Involvement Of Hsp70 Research Articles

DJ-1 Modulates α-Synuclein Aggregation State in a Cellular Model of Oxidative Stress: Relevance for Parkinson's Disease and Involvement of HSP70

Parkinson's disease (PD) is a neurodegenerative pathology whose molecular etiopathogenesis is not known. Novel contributions have come from familial forms of PD caused by alterations in genes with apparently unrelated physiological functions. The gene coding for alpha-synuclein (alpha-syn) (PARK1) has been investigated as alpha-syn is located in Lewy bodies (LB), intraneuronal inclusions in the substantia nigra (SN) of PD patients. A-syn has neuroprotective chaperone-like and antioxidant functions and is involved in dopamine storage and release. DJ-1 (PARK7), another family-PD-linked gene causing an autosomal recessive form of the pathology, shows antioxidant and chaperone-like activities too. The present study addressed the question whether alpha-syn and DJ-1 interact functionally, with a view to finding some mechanism linking DJ-1 inactivation and alpha-syn aggregation and toxicity. We developed an in vitro model of alpha-syn toxicity in the human neuroblastoma cell line SK-N-BE, influencing DJ-1 and alpha-syn intracellular concentrations by exogenous addition of the fusion proteins TAT-alpha-syn and TAT-DJ-1; DJ-1 was inactivated by the siRNA method. On a micromolar scale TAT-alpha-syn aggregated and triggered neurotoxicity, while on the nanomolar scale it was neuroprotective against oxidative stress (induced by H(2)O(2) or 6-hydroxydopamine). TAT-DJ-1 increased the expression of HSP70, while DJ-1 silencing made SK-N-BE cells more susceptible to oxidative challenge, rendering TAT-alpha-syn neurotoxic at nanomolar scale, with the appearance of TAT-alpha-syn aggregates. DJ-1 inactivation may thus promote alpha-syn aggregation and the related toxicity, and in this model HSP70 is involved in the antioxidant response and in the regulation of alpha-syn fibril formation.

Regulation of the human apoptotic DNase/RNase Endonuclease G: involvement of Hsp70 and ATP

Endonuclease G (EndoG) is a mitochondrial enzyme that becomes an apoptotic nuclease when released from the mitochondrial intermembrane space. EndoG will digest either DNA or RNA, but at physiological ionic strength, RNA is a much more favorable substrate as compared to chromatin. This indicates that EndoG's major in vivo function(s) may be: (i) an apoptotic RNase, and/or (ii) an apoptotic DNase in the presence of additional co-activators. In the present study we have searched for factors that modulate the activity of human EndoG on DNA substrates. We demonstrate that EndoG forms complexes with AIF and FEN-1 but not with PCNA. Interestingly, heat shock proteins 70 interact with EndoG and are involved in the regulation of its activity. Purified Hsp70 prevented stimulation of EndoG DNase activity by other nuclear factors in the ATP-dependent manner.

Protective effect of TAT-delivered alpha-synuclein: relevance of the C-terminal domain and involvement of HSP70.

Alpha-synuclein (alpha-syn) is a 140-amino acid presinaptic protein whose mutations A30P and A53T have been linked to familiar Parkinson's disease (PD). Many data suggest that alpha-syn aggregation is the key event that triggers alpha-syn-mediated neurotoxicity. Nevertheless, other lines of evidence proposed a protective role of alpha-syn against oxidative stress (a major feature of PD), even if the exact mechanism of this protective action and the role of the pathogenetic mutations to this respect have not been elucidated yet. To address these points, we developed an in vitro model of oxidative stress by exposing PC12 cells to hydrogen peroxide (H2O2) (150 microM) for 72 h, and we evaluated alpha-syn-mediated protection delivering increasing amounts of alpha-syn (wild type [WT] or mutated) inside cells using the fusion proteins TAT-alpha-syn (WT, A30P, and A53T). We found that nanomolar amounts of TAT-alpha-syn-mediated protected against oxidative stress and other cellular injuries (6-hydroxydopamine and serum deprivation), whereas micromolar amounts of the fusion proteins were intrinsically toxic to cells. The protective effect was independent from the presence of the mutations A30P and A53T, but no protection occurred when cells were challenged with the proteasome inhibitors lactacystin and MG132. We verified that the protection mechanism required the presence of the C-terminal domain of alpha-syn, as nanomolar amounts of the C-terminal truncated fusion protein TAT-alpha-syn (WT[1-97]) failed in preventing H2O2 toxicity. To further characterize the molecular mechanisms at the basis of alpha-syn protection, we investigated the possible involvement of the chaperone protein HSP70 that is widely implicated in neuroprotection. We found that, at nanomolar concentrations, TAT-alpha-syn was able to increase HSP70 protein level, whereas at the micromolar scale, TAT-alpha-syn decreased HSP70 at the protein level. These effects on HSP70 were independent from the presence of alpha-syn pathogenetic mutations but required the alpha-syn C-terminal domain. The implications for alpha-syn-mediated neurotoxicity and for PD pathogenesis and progression are discussed.

