular dystrophy and Hutchinson-Gilford progeria syndrome, both caused by mutations in LMNA [3, 4]. However, roles of mammalian SUN proteins in mitotic cell division and genomic stability are unknown. Here we report that the inner NE proteins SUN1 and SUN2 may play a redundant role in DDR. Mouse embryonic fibroblasts from Sun1(-/-)Sun2(-/-) mice displayed premature proliferation arrest in S phase of cell cycle, increased apoptosis and DNA damage, and decreased perinuclear heterochromatin, indicating genome instability. Furthermore, activation of ATM and H2A.X, early events in?DDR, were impaired in Sun1(-/-)Sun2(-/-) fibroblasts. A biochemical screen identified interactions between SUN1 and SUN2 and DNA-dependent protein kinase (DNAPK) complex that functions in DNA nonhomologous end joining repair and possibly in DDR [2, 5, 6]. Knockdown of DNAPK reduced ATM activation in NIH 3T3 cells, consistent with a potential role of SUN1- and SUN2-DNAPK interaction during DDR. SUN1 and SUN2 could affect DDR by localizing certain nuclear factors to the NE or by mediating communication between nuclear and cytoplasmic events.     

Overexpression of AtLEA3-3 confers resistance to cold stress in Escherichia coli and provides enhanced osmotic stress tolerance and ABA sensitivity in Arabidopsis thaliana

Previous studies have shown that the late embryogenesis abundant (LEA) group 3 proteins significantly respond to changes in environmental conditions. However, reports that demonstrate their biological role, especially in Arabidopsis, are notably limited. This study examines the functional roles of the Arabidopsis LEA group 3 proteins AtLEA3-3 and AtLEA3-4 in abiotic stress and ABA treatments. Expression of AtLEA3-3 and AtLEA3-4 is upregulated by ABA, high salinity, and osmotic stress. Results on the ectopic expression of AtLEA3-3 and AtLEA3-4 in E. coli suggest that both proteins play important roles in resistance to cold stress. Overexpression of AtLEA3-3 in Arabidopsis (AtLEA3-3-OE) confers salt and osmotic stress tolerance that is characterized during germination and early seedling establishment. However, AtLEA3-3-OE lines show sensitivity to ABA treatment during early seedling development. These results suggest that accumulation of AtLEA3-3 mRNA and/or proteins may help heterologous ABA re-initiate second dormancy during seedling establishment. Analysis of yellow fluorescent fusion proteins localization shows that AtLEA3-3 and AtLEA3-4 are mainly distributed in the ER and that AtLEA3-3 also localizes in the nucleus, and in response to salt, mannitol, cold, or BFA treatments, the localization of AtLEA3-3 and AtLEA3-4 is altered and becomes more condensed. Protein translocalization may be a positive and effective strategy for responding to abiotic stresses. Taken together, these results suggest that AtLEA3-3 has an important function during seed germination and seedling development of Arabidopsis under abiotic stress conditions.     

Discovery of Prognostic Biomarker Candidates of Lacunar Infarction by Quantitative Proteomics of Microvesicles Enriched Plasma

Background

Lacunar infarction (LACI) is a subtype of acute ischemic stroke affecting around 25% of all ischemic stroke cases. Despite having an excellent recovery during acute phase, certain LACI patients have poor mid- to long-term prognosis due to the recurrence of vascular events or a decline in cognitive functions. Hence, blood-based biomarkers could be complementary prognostic and research tools.

Methods and Finding

Plasma was collected from forty five patients following a non-disabling LACI along with seventeen matched control subjects. The LACI patients were monitored prospectively for up to five years for the occurrence of adverse outcomes and grouped accordingly (i.e., LACI-no adverse outcome, LACI-recurrent vascular event, and LACI-cognitive decline without any recurrence of vascular events). Microvesicles-enriched fractions isolated from the pooled plasma of four groups were profiled by an iTRAQ-guided discovery approach to quantify the differential proteome. The data have been deposited to the ProteomeXchange with identifier PXD000748. Bioinformatics analysis and data mining revealed up-regulation of brain-specific proteins including myelin basic protein, proteins of coagulation cascade (e.g., fibrinogen alpha chain, fibrinogen beta chain) and focal adhesion (e.g., integrin alpha-IIb, talin-1, and filamin-A) while albumin was down-regulated in both groups of patients with adverse outcome.

Conclusion

This data set may offer important insight into the mechanisms of poor prognosis and provide candidate prognostic biomarkers for validation on larger cohort of individual LACI patients.