Academic literature on the topic 'Cellular membrane thermostability'

Six red (Acer rubrum `Autumn Flame', `Fairview Flame', `Franksred', `Landsburg', `Northwood', and `October Glory') and three Freeman (A. × freemanii `Armstrong', `Celzam', and `Jeffersred') maple cultivars originating in different parts of the United States were grown in containers in 1995 and 1996 prior to laboratory procedures to determine root cell membrane thermostability. Electrolyte leakage from excised root tissue exposed for 30 min to temperatures ranging from 20 to 63 °C, was used to assess cellular injury of unsuberized, current season, fine roots. The critical killing temperatures of root tissue of cultivars evaluated ranged from 52.0 ± 0.8 °C to 53.3 ± 0.5 °C, indicating minimal differences in root membrane thermostability among the selections evaluated. Critical temperatures for cultivars selected from the northern part of the native range did not differ from cultivars originating elsewhere. Implications of these differences for container production will be discussed.

Experiments were carried out to study the mechanisms for heat tolerance in chili pepper (Capsicum annuum). To assess these mechanisms, six genotypes were evaluated for cellular membrane thermostability (CMT) and for HSP70 gene expression. The plants were grown in an experimental plant growth chamber. The mean value of CMT indicates that membrane integrity was not damaged by the high temperature treatment (50 °C) in most of the genotypes. The genotypes were classified as follows: heat-tolerant (greater than 60%), moderately tolerant (30% to 60%), and susceptible (less than 30%). The heat-tolerant plants recorded the highest CMTs at 89.27%, 88.03%, and 85.10% for AVPP0702, AVPP0116, and AVPP9905, respectively, which might be the reason for the change in their cell membrane thermostability. AVPP9703 and AVPP0002 showed CMTs of 15.87% and 18.43%, which might indicate their sensitivity to heat stress. Heat shock protein 70 kDa was identified and found to be differentially expressed under the heat stress. Under heat stress, significantly increased levels of the HSP70 gene were detected after 2 h of temperature treatment at 42 °C, which indicated that this gene is quickly and sharply induced by heat shock. This was true for all genotypes tested, which were significantly up-regulated by more than 36.9-, 7.10-, 3.87-, and 3-fold for AVPP0702, AVPP0116, AVPP0002, and AVPP9703, respectively. The HSP70 gene was found to be significantly down-regulated under heat stress in ‘Kulai’. AVPP0702, AVPP9905, and AVPP0116 could be considered as heat-tolerant genotypes, whereas ‘Kulai’ and AVPP9703 were found to be heat-sensitive genotypes in this investigation.

ABSTRACT The vaccinia virus F10 protein is one of two virally encoded protein kinases. A phenotypic analysis of infections involving a tetracycline-inducible recombinant (vΔiF10) indicated that F10 is involved in the early stages of virion morphogenesis, as previously reported for the mutants ts28 and ts15. The proteins encoded by ts28 and ts15 have primary defects in enzymatic activity and thermostability, respectively. Using a transient complementation assay, we demonstrated that the enzymatic activity of F10 is essential for its biological function and that both its enzymatic and biological functions depend upon N-terminal sequences that precede the catalytic domain. An execution point analysis indicated that in addition to its role at the onset of morphogenesis, F10 is also required at later stages, when membrane crescents surround virosomal contents and develop into immature virions. The F10 protein is phosphorylated in vivo, appears to be tightly associated with intracellular membranes, and can bind to specific phosphoinositides in vitro. When F10 is repressed or impaired, the phosphorylation of several cellular and viral proteins appears to increase in intensity, suggesting that F10 may normally intersect with cellular signaling cascades via the activation of a phosphatase or the inhibition of another kinase. These cascades may drive the F10-induced remodeling of membranes that accompanies virion biogenesis. Upon the release of ts28-infected cultures from a 40°C-induced block, a synchronous resumption of morphogenesis that culminates in the production of infectious virus can be observed. The pharmacological agents H89 and cerulenin, which are inhibitors of endoplasmic reticulum exit site formation and de novo lipid synthesis, respectively, block this recovery.

