A Voltage-Gated Calcium Channel Regulates Lysosomal Fusion with Endosomes and Autophagosomes and Is Required for Neuronal Homeostasis

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.     

Phosphorylated TAR DNA-binding protein-43: Aggregation and antibody-based inhibition

TAR DNA-binding protein-43 (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). Hyperphosphorylation and aggregation of TDP-43 contribute to pathology and are viable therapeutic targets for ALS. In vivo inhibition of TDP-43 aggregation was evaluated using anti-TDP-43 antibodies with promising outcomes. However, the exact mechanism of antibody-based inhibition targeting TDP-43 is not well understood but may lead to the identification of viable immunotherapies. Herein, the mechanism of in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Specifically, the aggregation of phosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) spectroscopy. The protein aggregates were insoluble, ThT-positive and characterized with heterogeneous morphologies (fibers, amorphous structures). Antibodies specific to epitopes 178-393 and 256-269, within the RRM2-CTD domain, reduced the formation of β-sheets and insoluble aggregates, at low antibody loading (antibody: protein ratio = 1 μg/mL: 45 μg/mL). Inhibition outcomes were highly dependent on the type and loading of antibodies, indicating dual functionality of such inhibitors, as aggregation inhibitors or aggregation promoters. Anti-SOD1 and anti-tau antibodies were not effective inhibitors against TDP-43 aggregation, indicating selective inhibition.     

Mutations of the Calcium Channel Gene cacophony Suppress Seizures in Drosophila

Bang sensitive (BS) Drosophila mutants display characteristic seizure-like phenotypes resembling, in some aspects, those of human seizure disorders such as epilepsy. The BS mutant para^bss1, caused by a gain-of-function mutation of the voltage-gated Na⁺ channel gene, is extremely seizure-sensitive with phenotypes that have proven difficult to ameliorate by anti-epileptic drug feeding or by seizure-suppressor mutation. It has been presented as a model for intractable human epilepsy. Here we show that cacophony (cac^TS2), a mutation of the Drosophila presynaptic Ca⁺⁺ channel α₁ subunit gene, is a particularly potent seizure-suppressor mutation, reverting seizure-like phenotypes for para^bss1 and other BS mutants. Seizure-like phenotypes for para^bss1 may be suppressed by as much as 90% in double mutant combinations with cac^TS2. Unexpectedly, we find that para^bss1 also reciprocally suppresses cac^TS2 seizure-like phenotypes. The cac^TS2 mutant displays these seizure-like behaviors and spontaneous high-frequency action potential firing transiently after exposure to high temperature. We find that this seizure-like behavior in cac^TS2 is ameliorated by 85% in double mutant combinations with para^bss1.     

Cloning and Aggregation Characterization of Rubber Elongation Factor and Small Rubber Particle Protein from <Emphasis Type="Italic">Ficus carica</Emphasis>

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are major latex proteins harvested from Hevea brasiliensis (the rubber tree; HbREF and HbSRPP, respectively). Their amino acid sequences exhibit high homology with each other. In the present study, we cloned two cDNAs encoding REF/SRPP-family proteins (FcREF/SRPP-1 and -2) from the laticifers of Ficus carica (fig tree). The amino acid sequences of these proteins showed high homology not only with each other but also with HbREF and HbSRPP. Recombinant FcREF/SRPP-1 and -2 were expressed in E. coli, and their aggregation properties were examined using a Congo red binding assay, agarose gel electrophoresis, and transmission electron microscopy. FcREF/SRPP-1 formed fibrils when incubated in PBS, and grew to micrometer-sized amorphous aggregates that precipitated rapidly. These aggregation properties of FcREF/SRPP-1 are quite similar to those of HbREF, although the growth rate and size of FcREF/SRPP-1 aggregates were inferior to those of HbREF. FcREF/SRPP-2 also formed aggregates during the incubation, but they did not precipitate, as has been reported for HbSRPP. Our results suggest that FcREF/SRPP-1 and -2 correspond to HbREF and HbSRPP, respectively. These aggregation properties could provide useful benchmarks for classifying REF/SRPP-family proteins as REF or SRPP.     

