All roads lead to Rome (but some may be harder to travel): SRP-independent translocation into the endoplasmic reticulum

Abstract

Translocation into the endoplasmic reticulum (ER) is the first biogenesis step for hundreds of eukaryotic secretome proteins. Over the past 30 years, groundbreaking biochemical, structural and genetic studies have delineated one conserved pathway that enables ER translocation- the signal recognition particle (SRP) pathway. However, it is clear that this is not the only pathway which can mediate ER targeting and insertion. In fact, over the past decade, several SRP-independent pathways have been uncovered, which recognize proteins that cannot engage the SRP and ensure their subsequent translocation into the ER. These SRP-independent pathways face the same challenges that the SRP pathway overcomes: chaperoning the preinserted protein while in the cytosol, targeting it rapidly to the ER surface and generating vectorial movement that inserts the protein into the ER. This review strives to summarize the various mechanisms and machineries which mediate these stages of SRP-independent translocation, as well as examine why SRP-independent translocation is utilized by the cell. This emerging understanding of the various pathways utilized by secretory proteins to insert into the ER draws light to the complexity of the translocational task, and underlines that insertion into the ER might be more varied and tailored than previously appreciated.     

Molecular Chaperones HscA/Ssq1 and HscB/Jac1 and Their Roles in Iron-Sulfur Protein Maturation

ABSTRACT

Genetic and biochemical studies have led to the identification of several cellular pathways for the biosynthesis of iron-sulfur proteins in different organisms. The most broadly distributed and highly conserved system involves an Hsp70 chaperone and J-protein co-chaperone system that interacts with a scaffold-like protein involved in [FeS]-cluster preassembly. Specialized forms of Hsp70 and their co-chaperones have evolved in bacteria (HscA, HscB) and in certain fungi (Ssq1, Jac1), whereas most eukaryotes employ a multifunctional mitochondrial Hsp70 (mtHsp70) together with a specialized co-chaperone homologous to HscB/Jac1. HscA and Ssq1 have been shown to specifically bind to a conserved sequence present in the [FeS]-scaffold protein designated IscU in bacteria and Isu in fungi, and the crystal structure of a complex of a peptide containing the IscU recognition region bound to the HscA substrate binding domain has been determined. The interaction of IscU/Isu with HscA/Ssq1 is regulated by HscB/Jac1 which bind the scaffold protein to assist delivery to the chaperone and stabilize the chaperone-scaffold complex by enhancing chaperone ATPase activity. The crystal structure of HscB reveals that the N-terminal J-domain involved in regulation of HscA ATPase activity is similar to other J-proteins, whereas the C-terminal domain is unique and appears to mediate specific interactions with IscU. At the present time the exact function(s) of chaperone-[FeS]-scaffold interactions in iron-sulfur protein biosynthesis remain(s) to be established. In vivo and in vitro studies of yeast Ssq1 and Jac1 indicate that the chaperones are not required for [FeS]-cluster assembly on Isu. Recent in vitro studies using bacterial HscA, HscB and IscU have shown that the chaperones destabilize the IscU[FeS] complex and facilitate cluster delivery to an acceptor apo-protein consistent with a role in regulating cluster release and transfer. Additional genetic and biochemical studies are needed to extend these findings to mtHsp70 activities in higher eukaryotes.     

C-terminus of Hsc70-interacting protein (CHIP) enhances stemness properties of human Wharton’s jelly mesenchymal stem cell

Mesenchymal stem cells are an attractive source of multipotent cells in part because they are easy to obtain. Several E3 ligases regulate the stability and functions of various factors in different adult stem cells through the ubiquitylation pathway. We investigated the C-terminus of Hsc70-interacting protein (CHIP) E3 ligase that regulates pluripotency of human Wharton’s jelly mesenchymal stem cells (hWJMSC). We found that CHIP increases protein kinase B (Akt) phosphorylation by decreased expression of phosphatase and tensin homolog (PTEN), which suggests improvement of the survival pathway by CHIP over-expression. We also found that increased CHIP expression induced Sox2 and NANOG, which can promote stem cell self-renewal and prevent oxidative stress-induced senescence of hWJMSC by decreased p21. We found that CHIP could be used to enhance the multiple functions of hWJMSC.     

Constitutive heat shock protein 70 interacts with α-enolase and protects cardiomyocytes against oxidative stress

Abstract

Constitutive heat shock protein 70 (Hsc70) is a molecular chaperone that has been shown to protect cardiomyocytes against oxidative stress. However, the molecular mechanism responsible for this protection remains uncertain. To understand the mechanism associated with the myocardial protective role of Hsc70, we have embarked upon a systematic search for Hsc70-interacting proteins. Using adenosine diphosphate (ADP) affinity chromatography and mass spectrometry, we have identified α-enolase, a rate-limiting enzyme in glycolysis, as a novel Hsc70-interacting protein in the myocardium of both sham and myocardial ischemia-reperfused Sprague–Dawley rat hearts. This interaction was confirmed by co-immunoprecipitation (IP) assays in the myocardial tissues and H9c2 cardiomyocytes and protein overlay assay (POA). It was further shown that Hsc70-overexpression alleviated the H₂O₂-induced decrease of α-enolase activity and cell damage, and Hsc70 deficiency aggravated the decrease of α-enolase activity and cell damage in H₂O₂ treated H9c2 cells. Our research suggests that the protective effect of Hsc70 on the cardiomyocytes against oxidative stress is partly associated with its interaction with α-enolase.     