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) entry into the host cell involves a cascade of events and currently represents one of most attractive targets in the search for new antiviral drugs. The fusion-active gp41 core structure is a stable six-helix bundle (6-HB) folded by its trimeric N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR). Peptides derived from the CHR region of HIV-1 gp41 are potent fusion inhibitors that target the NHR to block viral and cellular membrane fusion in a dominant negative fashion. However, all CHR peptides reported to date are derived primarily from residues 628 to 673 of gp41; little attention has been paid to the upstream sequence of the pocket binding domain (PBD) in the CHR. Here, we have identified a motif (621QIWNNMT627) located at the upstream region of the gp41 CHR, immediately adjacent to the PBD (628WMEWEREI635). Biophysical characterization demonstrated that this motif is critical for the stabilization of the gp41 6-HB core. The peptide CP621-652, containing the 621QIWNNMT627 motif, was able to interact with T21, a counterpart peptide derived from the NHR, to form a typical 6-HB structure with a high thermostability (thermal unfolding transition [T m ] value of 82°C). In contrast, the 6-HB formed by the peptides N36 and C34, which has been considered to be a core structure of the fusion-active gp41, had a T m of 64°C. Different from T-20 (brand name Fuseon), which is the first and only HIV-1 fusion inhibitor approved for clinical use, CP621-652 could efficiently block 6-HB formation in a dose-dependent manner. Significantly, CP621-652 had potent inhibitory activity against HIV-1-mediated cell-cell fusion and infection, especially against T-20- and C34-resistant virus. Therefore, our works provide important information for understanding the core structure of the fusion-active gp41 and for designing novel anti-HIV peptides.

ABSTRACT Arg-gingipains are extracellular cysteine proteases produced by the gram-negative periodontal pathogen Porphyromonas gingivalis and are encoded by rgpA and rgpB. Three Arg-gingipains, heterodimeric high-molecular-mass Arg-gingipain HRgpA comprising the α-catalytic chain and the β-adhesin chain, the monomeric soluble Arg-gingipain comprising only the α-catalytic chain (RgpAcat), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the α-catalytic chain (mt-RgPAcat), are derived from rgpA. The monomeric enzymes contain between 14 and 30% carbohydrate by weight. rgpB encodes two monomeric enzymes, RgpB and mt-RgpB. Earlier work indicated that rgpB is involved in the glycosylation process, since inactivation of rgpB results in the loss of not only RgpB and mt-RgpB but also mt-RgpAcat. This work aims to confirm the role of RgpB in the posttranslational modification of RgpAcat and the effect of aberrant glycosylation on the properties of this enzyme. Two-dimensional gel electrophoresis of cellular proteins from W50 and an inactivated rgpB strain (D7) showed few differences, suggesting that loss of RgpB has a specific effect on RgpA maturation. Inactivation of genes immediately upstream and downstream of rgpB had no effect on rgpA-derived enzymes, suggesting that the phenotype of the rgpB mutant is not due to a polar effect on transcription at this locus. Matrix-assisted laser desorption ionization-time of flight analysis of purified RgpAcat from W50 and D7 strains gave identical peptide mass fingerprints, suggesting that they have identical polypeptide chains. However, RgpAcat from D7 strain had a higher isoelectric point and a dramatic decrease in thermostability and did not cross-react with a monoclonal antibody which recognizes a glycan epitope on the parent strain enzyme. Although it had the same total sugar content as the parent strain enzyme, there were significant differences in the monosaccharide composition and linking sugars. These data suggest that RgpB is required for the normal posttranslational glycosylation of Arg-gingipains derived from rgpA and that this process is required for enzyme stabilization.