A Modified Lumry–Eyring Analysis for the Determination of the Predominant Mechanism Underlying the Diminution of Protein Aggregation by Glycerol

Aggregation of aspartate-β-semialdehyde dehydrogenase (ASD) was analyzed by applying modified Lumry–Eyring with nucleated polymerization (LENP) model. Intrinsic nucleation time scales were determined. In absence of glycerol, ASD undergoes concentration and time-dependent polymerization into low-molecular weight soluble aggregates and thereafter condensation into insoluble aggregates. In the presence of increasing solvent glycerol concentration, the aggregation becomes more and more nucleation dominated, with slower polymerization to low-molecular weights soluble aggregates, without any condensation into insoluble aggregates. Effective nucleus size as well as the number of monomers in each irreversible growth event were sensitive to the changes in solvent glycerol concentration. Glycerol-directed diminution of aggregation appears to be largely due to the inhibition of rearrangement (decreased nucleation rearrangement rate coefficient, K _r,x ) because of compaction induced due to preferential hydration, thus, preventing the soluble aggregates from locking into irreversible soluble nuclei. Appreciably decreased K _r,x (as compared to nucleation dissociation constant, K _d,x ), appears to be responsible for increased nucleus size at higher solvent glycerol concentration. This study explains how modified LENP model can be applied to determine the predominant mechanism responsible for the diminution of aggregation by polyhydric alcohols (glycerol).     

Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin

Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, U_hr) is characterized by a high rate of aggregation. Aggregation of U_hr leads to the formation of primary aggregates with the hydrodynamic radius (R_h,1) of 10.3 nm. The second form (low reactive unfolded form, U_lr) participates in the aggregation process by its attachment to the primary aggregates produced by the U_hr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (A_st). At complete exhaustion of U_lr, secondary aggregates with the hydrodynamic radius (R_h,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.     

Saliva-Induced Aggregation of Oral Streptococci

Cells of several species of oral microorganisms have been shown, in earlier studies, to be aggregated by saliva. In the present study some of the basic properties of the aggregation system are examined. The observation is made that the saliva-induced aggregates of Streptococcus sanguis and S. mitis can be dissociated to stable particles which consist of about 100 cells and have a median diameter of about 4.5 μm. It is proposed that these are subunits, or core aggregates, of the large primary aggregates. Counts of the core aggregates can be taken as a precise and accurate measure of aggregation. Experiments based on this procedure show that the aggregation of S. sanguis is maximal at 10 C and at 1 meq of Ca²⁺ ions per liter and is not affected by a change in pH between 3.9 and 8.7 or by a change in the phase of growth of the microorganisms. Core aggregates diminish in number with prolonged incubation, suggesting that the aggregating factors break down with time. Formalinized cells yield stable aggregates. However, with Formalinized cell aggregation is maximal between 20 and 30 C and proceeds in the absence of calcium ions. Evidence is presented that whole saliva contains separate aggregating factors for S. sanguis and S. mitis. The factors differ in their affinity for intact cells and for hydroxyapatite and differ in their stability to dialysis. These findings suggest that many different aggregating factors exist in saliva, each of which may be capable of interacting with cells of one or several bacterial species.     

Haptoglobin2-2 phenotype is an additional risk factor of retinopathy in type 2 diabetes mellitus

AIMS:

The aim of this study was to investigate the association between haptoglobin (Hp) phenotypes and risk of the development of diabetic retinopathy (DR) in patients of type 2 diabetes mellitus.

MATERIALS AND METHODS:

This cross-sectional study included 45 normotensive type 2 diabetic patients (duration more than 5 years) admitted in the hospital divided into two groups (with and without DR) on the basis of fundus examination by direct ophthalmoscopy. Serum samples of all patients were subjected for Hp phenotyping by polyacrylamide gel electrophoresis.

