hnRNP proteins interact with nascent Adenovirus RNA

By using Adenovirus 2 infected HeLa cells labeled during very brief pulses of (³H)Uridine, we have shown that nascent chains of heterogenous nuclear RNA (hnRNA) were already associated with proteins to form ribonucleoprotein particles (hnRNP). It was also shown that the small Ad2 specific VA RNA was not associated with these hnRNP.     

Protection against endotoxemia by HSP70 in rodent cardiomyocytes

Clinical and experimental studies have shown that myocardial dysfunction is an early event during endotoxemia or septic shock. Several reports have shown that rodents submitted to a mild heat shock become resistant to lipopolysaccharides (LPS) or sepsis. The most abundant of the heat shock proteins (HSP), the HSP70, has been postulated to be the principal mediator of the observed protection against endotoxemia. We have tested the hypothesis that a protective effect against endotoxemia is achievable by the increased presence of the HSP70 in rodent cardiomyocytes. We have found that a transgenic mouse line overexpressing the rat HSP70 gene in the heart exhibits an increased tolerance to LPS treatment (control estimated survival function [S(t)] = 0.538, transgenic S(t) = 0.787, P < 0.05). Interestingly, the increased presence of the HSP70 in the hearts of these mice results in a decrease in the activation of the inducible nitric oxide synthase (iNOS) after LPS treatment. We conclude that HSP70 protection against LPS is most probably mediated through the modulation of iNOS activation and the subsequent decreased synthesis of nitric oxide in cardiomyocytes.     

Transfection of human HSP27 in rodent cells: Absence of compensatory regulation between small heat shock proteins

1. 1. We examined rodent cells transfected with an expression plasmid encoding a human small heat shock protein for possible compensatory expression of endogenous heat shock genes. For these investigations, human hsp27 was transfected into CHO cells which express endogenous HSP25.

2. 2. Both endogenous HSP25 and transfected HSP27 were expressed and multiple phosphorylated isoforms were detected upon exposure to thermal stress.

3. 3. Levels of endogenous HSP70 and HSP25 did not appear to be altered by expression of the heterologous heat shock protein.

4. 4. These results suggest that compensatory interactions are not exhibited in the expression of the heat shock genes examined, and that independent regulation may exist not only between the large and small heat shock proteins, but also between individual small heat shock proteins as well.

     

Nuclear colocalization of cellular and viral myc proteins with HSP70 in myc-overexpressing cells.

The c-myc oncogene and its viral counterpart v-myc encode phosphoproteins which have been located within cell nuclei, excluding nucleoli. We have expressed the c-myc gene under the simian virus 40 early promoter and studied the distribution of its protein product in transient expression assays in COS, HeLa, and 293 cells. We found three distinct patterns of c-myc immunofluorescence in the transfected cells: one-third of the c-myc-positive cells displayed a diffuse nuclear distribution, and in two-thirds of the cells the c-myc fluorescence was accumulated either in small amorphous or in large multilobed phase-dense nuclear structures. Unexpectedly, these structures also stained for the HSP70 heat shock protein in both heat-shocked and untreated cells. Our results indicate that both transient and stable overexpression of either the c-myc or v-myc protein induces translocation of the endogenous HSP70 protein from the cytoplasm to the nucleus, where it becomes sequestered in structures containing the myc protein. Interestingly, the closely related N-myc protein does not stimulate substantial nuclear expression of the HSP70 protein. Studies with chimeric myc proteins revealed that polypeptide sequences encoded by the second exon of c-myc are involved in colocalization with HSP70.     

HSP70-related proteins in bovine skeletal muscle

Constitutive expression of HSP70-related proteins was detected in a variety of bovine tissues using a specific antibody. All tissues contained a 73 kilodalton protein. A lower molecular weight form (72 kilodaltons) that co-migrated on two-dimensional gels with the stressed-induced HSP70 was present in high levels in bovine skeletal muscle, but absent from rat skeletal muscle. Two-dimensional gel analysis revealed several isoforms for both the 73 and 72 kilodalton forms. Purification of HSP70-related proteins from bovine skeletal muscle, thymus gland and rat skeletal muscle demonstrated that the antibody recognized all the forms present in the tissue homogenates. The two proteins are similar but distinct as detected by one-dimensional peptide mapping. The lower molecular form was not present in fetal tissue but was detectable in newborn animals, suggesting that the levels are regulated during development.     

Cell cycle-dependent association of HSP70 with specific cellular proteins

In asynchronous populations of HeLa cells maintained at control or heat shock temperatures, HSP70 levels and its subcellular distribution exhibit substantial heterogeneity as demonstrated by indirect immunofluorescence with HSP70-specific monoclonal antibodies. Of particular interest is a subpopulation of cells in which the characteristic nuclear accumulation and nucleolar association of HSP70 is not detected after heat shock treatment. This apparent variation in the heat shock response is not observed when synchronized cells are examined. In this study, we demonstrate that three monoclonal antibodies to HSP70, in particular, do not detect nucleolar-localized HSP70 in heat-shocked G2 cells. This is not due to an inability of G2 cells to respond to heat shock as measured by increased HSP70 mRNA and protein synthesis, or due to a lack of accumulation of HSP70 after heat shock in G2. Rather the epitopes recognized by the various antibodies appear to be inaccessible, perhaps due to the association of HSP70 with other proteins. Non-denaturing immunoprecipitations with these HSP70-specific antibodies suggest that HSP70 may interact with other cellular proteins in a cell cycle-dependent manner.     

