Conserved central domains control the quaternary structure of type I and type II Hsp40 molecular chaperones

Heat shock protein (Hsp)40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large, and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved, centrally located cysteine-rich domain that is replaced by a glycine- and methionine-rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void, we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small-angle X-ray scattering, and ab initio protein modeling. Low-resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved cysteine-rich domain and glycine- and methionine-rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed.     

The response of the yeast Saccharomyces cerevisiae to sudden vs. gradual changes in environmental stress monitored by expression of the stress response protein Hsp12p

The response of the yeast Saccharomyces cerevisiae to sudden vs. gradual changes in different environmental stress conditions during both respiratory growth and aerobic fermentative growth in the presence of excess glucose was investigated by monitoring the level and rate of expression of the stress response protein Hsp12p using the fluorescent fusion construct Hsp12p-Gfp2p. The initial expression level and the rate of Hsp12p synthesis was significantly greater under glucose-limited conditions in the chemostat ( D <0.14 h⁻¹) compared with when excess glucose was present in the auxostat. Decreasing the dilution rate and the glucose concentration further in the A-stat resulted in increased Hsp12p expression, which was more marked when a rapid rather than a gradual change was affected. Common stress factors such as NaCl, ethanol and elevated temperature caused stress responses in both D-stat and auxo-accelerostat culture. The magnitude of the stress response depended on the stress factor, cultivation conditions as well as the rate of change of the stress factor. The rate of Hsp12p synthesis increased due to all applied stresses, with the observed increase between 2 and 20 times lower when the stress was applied gradually rather than rapidly. The results suggested that the Hsp12p expression rate is a good indicator of applied stress in S. cerevisiae .     

Effects of vanadium-containing compounds on membrane lipids and on microdomains used in receptor-mediated signaling

There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium (V) compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of V compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving V complexes such as [VO(2)(dipic)](-) (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V(10)O(28)(6-), V(10)), BMOV (bis(maltolato)oxovanadium(IV)), and [VO(saltris)](2) (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between V-containing compounds and model lipid systems, an evaluation of the effects of V compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of V-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms.     

Characterizing Escherichia coli DH5alpha growth and metabolism in a complex medium using genome-scale flux analysis

Genome-scale flux analysis of Escherichia coli DH5alpha growth in a complex medium was performed to investigate the relationship between the uptake of various nutrients and their metabolic outcomes. During the exponential growth phase, we observed a sequential consumption order of serine, aspartate and glutamate in the complex medium as well as the complete consumption of key carbohydrate nutrients, glucose and trehalose. Based on the consumption and production rates of the measured metabolites, constraints-based flux analysis of a genome-scale E. coli model was then conducted to elucidate their utilization in the metabolism. The in silico analysis revealed that the cell exploited biosynthetic precursors taken up directly from the complex medium, through growth-related anabolic pathways. This suggests that the cell could be functioning in an energetically more efficient manner by reducing the energy needed to produce amino acids. The in silico simulation also allowed us to explain the observed rapid consumption of serine: excessively consumed external serine from the complex medium was mainly converted into pyruvate and glycine, which in turn, led to the acetate accumulation. The present work demonstrates the application of an in silico modeling approach to characterizing microbial metabolism under complex medium condition. This work further illustrates the use of in silico genome-scale analysis for developing better strategies related to improving microbial growth and enhancing the productivity of desirable metabolites.     

The signaling role of extracellular ATP and its dependence on Ca2+ flux in elicitation of Salvia miltiorrhiza hairy root cultures

The application of a polysaccharide elicitor from yeast extract,YE, to Salvia miltiorrhiza hairy root cultures induced transientrelease of ATP from the roots to the medium, leading to a dose-dependentincrease in the extracellular ATP (eATP) level. The eATP levelrose to a peak (about 6.5 nM with 100 mg l^–1 YE) at about10 h after YE treatment, but dropped to the control level 6h later. The elicitor-induced ATP release was dependent on membraneCa²⁺ influx, and abolished by the Ca²⁺ chelator EGTA or thechannel blocker La³⁺. The YE-induced H₂O₂ production was stronglyinhibited by reactive blue (RB), a specific inhibitor of membranepurinoceptors. On the other hand, the application of exogenousATP at 10–100 µM to the cultures also induced rapidand dose-dependent increases in H₂O₂ production and medium pH,both of which were effectively blocked by RB and EGTA. The non-hydrolyzableATP analog ATPS was as effective as ATP, but the hydrolyzedderivatives ADP or AMP were not so effective in inducing thepH and H₂O₂ increases. Our results suggest that ATP releaseis an early event and that eATP plays a signaling role in theelicitation of plant cell responses; Ca²⁺ is required for activationof the elicitor-induced ATP release and the eATP signal transduction.This is the first report on ATP release induced by a fungalelicitor and its involvement in the elicitor-induced responsesin plant cells.     