Induced expression and localization to nuclear-inclusion bodies of hsp70 in varicella-zoster virus-infected human diploid fibroblasts.

The expression and subcellular localization of cellular heat-shock protein hsp70 were examined in varicella-zoster virus (VZV)-infected human diploid fibroblasts. Infection with VZV elevated the steady-state levels of hsp70 mRNA by 24 hr post-infection (hpi). Western blotting analysis revealed an increase in accumulation of hsp70 from 24 hpi. Subcellular localization of the hsp70 in VZV-infected cells was examined by indirect immunofluorescence. In most VZV-infected cells, hsp70 was localized to inclusion bodies induced in the cell nucleus by infection with VZV. In some cells, however, the remaining parts of the cell nucleus and the cytoplasm were also stained with anti-hsp70 antibody. These results indicate that infection with VZV induces the expression of hsp70 and its localization to VZV-specific inclusion bodies, which suggests the involvement of hsp70 in molecular events within inclusion bodies.

Transcription of Amphibian Lampbrush Chromosomes Is Disturbed by Microinjection of HSP70 Monoclonal Antibodies

To investigate the possible involvement of HSP70 in nuclear transport of proteins associated with the transcription process in amphibian oocyte lampbrush chromosomes, we examined the effect of anti-HSP70 mouse monoclonal antibodies on the transcriptional activity of lampbrush chromosomes. When injected into the oocyte cytoplasm, anti-HSP70 induced disorganization followed by retraction of chromosomal lateral loops. This retraction reflected inhibition of transcription, which was confirmed by our electron microscopic observations. At the same time, while nucleoli were morphologically disorganized, nucleolar transcription persisted. These modifications were completely reversible. In contrast, no modifications were observed when antibodies were directly microinjected into nuclei. Similar observations were registered at the level of lampbrush chromosomes when wheat germ agglutinin was injected into oocyte cytoplasm. All of these results suggest that HSP70 mediates the nuclear transport of proteins involved in the lampbrush chromosome transcriptional process.

Evidence That Heat Shock Protein-70 Associated with Progesterone Receptors Is not Involved in Receptor-DNA Binding

In the absence of hormone, human progesterone receptors (PR) are recovered in the cytosolic fraction of cell lysates as a multimeric complex containing the steroid-binding polypeptide, heat shock protein-90 (hsp90), and heat shock protein-70 (hsp70). Activated forms of human PR that acquire the ability to bind to DNA are dissociated from hsp90, but retain association with hsp70. The present study has examined whether associated hsp70 has a function in receptor-DNA binding. When activated PR was bound to specific target DNA in a gel shift assay, no hsp70 was detectable in the PR-DNA complex, as evidenced by the failure of several antibodies to hsp70 to affect the mobility or the amount of complexes. To determine whether hsp70 might indirectly influence DNA-binding activity, we have examined the effect of hsp70 dissociation on PR-DNA-binding activity. Dissociation was achieved either by treatment of immunoaffinity-purified immobilized PR complexes with ATP or by the binding of PR complexes to ATP-agarose, followed by elution with high salt. Under both conditions, dissociation from hsp70 neither enhanced nor impaired the ability of PR to bind to specific DNA. These results suggest that hsp70 is not involved in PR binding to DNA, either directly by participating in DNA binding or indirectly by modulating PR-DNA-binding activity. This implies that hsp70 functions at an earlier stage in the receptor activation pathway. Consistent with the known involvement of hsp70 in stabilizing unfolded states of other target proteins, we propose that hsp70 may assist in nuclear transport of PR or in assembly-disassembly of the 8-10S multimeric complex.