Phosphorylation in the Charged Linker Modulates Interactions and Secretion of Hsp90β

Hsp90β is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90β for proper folding and/or activity. Regulation of Hsp90β is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90β, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90β is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90β activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90β globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90β and its secretion, through changes in the conformation of the chaperone.

A Rotavirus Spike Protein Conformational Intermediate Binds Lipid Bilayers

ABSTRACT During rotavirus entry, a virion penetrates a host cell membrane, sheds its outer capsid proteins, and releases a transcriptionally active subviral particle into the cytoplasm. VP5*, the rotavirus protein believed to interact with the membrane bilayer, is a tryptic cleavage product of the outer capsid spike protein, VP4. When a rotavirus particle uncoats, VP5* folds back, in a rearrangement that resembles the fusogenic conformational changes in enveloped-virus fusion proteins. We present direct experimental evidence that this rearrangement leads to membrane binding. VP5* does not associate with liposomes when mounted as part of the trypsin-primed spikes on intact virions, nor does it do so after it has folded back into a stably trimeric, low-energy state. But it does bind liposomes when they are added to virions before uncoating, and VP5* rearrangement is then triggered by addition of EDTA. The presence of liposomes during the rearrangement enhances the otherwise inefficient VP5* conformational change. A VP5* fragment, VP5CT, produced from monomeric recombinant VP4 by successive treatments with chymotrypsin and trypsin, also binds liposomes only when the proteolysis proceeds in their presence. A monoclonal antibody that neutralizes infectivity by blocking a postattachment entry event also blocks VP5* liposome association. We propose that VP5* binds lipid bilayers in an intermediate conformational state, analogous to the extended intermediate conformation of enveloped-virus fusion proteins.

Catecholaminergic polymorphic ventricular tachycardia-related mutations R33Q and L167H alter calcium sensitivity of human cardiac calsequestrin

Two missense mutations, R33Q and L167H, of hCASQ2 (human cardiac calsequestrin), a protein segregated to the lumen of the sarcoplasmic reticulum, are linked to the autosomal recessive form of CPVT (catecholaminergic polymorphic ventricular tachycardia). The effects of these mutations on the conformational, stability and Ca2+ sensitivity properties of hCASQ2, were investigated. Recombinant WT (wild-type) and mutant CASQ2s were purified to homogeneity and characterized by spectroscopic (CD and fluorescence) and biochemical (size-exclusion chromatography and limited proteolysis) methods at 500 and 100 mM KCl, with or without Ca2+ at a physiological intraluminal concentration of 1 mM; Ca2+-induced polymerization properties were studied by turbidimetry. In the absence of Ca2+, mutations did not alter the conformation of monomeric CASQ2. For L167H only, at 100 mM KCl, emission fluorescence changes suggested tertiary structure alterations. Limited proteolysis showed that amino acid substitutions enhanced the conformational flexibility of CASQ2 mutants, which became more susceptible to tryptic cleavage, in the order L167H>R33Q>WT. Ca2+ at a concentration of 1 mM amplified such differences: Ca2+ stabilized WT CASQ2 against urea denaturation and tryptic cleavage, whereas this effect was reduced in R33Q and absent in L167H. Increasing [Ca2+] induced polymerization and precipitation of R33Q, but not that of L167H, which was insensitive to Ca2+. Based on CASQ2 models, we propose that the Arg33→Gln exchange made the Ca2+-dependent formation of front-to-front dimers more difficult, whereas the Leu167→His replacement almost completely inhibited back-to-back dimer interactions. Initial molecular events of CPVT pathogenesis begin to unveil and appear to be different depending upon the specific CASQ2 mutation.

Circumventing the Effect of Product Toxicity: Development of a Novel Two-Stage Production Process for the Lantibiotic Gallidermin

ABSTRACT Lantibiotics such as gallidermin are lanthionine-containing polypeptide antibiotics produced by gram-positive bacteria that might become relevant for the treatment of various infectious diseases. So far, self-toxicity has prevented the isolation of efficient overproducing strains, thus hampering their thorough investigation and preventing their exploitation in fields other than the food area. We wanted to investigate the effect of lantibiotic precursor peptides on the producing strains in order to evaluate novel strategies for the overproduction of these promising peptides. In this study, gallidermin was chosen as a representative example of the type A lantibiotics. A Staphylococcus gallinarum Tü3928 mutant, whose gene for the extracellular pregallidermin protease GdmP was replaced by a kanamycin-resistance gene, was constructed. Mass spectrometry (MS) analysis indicated that this mutant produced fully posttranslationally modified gallidermin precursors with truncated versions of the leader peptide, but not the entire leader as predicted from the gdmA sequence. In filter-on-plate assays, these truncated pregallidermins showed no toxicity against Staphylococcus gallinarum Tü3928 up to a concentration of 8 g/liter (corresponding to approximately 2.35 mM), while gallidermin produced clear inhibitory zones at concentrations as low as 0.25 g/liter (0.12 mM). We showed that the lack of toxicity is due entirely to the presence of the truncated leader, since MS as well as bioassay analysis showed that the peptides resulting from tryptic cleavage of pregallidermins and gallidermin produced by S. gallinarum Tü3928 had identical masses and approximately the same specific activity. This demonstrates that even a shortened leader sequence is sufficient to prevent the toxicity of mature gallidermin. In nonoptimized fermentations, the gdmP mutant produced pregallidermin to a 50%-higher molar titer, suggesting that the absence of self-toxicity has a beneficial effect on gallidermin production and giving a first confirmation of the suitability of the overproduction strategy.