RESULTS:

DR was associated significantly in diabetic patients with Hp2-2 phenotype (79.31%) than diabetic patients with Hp2-1 phenotype (43.75%) and Hp2-2 had higher odds ratio (OR) for DR in univariate analysis (OR 4.929, [95% confidence interval [CI] (1.297-18.733)], P = 0.016) and multivariate analysis (OR 7.704 [95% CI (0.887-66.945)], P = 0.064). Furthermore, Hp2-2 was associated significantly with severe forms of DR.

CONCLUSION:

Hp2-2 phenotype is associated with susceptibility to DR showing a graded risk relationship to the number of Hp2 alleles. Determination of Hp phenotype may be useful in the risk assessment and management of DR.     

Effect of Arginine on Pre-nucleus Stage of Interferon Beta-1b Aggregation

Understanding the mechanism of aggregation of a therapeutic protein would not only ease the manufacturing processing but could also lead to a more stable finished product. Aggregation of recombinant interferon (IFNβ-1b) was studied by heating, oxidizing, or seeding of unformulated monomeric solution. The formation of aggregates was monitored by dynamic light scattering (DLS) and UV spectroscopy. The autocatalytic monomer loss model was used to fit the data on aggregation rates. The influence of pre-nucleation on aggregation step was demonstrated by inducing the liquid samples containing a monomer form of folded IFNβ-1b by heat and also an oxidizing agent. Results tend to suggest that the nucleus includes a single protein molecule which has been probably deformed. Seeding tests showed that aggregation of IFNβ-1b was probably initiated when 1.0% (w/w) of monomers converted to nucleus form. Chemiluminescence spectroscopy analysis of the sample indicated the generation of 3.0 μM of hydrogen peroxide (H₂O₂) during nucleation stage of IFNβ-1b aggregation. Arginine with a concentration of 200 mM was sufficient to suppress aggregation of IFNβ-1b by decreasing the rate of pre-nucleation step. We proposed the formation of pre-nucleus structures prior to nucleation as the mechanism of aggregation of IFNβ-1b. Furthermore, we have showed the positive anti-aggregation effect of arginine on pre-nucleation step.KEY WORDS: aggregation, arginine, interferon beta-1b, mechanism, pre-nucleus     

Hir Proteins Are Required for Position-Dependent Gene Silencing in Saccharomyces cerevisiae in the Absence of Chromatin Assembly Factor I

Chromatin assembly factor I (CAF-I) is a three-subunit histone-binding complex conserved from the yeast Saccharomyces cerevisiae to humans. Yeast cells lacking CAF-I (cacΔ mutants) have defects in heterochromatic gene silencing. In this study, we showed that deletion of HIR genes, which regulate histone gene expression, synergistically reduced gene silencing at telomeres and at the HM loci in cacΔ mutants, although hirΔ mutants had no silencing defects when CAF-I was intact. Therefore, Hir proteins are required for an alternative silencing pathway that becomes important in the absence of CAF-I. Because Hir proteins regulate expression of histone genes, we tested the effects of histone gene deletion and overexpression on telomeric silencing and found that alterations in histone H3 and H4 levels or in core histone stoichiometry reduced silencing in cacΔ mutants but not in wild-type cells. We therefore propose that Hir proteins contribute to silencing indirectly via regulation of histone synthesis. However, deletion of combinations of CAC and HIR genes also affected the growth rate and in some cases caused partial temperature sensitivity, suggesting that global aspects of chromosome function may be affected by the loss of members of both gene families.     

