Interaction of heat shock protein 70 with membranes depends on the lipid environment

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER-Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. Since Hsp70, the major inducible hsp, has been detected on surface of stressed cells, we propose that membrane interaction is the first step in the export process. The question that emerges is how does this charged cytosolic protein interact with lipid membranes? Prior studies have shown that Hsp70 formed ion conductance pathways within artificial lipid bilayers. These early observations have been extended herewith using a liposome insertion assay. We showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high-molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments and the region embedded into the lipid membrane was mapped toward the C-terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted into a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.     

Spectrophotometric titrations of micellar Schiff''s bases prepared from pyridoxal 5′-phosphate

Electron absorption and equilibrium of the Schiffs bases prepared between pyridoxal 5′-phosphate (PLP) and dodecylamine (DODA) or some other shorter chain amines have been studied in nonionic and cationic micellar solutions with various pH of the bulk solution. In the presence of the nonionic (Triton X-100) micelles the Schiffs bases formed between PLP and DODA were embedded into the micelles because the absorption occured at 335 nm, indicative of the nonpolar milieu. This absorption was constant at pH 5–10. At pH 3–5, the tautomeric form absorbing at 415 nm appeared. This resembles the titration of glycogen phosphorylate or that of Schiffs bases in methanol. Short chain amines absorbed at 415 nm, which is typical of Schiffs bases in aqueous solutions. Tryptophan also absorbed first at 415 nm but the absorption changed to 325 nm with a half-time of ～20 min. This was interpreted as being due to formation of the cyclic structure catalysed by micelles. The pH-dependent equilibrium constant of the reaction between PLP and DODA in Triton X-100 solution had a maximum at pH9, the value being 3500 M^?1, about ten times greater than the value of ethylamine at the same pH. Spectral properties of PLP-DODA imines in the cationic micelles (cetyltrimethylammonium bromide) resembled those in the nonionic micelles, except that at low pH the absorption peak in the 415 nm region did not appear. The equilibrium constant of PLP-DODA had maximum at pH 9, the value being as high as 118000 M^?1. Different properties of nonionic and cationic micelles and the design of micellar model systems of PLP enzymes are discussed.     

Nucleotide-induced conformational changes of PMP70, an ATP binding cassette transporter on rat liver peroxisomal membranes

Nucleotide-induced conformational changes of the 70-kDa peroxisomal membrane protein (PMP70) were investigated by means of limited-trypsin digestion. Rat liver peroxisomes preincubated with various nucleotides were subsequently digested by trypsin. The digestion products were subjected to immunoblot analysis with an anti-PMP70 antibody that recognizes the carboxyl-terminal 15 amino acids of the protein. PMP70 was initially cleaved in the boundary region between the transmembrane and nucleotide-binding domains and a carboxyl-terminal 30-kDa fragment resulted. The fragment in turn was progressively digested at the helical domain between the Walker A and B motifs. The fragment, however, could be stabilized with MgATP or MgADP. In contrast to MgATP, MgATP-gammaS protected whole PMP70 as well as the fragment. The 30-kDa fragment processed by trypsin was recovered in the post-peroxisomal fraction as a complex with a molecular mass of about 60 kDa irrespective of the presence of MgATP. These results suggest that PMP70 exists as a dimer on the peroxisomal membranes and the binding and hydrolysis of ATP induce conformational changes in PMP70 close to the boundary between the transmembrane and nucleotide binding domains and the helical domain between the Walker A and B motifs.     

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.     

DnaJ/Hsc70 chaperone complexes control the extracellular release of neurodegenerative‐associated proteins

It is now known that proteins associated with neurodegenerative disease can spread throughout the brain in a prionlike manner. However, the mechanisms regulating the trans‐synaptic spread propagation, including the neuronal release of these proteins, remain unknown. The interaction of neurodegenerative disease‐associated proteins with the molecular chaperone Hsc70 is well known, and we hypothesized that much like disaggregation, refolding, degradation, and even normal function, Hsc70 may dictate the extracellular fate of these proteins. Here, we show that several proteins, including TDP‐43, α‐synuclein, and the microtubule‐associated protein tau, can be driven out of the cell by an Hsc70 co‐chaperone, DnaJC5. In fact, DnaJC5 overexpression induced tau release in cells, neurons, and brain tissue, but only when activity of the chaperone Hsc70 was intact and when tau was able to associate with this chaperone. Moreover, release of tau from neurons was reduced in mice lacking the DnaJC5 gene and when the complement of DnaJs in the cell was altered. These results demonstrate that the dynamics of DnaJ/Hsc70 complexes are critically involved in the release of neurodegenerative disease proteins.     

Sindbis virus replication, is insensitive to rapamycin and torin1, and suppresses Akt/mTOR pathway late during infection in HEK cells

Genetically engineered Sindbis viruses (SIN) are excellent oncolytic agents in preclinical models. Several human cancers have aberrant Akt signaling, and kinase inhibitors including rapamycin are currently tested in combination therapies with oncolytic viruses. Therefore, it was of interest to delineate possible cross-regulation between SIN replication and PI3K/Akt/mTOR signaling. Here, using HEK293T cells as host, we report the following key findings: (a) robust SIN replication occurs in the presence of mTOR specific inhibitors, rapamycin and torin1 or Ly294002 – a PI3K inhibitor, suggesting a lack of requirement for PI3K/Akt/mTOR signaling; (b) suppression of phosphorylation of Akt, mTOR and its effectors S6, and 4E-BP1 occurs late during SIN infection: a viral function that may be beneficial in counteracting cellular drug resistance to kinase inhibitors; (c) Ly294002 and SIN act additively to suppress PI3K/Akt/mTOR pathway with little effect on virus release; and (d) SIN replication induces host translational shut off, phosphorylation of eIF2α and apoptosis. This first report on the potent inhibition of Akt/mTOR signaling by SIN replication, bolsters further studies on the development and evaluation of engineered SIN genotypes in vitro and in vivo for unique cytolytic functions.     

Free energy analysis of ω-transaminase reactions to dissect how the enzyme controls the substrate selectivity

ω-Transaminase (ω-TA) is the only naturally occurring enzyme allowing asymmetric amination of ketones for production of chiral amines. The active site of the enzyme was proposed to consist of two differently sized substrate binding pockets and the stringent steric constraint in the small pocket has presented a significant challenge to production of structurally diverse chiral amines. To provide a mechanistic understanding of how the (S)-specific ω-TA from Paracoccus denitrificans achieves the steric constraint in the small pocket, we developed a free energy analysis enabling quantification of individual contributions of binding and catalytic steps to changes in the total activation energy caused by structural differences in the substrate moiety that is to be accommodated by the small pocket. The analysis exploited kinetic and thermodynamic investigations using structurally similar substrates and the structural differences among substrates were regarded as probes to assess how much relative destabilizations of the reaction intermediates, i.e. the Michaelis complex and the transition state, were induced by the slight change of the substrate moiety inside the small pocket. We found that ≈80% of changes in the total activation energy resulted from changes in the enzyme-substrate binding energy, indicating that substrate selectivity in the small pocket is controlled predominantly by the binding step (K_M) rather than the catalytic step (k_cat). In addition, we examined the pH dependence of the kinetic parameters and the pH profiles of the K_M and k_cat values suggested that key active site residues involved in the binding and catalytic steps are decoupled. Taken together, these findings suggest that the active site residues forming the small pocket are mainly engaged in the binding step but not significantly involved in the catalytic step, which may provide insights into how to design a rational strategy for engineering of the small pocket to relieve the steric constraint toward bulky substituents.