An investigation into the potential use of serum Hsp70 as a novel tumour biomarker for Hsp90 inhibitors

Hsp90 inhibitors are under investigation in multiple human clinical trials for the treatment of cancers, including myeloma, breast cancer, prostate, lung, melanoma, gastrointestinal stromal tumour and acute myeloid leukaemia. The pharmacodynamic activity of Hsp90 inhibitors in the clinic is currently assessed by Hsp70 induction in peripheral blood mononuclear cells using Western blot analysis, a method that is laborious, semiquantitative and difficult to implement in the clinic. Since Hsp70 was reported to be secreted by tumour cells and elevated in sera of cancer patients, serum Hsp70 has been evaluated as a potentially more robust, easily and reproducibly measured biomarker of Hsp90 inhibition as an alternative to cytosolic Hsp70. A highly sensitive and specific electrochemiluminescent ELISA was developed to measure serum Hsp70 and employed to evaluate Hsp70 levels in both ex vivo and xenograft samples. In ex vivo studies, maximal secretion of Hsp70 by tumour cells was observed between 48 and 72?h after exposure to Hsp90 inhibitors. In in vivo studies a 3–4-fold increase in serum Hsp70 was observed following treatment with BIIB021 in tumour-bearing mice. Strikingly, secreted Hsp70 was detectable in mice transplanted with human tumours but not in naive mice indicating a direct origination from the transplanted tumours. Analysis of clinical samples revealed low baseline levels (2–15?ng ml^?1) of Hsp70 in the serum of cancer patients and normal donors. Together these findings in laboratory studies and archived cancer patient sera suggest that serum Hsp70 could be a novel biomarker to assess reliably the pharmacological effects of Hsp90 inhibitors in clinical trials, especially under conditions where collection of tumour biopsies is not feasible.     

Toxicity of cypermethrin: hsp70 as a biomarker of response in transgenic Drosophila

Heat shock protein induction is often associated with a cellular response to a harmful environment or to adverse life conditions. The main aims of our study were (1) to evaluate the cytotoxic potential of cypermethrin; and (2) to investigate the suitability of stress-induced heat shock protein Hsp70 as a biomarker for environmental pollutants in transgenic Drosophila melanogaster (Hsp70-lacZ)Bg⁹. Different concentrations of cypermethrin (0.002, 0.2, 0.5 and 50.0 p.p.m.) were mixed with food. Third instar larvae of transgenic Drosophila melanogaster were allowed to feed on these mixtures for different time intervals (2, 4, 6, 12, 24 and 48h). Following feeding, hsp70 induction and tissue damage were evaluated. In the highest concentration treatment group (50 p.p.m.), 100% larval mortality was recorded after 12 h exposure. Hsp70 was found to be induced even at the lowest concentration (0.002 p.p.m.) of the insecticide, while tissue damage was observed in the larvae exposed for 48 h. While an insignificant decline in hsp70 expression was observed in the larvae exposed to cypermethrin at a dietary concentration of 0.002 p.p.m. after 48 h compared with those exposed for 24 h, in the next two higher concentrations of the toxicant, a similar but significant decline in hsp70 expression was evident in the exposed larvae after 48 h. The present study reveals the cytotoxic potential of cypermethrin and further proposes that hsp70 induction in transgenic Drosophila melanogaster could be used as a sensitive biomarker in risk assessment.     

Sine Wave Extremely Low Frequency Magnetic Fields Protect Chick Embryos Against UV-Induced Death

Four-day-old chicken embryos were exposed to extremely low frequency (ELF) magnetic fields (MF) prior to UV exposure (75 min, predominantly UV-C, 0.4 mW/cm²) to investigate possible MF-mediated protection against lethal effects of UV. The UV exposure typically resulted in a 20% survival rate (as judged by beating hearts) in sham-exposed embryos 3 h postexposure. In contrast, exposure to a 50 (10, 50, or 100 µT) or 60 Hz (10 µT) vertical MF caused a significant increase in survival rate, observed only 30 min after UV exposure. No difference in protection levels was seen between these exposure intensities. A horizontal 50 Hz MF (10, 50, or 100 µT) did not result in the general protection against UV-induced death observed for vertical fields, suggesting that the size of the induced electric field (which differs between horizontal and vertical exposure) is important for the MF-induced protection. To explore the molecular mechanisms involved in this effect, immunoblotting experiments with an antibody against the inducible form of hsp70 were performed. These showed that application of MF (50 Hz, 200 µT, 1 h) induced hsp70 expression in human K562 cells.     

Cep70 contributes to angiogenesis by modulating microtubule rearrangement and stimulating cell polarization and migration

Centrosomal proteins intricately regulate diverse microtubule-mediated cellular activities, including cell polarization and migration. However, the direct participation of these proteins in angiogenesis, which involves vascular endothelial cell migration from preexisting blood vessels, remains elusive. Here we show that the centrosomal protein Cep70 is necessary for angiogenic response in mice. This protein is also required for tube formation and capillary sprouting in vitro from vascular endothelial cells. Wound healing and transwell assays reveal that Cep70 plays a significant role in endothelial cell migration. Depletion of Cep70 results in severe defects in membrane ruffling and centrosome reorientation, indicating a requirement for this protein in cell polarization. In addition, Cep70 is critically involved in microtubule rearrangement in response to the migratory stimulus. Our data further demonstrate that Cep70 is important for Cdc42 and Rac1 activation to promote angiogenesis. These findings thus establish Cep70 as a crucial regulator of the angiogenic process and emphasize the significance of microtubule rearrangement and cell polarization and migration in angiogenesis.