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PubMed2023

Molecular Characterization of the ClpC AAA+ ATPase in the Biology of Chlamydia trachomatis.

Bacterial AAA+ unfoldases are crucial for bacterial physiology by recognizing specific substrates and, typically, unfolding them for degradation by a proteolytic component. The caseinolytic protease (Clp) system is one example where a hexameric unfoldase (e.g., ClpC) interacts with the tetradecameric proteolytic core ClpP. Unfoldases can have both ClpP-dependent and ClpP-independent roles in protein homeostasis, development, virulence, and cell differentiation. ClpC is an unfoldase predominantly found in Gram-positive bacteria and mycobacteria. Intriguingly, the obligate intracellular Gram-negative pathogen Chlamydia, an organism with a highly reduced genome, also encodes a ClpC ortholog, implying an important function for ClpC in chlamydial physiology. Here, we used a combination of in vitro and cell culture approaches to gain insight into the function of chlamydial ClpC. ClpC exhibits intrinsic ATPase and chaperone activities, with a primary role for the Walker B motif in the first nucleotide binding domain (NBD1). Furthermore, ClpC binds ClpP1P2 complexes via ClpP2 to form the functional protease ClpCP2P1 in vitro, which degraded arginine-phosphorylated β-casein. Cell culture experiments confirmed that higher order complexes of ClpC are present in chlamydial cells. Importantly, these data further revealed severe negative effects of both overexpression and depletion of ClpC in Chlamydia as revealed by a significant reduction in chlamydial growth. Here, again, NBD1 was critical for ClpC function. Hence, we provide the first mechanistic insight into the molecular and cellular function of chlamydial ClpC, which supports its essentiality in Chlamydia. ClpC is, therefore, a potential novel target for the development of antichlamydial agents. IMPORTANCE Chlamydia trachomatis is an obligate intracellular pathogen and the world's leading cause of preventable infectious blindness and bacterial sexually transmitted infections. Due to the high prevalence of chlamydial infections along with negative effects of current broad-spectrum treatment strategies, new antichlamydial agents with novel targets are desperately needed. In this context, bacterial Clp proteases have emerged as promising new antibiotic targets, since they often play central roles in bacterial physiology and, for some bacterial species, are even essential for survival. Here, we report on the chlamydial AAA+ unfoldase ClpC, its functional reconstitution and characterization, individually and as part of the ClpCP2P1 protease, and establish an essential role for ClpC in chlamydial growth and intracellular development, thereby identifying ClpC as a potential target for antichlamydial compounds.

Humans↗

PubMed1995

The stroma of higher plant plastids contain ClpP and ClpC, functional homologs of Escherichia coli ClpP and ClpA: an archetypal two-component ATP-dependent protease.

A cDNA representing the plastid-encoded homolog of the prokaryotic ATP-dependent protease ClpP was amplified by reverse transcription-polymerase chain reaction, cloned, and sequenced. ClpP and a previously isolated cDNA designated ClpC, encoding an ATPase related to proteins encoded by the ClpA/B gene family, were expressed in Escherichia coli. Antibodies directed against these recombinant proteins recognized proteins in a wide variety of organisms. N-terminal analysis of the Clp protein isolated from crude leaf extracts showed that the N-terminal methionine is absent from ClpP and that the transit peptide is cleaved from ClpC. A combination of chloroplast subfractionation and immunolocalization showed that in Arabidopsis, ClpP and ClpC localize to the stroma of the plastid. Immunoblot analyses indicated that ClpP and ClpC are constitutively expressed in all tissues of Arabidopsis at levels equivalent to those of E. coli ClpP and ClpA. ClpP, immunopurified from tobacco extracts, hydrolyzed N-succinyl-Leu-Tyr-amidomethylcoumarin, a substrate of E. coli ClpP. Purified recombinant ClpC facilitated the degradation of 3H-methylcasein by E. coli ClpP in an ATP-dependent fashion. This demonstrates that ClpC is a functional homolog of E. coli ClpA and not of ClpB or ClpX. These data represent the only in vitro demonstration of the activity of a specific ATP-dependent chloroplast protease reported to date.

ATP-Dependent Proteases↗

PubMed1994

Stress induction of clpC in Bacillus subtilis and its involvement in stress tolerance.

A member of the clpC subfamily of stress response-related Clp ATPases was cloned from Bacillus subtilis. The B. subtilis clpC gene was induced in response to various stresses, including heat shock. Its product was identified as a general stress protein (Gsp12) described previously. A dramatic increase in the amount of clpC mRNA immediately after exposure to multiple stresses suggested regulation on a transcriptional level. Induction by heat shock was independent of the alternative sigma factor SigB, indicating a new mechanism of heat shock induction in B. subtilis. A clpC insertional mutant had an impaired tolerance for heat shock and salt stress. Furthermore, the mutation triggered the formation of elongated cells, a phenomenon particularly pronounced during stress.

Adaptation, Biological↗

PubMed1994

MecB of Bacillus subtilis, a member of the ClpC ATPase family, is a pleiotropic regulator controlling competence gene expression and growth at high temperature.