Highly Oxidized Peroxisomes Are Selectively Degraded via Autophagy in Arabidopsis

The positioning of peroxisomes in a cell is a regulated process that is closely associated with their functions. Using this feature of the peroxisomal positioning as a criterion, we identified three Arabidopsis thaliana mutants (peroxisome unusual positioning1 [peup1], peup2, and peup4) that contain aggregated peroxisomes. We found that the PEUP1, PEUP2, and PEUP4 were identical to Autophagy-related2 (ATG2), ATG18a, and ATG7, respectively, which are involved in the autophagic system. The number of peroxisomes was increased and the peroxisomal proteins were highly accumulated in the peup1 mutant, suggesting that peroxisome degradation by autophagy (pexophagy) is deficient in the peup1 mutant. These aggregated peroxisomes contained high levels of inactive catalase and were more oxidative than those of the wild type, indicating that peroxisome aggregates comprise damaged peroxisomes. In addition, peroxisome aggregation was induced in wild-type plants by exogenous application of hydrogen peroxide. The cat2 mutant also contained peroxisome aggregates. These findings demonstrate that hydrogen peroxide as a result of catalase inactivation is the inducer of peroxisome aggregation. Furthermore, an autophagosome marker, ATG8, frequently colocalized with peroxisome aggregates, indicating that peroxisomes damaged by hydrogen peroxide are selectively degraded by autophagy in the wild type. Our data provide evidence that autophagy is crucial for quality control mechanisms for peroxisomes in Arabidopsis.     

Aggregation Properties of the Small Nuclear Ribonucleoprotein U1-70K in Alzheimer Disease

Recent evidence indicates that U1-70K and other U1 small nuclear ribonucleoproteins are Sarkosyl-insoluble and associate with Tau neurofibrillary tangles selectively in Alzheimer disease (AD). Currently, the mechanisms underlying the conversion of soluble nuclear U1 small nuclear ribonucleoproteins into insoluble cytoplasmic aggregates remain elusive. Based on the biochemical and subcellular distribution properties of U1-70K in AD, we hypothesized that aggregated U1-70K itself or other biopolymers (e.g. proteins or nucleic acids) interact with and sequester natively folded soluble U1-70K into insoluble aggregates. Here, we demonstrate that total homogenates from AD brain induce soluble U1-70K from control brain or recombinant U1-70K to become Sarkosyl-insoluble. This effect was not dependent on RNA and did not correlate with detergent-insoluble Tau levels as AD homogenates with reduced levels of these components were still capable of inducing U1-70K aggregation. In contrast, proteinase K-treated AD homogenates and Sarkosyl-soluble AD fractions were unable to induce U1-70K aggregation, indicating that aggregated proteins in AD brain are responsible for inducing soluble U1-70K aggregation. It was determined that the C terminus of U1-70K, which harbors two disordered low complexity (LC) domains, is necessary for U1-70K aggregation. Moreover, both LC1 and LC2 domains were sufficient for aggregation. Finally, protein cross-linking and mass spectrometry studies demonstrated that a U1-70K fragment harboring the LC1 domain directly interacts with aggregated U1-70K in AD brain. Our results support a hypothesis that aberrant forms of U1-70K in AD can directly sequester soluble forms of U1-70K into insoluble aggregates.     

Diffusible fruiting factor in Myxococcus coralloides and Myxococcus fulvus

Myxococcus coralloides ATCC 25202 and Myxococcus fulvus ATCC 23093 produced an extracellular diffusible factor during fruiting body formation. This factor acted to shorten the lag time before the appearance of aggregates and to increase the number of aggregates for both species. The factor from M. coralloides presented a more selective activity on the aggregation of this species.     