Abstract Pyruvate kinase deficiency (PKD) is an autosomal recessive enzymopathy that is the most common cause of hereditary nonspherocytic hemolytic anemia (HNSHA). PKD is a rare disease characterized by a life-long chronic hemolysis with severe co-morbidities. It is hypothesized that insufficient energy production to maintain red cell membrane homeostasis promotes the chronic hemolysis. Treatment is generally palliative, focusing on the resultant anemia, and there are no approved drugs that directly target mutated pyruvate kinase. Here, we describe the mechanism of action and cellular effects of AG-348, an allosteric activator of the red cell isoform of pyruvate kinase (PKR). Hundreds of mutant alleles of PKR have been identified and are known to have deleterious effects on catalytic activity, protein stability, or protein expression. We demonstrate that AG-348 can potently activate a spectrum of recombinantly expressed PKR mutant proteins, including mutations that span distinct subdomains of the enzyme. The R532W mutation is quite sensitive to AG-348 modulation, with over 4-fold activation of the enzyme activity, even as the mutation renders PKR insensitive to stimulation by its endogenous allosteric regulator fructose 1,6-bisphosphate (FBP) (Figure A). Crystallographic analysis reveals that very few mutations associated with PKD occur within the AG-348 binding pocket, accounting for its broad activity. The binding of AG-348 attenuates the thermostability defect of several mutant alleles of PKR, including the commonly observed R510Q mutant that has a half-life of ∼2% of the half-life of wild-type PKR when incubated at 53°C. Pre-incubation of the R510Q protein with AG-348 restores the half-life to ∼70% that of the wild-type enzyme (Figure B). PKD red cells are characterized by changes in metabolism associated with defective glycolysis, including a build-up of the PKR substrate phosphenolpyruvate (PEP) and deficiency in the PKR product adenosine triphosphate (ATP). PKD red cells from several patients with distinct compound heterozygous PKR mutations exposed to AG-348 had increased PKR enzyme activity (up to 4-fold over control) and showed consistent dose and time-dependent metabolic responses (Figure C), including sharp reductions in PEP (up to 70% compared to control) and increases in ATP levels (up to 100% over control). These responses were observed in patient samples harboring PKR mutations that we had studied biochemically (including R486W and R510Q), but also in an instance where the mutation had not previously been biochemically characterized (A495V). In these ex-vivo settings, ATP levels in AG-348 treated cells can reach levels that are typical of normal, non-PKD red cells. These data support the hypothesis that drug intervention with AG-348 may restore glycolytic pathway activity and normalize red cell metabolism in vivo. This therapeutic approach may be an effective way to correct the underlying pathology of PKD and, importantly, provide clinical benefit to patients. Disclosures: Kung: Agios Pharmaceuticals: Employment, Equity Ownership. Hixon:Agios Pharmaceuticals: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals: Employment, Equity Ownership. Histen:Agios Pharmaceuticals: Employment, Equity Ownership. Hill:Agios Pharmaceuticals: Employment, Equity Ownership. Si:Agios Pharmaceuticals: Employment, Equity Ownership. Kernytsky:Agios Pharmaceuticals: Employment, Equity Ownership. Chen:Agios Pharmaceuticals: Employment, Equity Ownership. DeLaBarre:Agios Pharmaceuticals: Employment, Equity Ownership. Clasquin:Agios Pharmaceuticals: Employment, Equity Ownership. Ho:Agios Pharmaceuticals: Employment, Equity Ownership. Salituro:Agios Pharmaceuticals: Employment, Equity Ownership. Popovici-Muller:Agios Pharmaceuticals: Employment, Equity Ownership. Agresta:Agios Pharmaceuticals: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals: Employment, Equity Ownership. Dang:Agios Pharmaceuticals: Employment, Equity Ownership.

There is circumstantial evidence that protein denaturation occurs in cells during heat shock at hyperthermic temperatures and that denatured or damaged protein is the primary inducer of the heat shock response. However, there is no direct evidence regarding the extent of denaturation of normal cellular proteins during heat shock. Differential scanning calorimetry (DSC) is the most direct method of monitoring protein denaturation or unfolding. Due to the fundamental parameter measured, heat flow, DSC can be used to detect and quantitate endothermic transitions in complex structures such as isolated organelles and even intact cells. DSC profiles with common features are obtained for isolated rat hepatocytes, liver homogenate, and Chinese hamster lung V79 fibroblasts. Five main transitions (A-E), several of which are resolvable into subcomponents, are observed with transition temperatures (Tm) of 45-98 degrees C. The onset temperature is approximately 40 degrees C, but some transitions may extend as low as 37-38 degrees C. In addition to acting as the primary signal for heat shock protein synthesis, the inactivation of critical proteins may lead to cell death. Critical target analysis implies that the rate limiting step of cell killing for V79 cells is the inactivation of a protein with Tm = 46 degrees C within the A transition. Isolated microsomal membranes, mitochondria, nuclei, and a cytosolic fraction from rat liver have distinct DSC profiles that contribute to different peaks in the profile for intact hepatocytes. Thus, the DSC profiles for intact cells appears to be the sum of the profiles of all subcellular organelles and components. The presence of endothermic transitions in the isolated organelles is strong evidence that they are due to protein denaturation. Each isolated organelle has an onset for denaturation near 40 degrees C and contains thermolabile proteins denaturing at the predicted Tm (46 degrees C) for the critical target. The extent of denaturation at any temperature can be approximately by the fractional calorimetric enthalpy. After scanning to 45 degrees C at 1 degree C/min and immediately cooling, a relatively mild heat shock, an estimated fraction denaturation of 4-7% is found in hepatocytes, V79 cells, and the isolated organelles other than nuclei, which undergo only 1% denaturation because of the high thermostability of chromatin. Thus, thermolabile proteins appear to be present in all cellular organelles and components, and protein denaturation is widespread and extensive after even mild heat shock.