Antimutagenic activity of serotoninergic system and underlying mechanisms in fry of sturgeon and goldfish

The paper deals with antimutagenic body activity and its underlying mechanisms. The experiments carried out on the one-year old sturgeons (Acipenser gueldenstaedti persicus) and goldfish (Carassius auratus) have shown that intramuscular administration of serotonin-modulated anticonsolidation protein (SMAP) leads to a twofold decrease of erythrocyte mutagenic alterations (the micronuclear test, p < 0.01) caused by action of benthic deposits (0.8 ml/l, 3 days) polluted with industrial wastes. Exposure of goldfish in water contaminated with crude oil (500 mg/l, 3 days) led to a sharp rise of the content of the 70 kDa brain protein fraction (p < 0.001); these water-soluble proteins are assumed to belong to heat shock proteins (HSP). At the same time, in the brain of the studied animals there was observed a simultaneous increase of the SMAP content (p < 0.001). After 3 h, intracerebral SMAP administration to goldfish increased significantly the 90 kDa protein fraction content (p < 0.01), probably HSP90, in the electrophoretic profiles of the brain water-soluble proteins. Thus, the obtained results indicate that the body serotoninergic system has the antimutagenic activity providing protection of cells from action of harmful environmental factors by an enhancement of synthesis of proteins suggested to belong to HSP.     

Differential expression of two small Hsps during diapause in the corn stalk borer Sesamia nonagrioides (Lef.)

We isolated and characterized two members of the α-crystallin/sHsp family, SnoHsp19.5 and SnoHsp20.8 from Sesamia nonagrioides (Lepidoptera: Noctuidae). The cDNAs encoded proteins of 174 and 185 amino acids, with calculated molecular weights of 19.5 and 20.8 kDa, respectively. The deduced amino acid sequences of SnoHsp19.5 and SnoHsp20.8 showed highest homology to Hsp19.7 of Mamestra brassicae and to Bombyx mori Hsp20.4, respectively. Expression patterns of SnoHsp19.5 and SnoHsp20.8 in non-diapausing individuals under different environmental conditions (heat or cold) showed different accumulation profiles for the two genes after heat and cold treatment. SnoHsp19.5 was consistently expressed, while SnoHsp20.8 gene was down-regulated in deep diapause and was up-regulated at the termination of diapause. Our results suggest that these two genes play distinctive roles in the regulation of diapause.     

In vivo D2O labeling to quantify static and dynamic changes in cholesterol and cholesterol esters by high resolution LC/MS

High resolution LC/MS-MS and LC/APPI-MS methods have been established for the quantitation of flux in the turnover of cholesterol and cholesterol ester. Attention was directed toward quantifying the monoisotopic mass (M0) and that of the singly deuterated labeled (M+1) isotope. A good degree of isotopic dynamic range has been achieved by LC/MS-MS ranging from 3-4 orders of magnitude. Correlation between the linearity of GC/MS and LC atmospheric pressure photoionization (APPI)-MS are complimentary (r² = 0.9409). To prove the viability of this particular approach, male C57Bl/6 mice on either a high carbohydrate (HC) or a high fat (HF) diet were treated with ²H₂O for 96 h. Gene expression analysis showed an increase in the activity of stearoyl-CoA desaturase (Scd1) in the HC diet up to 69-fold (P < 0.0008) compared with the HF diet. This result was supported by the quantitative flux measurement of the isotopic incorporation of ²H into the respective cholesterol and cholesterol ester (CE) pools. We concluded that it is possible to readily obtain static and dynamic measurement of cholesterol and CEs in vivo by coupling novel LC/MS methods with stable isotope-based protocols.     

PKN-1, a homologue of mammalian PKN, is involved in the regulation of muscle contraction and force transmission in C. elegans

To examine the in vivo functions of protein kinase N (PKN), one of the effectors of Rho small guanosine triphosphatases (GTPases), we used the nematode Caenorhabditis elegans as a genetic model system. We identified a C. elegans homologue (pkn-1) of mammalian PKN and confirmed direct binding to C. elegans Rho small GTPases. Using a green fluorescent protein reporter, we showed that pkn-1 is mainly expressed in various muscles and is localized at dense bodies and M lines. Overexpression of the PKN-1 kinase domain and loss-of-function mutations by genomic deletion of pkn-1 resulted in a loopy Unc phenotype, which has been reported in many mutants of neuronal genes. The results of mosaic analysis and body wall muscle-specific expression of the PKN-1 kinase domain suggests that this loopy phenotype is due to the expression of PKN-1 in body wall muscle. The genomic deletion of pkn-1 also showed a defect in force transmission. These results suggest that PKN-1 functions as a regulator of muscle contraction-relaxation and as a component of the force transmission mechanism.