Sponge cell aggregation

Summary Dissociated sponge cell system has proved to be a useful model to study the process of cell aggregation both on cellular and subcellular level. The purpose of this review is to discuss recent results obtained from experiments with the marine sponge Geodia cydonium.Dissociated cells form functional aggregates during a process which can be sub-divided into three phases: first, formation of small primary aggregates in the presence of Ca²⁺; second, formation of secondary aggregates in the presence of an aggregation factor and third, reconstitution of a functional system of watercontaining channels by rearrangement in the secondary aggregates.On subcellular level a series of macromolecules are known which are involved in the control of aggregation and separation of sponge cells: Aggregation factor, aggregation receptor, anti-aggregation receptor, glucuronidase, ß-glucuronosyltransferase, ß- ß-galactosidase and a lectin. These components might be linked in the following sequence: (a) Activation of the aggregation receptor by its enzymic glucuronylation; (b) Adhesive recognition of the cells, mediated by the aggregation factor and the glucuronylated aggregation receptor; (c) Inactivation of the aggregation receptor by its deglucuronylation with the membrane-associated ß-glucuronidase; (d) Cell separation due to either the loss of the recognition site (glucuronic acid) of the aggregation receptor for the aggregation factor or to an inactivation of the aggregation factor by the anti-aggregation receptor. The activity of the anti-aggregation receptor is most likely controlled by the Geodia lectin.The events leading to cell-cell recognition cause a change in the following metabolic events: Increase of oxygen uptake, decrease of cyclic AMP level, increase of cyclic GMP level and stimulation of programmed syntheses.Abbreviations AF aggregation factor - CPP circular proteid particle - AR aggregation receptor - aAR anti-aggregation receptor - CMF calcium- and magnesium-free artificial sea water - ASW calcium- and magnesium-containing artificial sea water This paper is dedicated to PROF. DR. R. K. ZAHN on the occasion of his 60^th birthday.     

Functional and genetic interactions of TOR in the budding yeast Saccharomyces cerevisiae with myosin type II-deficiency (myo1Δ)

Background

Protein misfolding and subsequent aggregation are hallmarks of several human diseases. The cell has a variety of mechanisms for coping with misfolded protein stress, including ubiquitin-mediated protein degradation. In fact, the presence of ubiquitin at protein aggregates is a common feature of protein misfolding diseases. Ubiquitin conjugating enzymes (UBCs) are part of the cascade of enzymes responsible for the regulated attachment of ubiquitin to protein substrates. The specific UBC used during ubiquitination can determine the type of polyubiquitin chain linkage, which in turn plays an important role in determining the fate of the ubiquitinated protein. Thus, UBCs may serve an important role in the cellular response to misfolded proteins and the fate of protein aggregates.

Results

The Q82 strain of C. elegans harbors a transgene encoding an aggregation prone tract of 82 glutamine residues fused to green fluorescent protein (Q82::GFP) that is expressed in the body wall muscle. When measured with time-lapse microscopy in young larvae, the initial formation of individual Q82::GFP aggregates occurs in approximately 58 minutes. This process is largely unaffected by a mutation in the C. elegans E1 ubiquitin activating enzyme. RNAi of ubc-22, a nematode homolog of E2-25K, resulted in higher pre-aggregation levels of Q82::GFP and a faster initial aggregation rate relative to control. Knockdown of ubc-1 (RAD6 homolog), ubc-13, and uev-1 did not affect the kinetics of initial aggregation. However, RNAi of ubc-13 decreases the rate of secondary growth of the aggregate. This result is consistent with previous findings that aggregates in young adult worms are smaller after ubc-13 RNAi. mCherry::ubiquitin becomes localized to Q82::GFP aggregates during the fourth larval (L4) stage of life, a time point long after most aggregates have formed. FLIP and FRAP analysis indicate that mCherry::ubiquitin is considerably more mobile than Q82::GFP within aggregates.

Conclusions

These data indicate that initial formation of Q82::GFP aggregates in C. elegans is not directly dependent on ubiquitination, but is more likely a spontaneous process driven by biophysical properties in the cytosol such as the concentration of the aggregating species. The effect of ubiquitination appears to be most significant in later, secondary aggregate growth.     