The Bacillus subtilis DegS-DegU histidine kinase-response regulator pair controls the expression of genes encoding degradative enzymes such as levansucrase (sacB) and of genes involved in genetic competence. The mecA and mecB mutations were previously isolated as allowing competence gene expression in complex media. We have shown that the mec mutations also lead to overexpression of sacB, bypassing the DegS-DegU requirement. This expression was shown to be entirely dependent upon ComK, a positive regulator of competence gene expression. The mecB gene was cloned and its nucleotide sequence was determined. The predicted MecB protein show very high similarity over its entire length with members of the ClpC family of ATPases (60% identity). MecB is essential for growth of B. subtilis at high temperature. MecB also acts as a negative regulator of ComK synthesis, thus preventing late competence gene expression. We suggest that under these conditions MecB may interact with MecA to sequester or otherwise inactivate ComK. In response to an unknown signal, active ComK would accumulate through a positive feedback loop, leading to expression of competence genes allowing DNA uptake.

Adenosine Triphosphatases↗

PubMed1984

An immunological study of papillary conjunctivitis due to contact lenses.

Serum and tear levels of IgE were compared in patients with contact lens papillary conjunctivitis (CLPC), vernal keratoconjunctivitis (VC), healthy contact lens wearers (CLW), and healthy controls without lenses. The mean of serum IgE was elevated only in the VC group. Tear IgE levels were significantly higher for CLPC and VC than for CLW and healthy controls. Tear IgG levels were also increased in CLPC patients. With serum albumin (HSA) as a marker for leakage of proteins from the circulation to the tear fluid, the data indicated, that tear IgG was blood-borne whereas tear IgE was essentially a product of local synthesis. In one of the 10 CLPC patients, high titers of IgE type antibodies to housedust mites and cat epithelium were demonstrated in both serum and tears. We conclude that CLPC is usually an IgE mediated reaction to the lens material or to contaminations of allergenic material that sticks to the lenses.

Adolescent↗

PubMed1983

Monoclonal antibodies against T cell differentiation antigens initiate stimulation of monocyte/macrophage oxidative metabolism.

Within the first minute after incubation with the mouse anti-human T cell orthoclone monoclonal antibodies OKT3, OKT4, and OKT8, and in the absence of complement, human monocytes generate a burst of highly reactive oxygen metabolites as detected by a luminol-dependent photometric chemiluminescence (CL) assay. The kinetics of the CL responses to these antibodies are identical to that induced by OKM1, the monoclonal antibody to human monocytes and granulocytes. With regard to CL response intensities, OKM1 induces the maximal response and those of OKT3, OKT4, and OKT8 closely reflect the proportion of T cell subsets recognized by these antibodies in peripheral blood. This reaction is also observed when monoclonal antibodies against mouse Lyt surface determinants (Lyt-1 and Lyt-2) and Thy-1 antigen are tested against murine spleen cells. This murine model was further used to investigate the specificity and the mechanism of this reaction. It was demonstrated that the CL response is Lyt antigen specific, occurs upon addition of monoclonal IgG but not IgM antibodies, requires the concomitant presence of CL-producing cells (CLPC) (promonocytes, monocytes, macrophages, and/or granulocytes) and of fully differentiated T cells, and lastly, is mediated via a T cell opsonization process. Selective blockade of bone marrow cell Fc receptors (FcR II) with monoclonal anti-mouse FcR II antibody inhibits the CL response to IgG2b anti-T cell antibody-coated thymocytes and thus strongly suggests that the stimulation of CLPC oxidative metabolism in this model results from the binding of opsonized T cells to plasma membrane Fc receptors. These observations lend additional support to increasing evidence that the initiation of effector functions by monoclonal anti-T cell antibodies may be strictly dependent upon the presence of monocytes and/or macrophages.

Animals↗

PubMed1978

Does personal future outlook cause self-esteem?

Melges et al. (1971) presented evidence that a client's sense of self-worth is highly correlated with his degree of optimism about his personal future. In the present study, the cross-lagged panel correlation (CLPC) technique was used to test their hypothesis that changes in personal future outlook lead to changes in self-esteem. Ninety-four outpatients in the private psychiatric practice of the senior author filled out the Decision-Making Evaluation Instrument used by Melges et al. to assess these two constructs as well as decision-making and outcome. Results did not support their implication that personal future outlook is causally predominant over self-esteem, nor did the results support their assumption that they were measuring a distinct construct of self-esteem.

Adolescent↗

PubMed1989

The Glenn A. Fry Award lecture 1988: the ocular response to contact lens wear.

Contact lens materials over the last 20 years have progressed from polymethyl methacrylate (PMMA) through hydrogels to high Dk rigid and semi-soft lenses. Our understanding of how and why contact lenses affect the eye has also advanced considerably. We now know that: (1) the oxygen demands of the eye are high and (2) carbon dioxide accumulates and corneal acidosis occurs with most current contact lenses, especially with extended wear (EW). The effects of these perturbations on the epithelium include decreased metabolic rate, thinning, microcysts, and reduced adhesion. In addition, stromal edema and thinning and, with pressure, distortion occur with the long-term use of low to moderate oxygen transmissible lenses. Endothelial polymegethism also occurs and is a relatively permanent effect of inadequate oxygen permeability (Dk). The major consequence of chronic hypoxia and acidosis is, for a significant number of patients, corneal intolerance and discontinuation of lens wear. High permeability materials (100 Dk) that provide adequate gaseous exchange for daily wear and minimal changes in EW are now available and should be used where possible. The more obvious problems of contact lens-induced chronic inflammation, e.g., contact lens-induced papillary conjunctivitis (CLPC), and acute inflammation, e.g., acute red eye (ARE), are less well understood. Protein deposits, lens ageing, occlusion, mechanical effects, and bacterial contamination have all been implicated. The remaining frontiers include understanding and avoiding the stimuli to low grade irritation and inflammation by making contact lenses more comfortable and improving their compatibility with the ocular surfaces.

Acidosis↗