HSP90, HSP70, and GAPDH directly interact with the cytoplasmic domain of macrophage scavenger receptors.

The macrophage scavenger receptor (MSR) is a trimeric membrane protein which binds to modified low-density lipoprotein (LDL) and has been indicated in the development of atherosclerosis. It has recently been demonstrated that the N-terminal cytoplasmic domain of MSR has an important role in the efficient internalization and cell-surface expression of the receptor. This study shows that the N-terminal cytoplasmic domain in bovine was constructed using a peptide architecture technique in which the peptide chain was bundled at their C-terminus to yield a trimeric form and that this did not form an ordered structure. Furthermore, the binding proteins to the cytoplasmic domain of MSR were determined for the first time using a peptide affinity column. Sequence analyses of the specific binding proteins in bovine revealed that heat shock protein 90 (HSP90), heat shock protein 70 (HSP70), leucine aminopeptidase (LAP), adenocylhomocysteinase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were included. GST-pull-down assay and immunoprecipitation analyses on HSP90, HSP70, and GAPDH showed that all these proteins could bind to the cytoplasmic domain of MSR in vitro and in vivo. These proteins interact with the cytoplasmic domain directly and may have an effect on the functions of MSR such as internalization, cell-surface expression, and signal transduction.     

c-Src and HSP72 interact in ATP-depleted renal epithelial cells

Disruption of cell contact sites during ischemiacontributes to the loss of organ function in acute renal failure.Because prior heat stress protects cell contact sites in ATP-depleted renal epithelial cells in vitro, we hypothesized that heat shock protein 72 (HSP72), the major inducible cytoprotectant in mammalian cells, interacts with protein kinases that regulate cell-cell andcell-matrix interactions. ATP depletion increased the content ofTyr⁴¹⁶ Src, the activated form of this kinase. c-Srcactivation was associated with an increase in the tyrosinephosphorylation state of -catenin, paxillin, and vinculin, threec-Src substrate proteins that localize to and regulate cell contactsites. Prior heat stress inhibited c-Src activation and decreased thedegree of tyrosine phosphorylation of all three Src substrates duringATP depletion and/or early recovery. HSP72 coimmunoprecipitated withc-Src only in cells subjected to heat stress. ATP depletion markedlyincreased the interaction between HSP72 and c-Src, supporting thehypothesis that HSP72 regulates Src kinase activity. These resultssuggest that alterations in the tyrosine phosphorylation state ofproteins located at the cell-cell and cell-matrix interface mediate, at least in part, the functional state of these structures during ATPdepletion and may be modulated by interactions between HSP72 and c-Src.

     

人皮肤成纤维细胞在紫外线B照射后的TGF—β和HSP70表达

目的和方法：采用人体皮肤成纤维细胞,观察了紫外线照射后细胞TGF－β表达与HSP70表达水平的相关性。结果：①经不同剂量的紫外线B照射后,TGF－βmRNA表达与HSP70表达水平呈正相关（r=0.906);②应用抗TGF－βⅡ型受体抗体后,在紫外线B照射后细胞培养上清液中的TGF－β含量与细胞HSP70表达水平呈负相关（r=-0.995)。结论：在紫外线B照射诱导人皮肤成纤维细胞表达HSP70的反应过程另TGF－β参与其信号转导。     

Structural analysis of antiviral agents that interact with the capsid of human rhinoviruses

X-Ray diffraction data have been obtained for nine related antiviral agents ("WIN compounds") while bound to human rhinovirus 14 (HRV14). These compounds can inhibit both viral attachment to host cells and uncoating. To calculate interpretable electron density maps it was necessary to account for (1) the low (approximately 60%) occupancies of these compounds in the crystal, (2) the large (up to 7.9 A) conformational changes induced at the attachment site, and (3) the incomplete diffraction data. Application of a density difference map technique, which exploits the 20-fold noncrystallographic redundancy in HRV14, resulted in clear images of the HRV14:WIN complexes. A real-space refinement procedure was used to fit atomic models to these maps. The binding site of WIN compounds in HRV14 is a hydrophobic pocket composed mainly from residues that form the beta-barrel of VP1. Among rhinoviruses, the residues associated with the binding pocket are far more conserved than external residues and are mostly contained within regular secondary structural elements. Molecular dynamics simulations of three HRV14:WIN complexes suggest that portions of the WIN compounds and viral protein near the entrance of the binding pocket are more flexible than portions deeper within the beta-barrel.     

Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells

Heat shock induces in cells the synthesis of specific proteins called heat shock proteins (HSPs) and a transient state of thermotolerance. The putative role of one of the HSPs, HSP27, as a protective molecule during thermal stress has been directly assessed by measuring the resistance to hyperthermia of Chinese hamster and mouse cells transfected with the human HSP27 gene contained in plasmid pHS2711. One- and two-dimensional gel electrophoresis of [3H]leucine- and [32P]orthophosphate-labeled proteins, coupled with immunological analysis using Ha27Ab and Hu27Ab, two rabbit antisera that specifically recognize the hamster and the human HSP27 protein respectively, were used to monitor expression and inducibility of the transfected and endogenous proteins. The human HSP27 gene cloned in pHS2711 is constitutively expressed in rodent cells, resulting in accumulation of the human HSP27 and all phosphorylated derivatives. No modification of the basal or heat-induced expression of endogenous HSPs is detected. The presence of additional HSP27 protein provides immediate protection against heat shock administered 48 h after transfection and confers a permanent thermoresistant phenotype to stable transfectant Chinese hamster and mouse cell lines. Mild heat treatment of the transfected cells results in an induction of the full complement of the endogenous heat shock proteins and a small increase in thermoresistance, but the level attained did not surpass that of heat-induced thermotolerant control cells. These results indicate that elevated levels of HSP27 is sufficient to give protection from thermal killing. It is concluded that HSP27 plays a major role in the increased thermal resistance acquired by cells after exposure to HSP inducers.     

Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells.

Members of the human heat shock (HSP) family of related proteins are involved in the intracellular folding, transport, and assembly of proteins and protein complexes. We have observed that human heat shock protein 70 (HSP70) is associated with the capsid precursor P1 of poliovirus and coxsackievirus B1 in infected HeLa cells. Antiserum generated against HSP70 coimmunoprecipitated the poliovirus protein P1, an intermediate in capsid assembly. Similarly, alpha-virion serum coimmunoprecipitated HSP70 from virus-infected cell extracts, but not from mock-infected cell extracts. The HSP70-P1 complex was stable in high-salt medium but was sensitive to incubation with 2 mM ATP, which is a characteristic of other known functional complexes between HSP70 and cellular proteins. The P1 in the complex was predominantly newly synthesized, and the half-life of complexed P1 was nearly twice as long as that of total P1. The HSP70-P1 complex was found to sediment at 3S to 6S, suggesting that it may be part of, or a precursor to, the "5S promoter particles" thought to be an assembly intermediate of picornaviruses. The finding that HSP70 was associated with the capsid precursors of at least two enteroviruses may suggest a functional role of these complexes in the viral life cycles.     

In vivo and in vitro association of hsc70 with polyomavirus capsid proteins.

Members of the 70-kDa family of cellular stress proteins assit in protein folding by preventing inappropriate intra- and intermolecular interactions during normal protein synthesis and transport and when cells are exposed to a variety of environmental stresses. During infection of A31 mouse fibroblasts with polyomavirus, the constitutive form of hsp70, hsc70, coimmunoprecipitated with all three viral capsid proteins (VP1, VP2, and VP3). In addition, the subcellular location of hsc70 changed from cytoplasmic to nuclear late in polyomavirus infection, coincident with the nuclear localization of the viral capsid proteins. VP1 and VP2 expressed in Sf9 insect cells with recombinant baculovirus vectors also coimmunoprecipitated with an hsp70-like protein, and VP1 expressed in Escherichia coli coimmunoprecipitated with the hsp70 homolog DnaK. Capsid proteins expressed by in vitro translation coimmunoprecipitated with the hsc70 protein present in the reticulocyte translation extract. Therefore, the polyomavirus capsid proteins associate with hsc70 during virus infection as well as in recombinant protein expression systems. This association may play a role in preventing the premature assembly of capsids in the cytosol and/or in facilitating the nuclear transport of capsid protein complexes.     

Study of the properties of exogenous recombinant human heat shock protein HSP70 using a rodent sepsis model

The protective effects of the exogenous recombinant human heat shock protein with a molecular mass of 70 kDa (rhHSP70) were demonstrated on blood laboratory indices following exogenous injection into Sprague-Dawley rats with induced gram-positive sepsis. Acute toxicity of the protein was studied on CD-1 rats.     

Precursor proteins in transit through mitochondrial contact sites interact with hsp70 in the matrix

We previously reported that hsp70 in the mitochondrial matrix (mt-hsp 70 = Ssclp) is required for import of precursor proteins destined for the matrix or intermembrane space. Here we show that mt-hsp70 is also needed for the import of mitochondrial inner membrane proteins. In particular, the precursor of ADP/ATP carrier that is known not to interact with hsp60 on its assembly pathway requires functional mt-hsp70 for import, suggesting a general role of mt-hsp70 in membrane translocation of precursors. Moreover, a precursor arrested in contact sites was specifically co-precipitated with antibodies directed against mt-hsp70. We conclude that mt-hsp70 is directly involved in the translocation of many, if not all, precursor proteins that are transported across the inner membrane.