Contribution of calystegine catabolic plasmid to competitive colonization of the rhizosphere of calystegine-producing plants by Sinorhizobium meliloti Rm41

Calystegines are plant secondary metabolites produced by the roots of a few plant species, and the ability to catabolize calystegines is infrequent in rhizosphere bacteria. In Sinorhizobium meliloti Rm41, the endosymbiont of the legume Medicago sativa, this ability results from the presence of the genes cac (for calystegine catabolism) located on the nonsymbiotic plasmid pRme41a. The effect of the cac catabolic plasmid pRme41a on the ability of Rm41 to colonize the rhizosphere of calystegine-positive plants was studied using derivatives of Rm41 with or without cac catabolic plasmid. When strains were inoculated alone, the presence of a cac catabolic plasmid had no effect on their colonization of the rhizosphere, regardless of whether plants produced calystegines or not. However, a spontaneous rifampicin-resistant mutant of Rm41 containing a cac catabolic plasmid reached population levels in the rhizosphere of calystegine-positive plants that were several orders of magnitude higher than those of the same strain without the plasmid, when each was co-inoculated with a derivative of Rm41 cured of pRme41a. In contrast, the cac catabolic plasmid provided little or no selective advantage in the rhizosphere of calystegine-negative plants. In conclusion, the cac catabolic plasmid pRme41a can contribute to the ability of S. meliloti Rm41 to colonize the rhizosphere of alternative, nonlegume plant hosts producing calystegines.     

AggLb Is the Largest Cell-Aggregation Factor from Lactobacillus paracasei Subsp. paracasei BGNJ1-64, Functions in Collagen Adhesion,and Pathogen Exclusion In Vitro

Eleven Lactobacillus strains with strong aggregation abilities were selected from a laboratory collection. In two of the strains, genes associated with aggregation capability were plasmid located and found to strongly correlate with collagen binding. The gene encoding the auto-aggregation-promoting protein (AggLb) of Lactobacillus paracasei subsp. paracasei BGNJ1-64 was cloned using a novel, wide-range-host shuttle cloning vector, pAZILSJ. The clone pALb35, containing a 11377-bp DNA fragment, was selected from the SacI plasmid library for its ability to provide carriers with the aggregation phenotype. The complete fragment was sequenced and four potential ORFs were detected, including the aggLb gene and three surrounding transposase genes. AggLb is the largest known cell-surface protein in lactobacilli, consisting of 2998 aa (318,611 Da). AggLb belongs to the collagen-binding superfamily and its C-terminal region contains 20 successive repeats that are identical even at the nucleotide level. Deletion of aggLb causes a loss of the capacity to form cell aggregates, whereas overexpression increases cellular aggregation, hydrophobicity and collagen-binding potential. PCR screening performed with three sets of primers based on the aggLb gene of BGNJ1-64 enabled detection of the same type of aggLb gene in five of eleven selected aggregation-positive Lactobacillus strains. Heterologous expression of aggLb confirmed the crucial role of the AggLb protein in cell aggregation and specific collagen binding, indicating that AggLb has a useful probiotic function in effective colonization of host tissue and prevention of pathogen colonization.     

Characterization of the chaperone-like activity of HtrA (DegP) protein from Escherichia coli under the conditions of heat shock

The protective action of chaperone-like activity of HtrA protease against protein aggregation was studied. High levels of proteolytically inactive HtrAS210A (active center serine replaced by alanine) suppressed the temperature-sensitive phenotype of the htrA mutants. The ability of HtrAS210A to alleviate the lethality of htrA bacteria at high temperatures correlated well with the observed decrease of cellular level of large protein aggregates in cells overproducing HtrAS210A. The in vitro experiments proved that HtrA was very efficient in inhibiting the unfolded substrate (lysozyme) aggregation over a wide range of temperatures (30-45 °C). HtrA was able to bind to the denatured polypeptides and as a consequence limited their ability to form large aggregates. Our results suggest that HtrA may protect the bacterial cells from deleterious effects of heat shock not only by degrading the damaged proteins but by combination of the proteolytic and chaperoning activities.