By using an internal part of the dnaK gene from BaciUlus megaterium as a probe, a 5.2-kb Hindlll fragment of chromosomal DNA of BaciUlus subtilis was cloned. Downstream sequences were isolated by in vivo chromosome walking. Sequencing of 5,085 bp revealed four open reading frames in the order orf9-grpEdnaK-dnaJ. orJ39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases. Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins. Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species. Primer extension studies revealed three potential transcription start sites, two preceding orJ39 (Si and S2) and a third one in front of grpE (S3). S2 and S3 were activated at a high temperature. Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb. RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease. A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in a-amylase activity after heat induction. A 9-bp inverted repeat was detected in front of the coding region of orJ39. This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria. The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed. The dnaK locus was mapped at about 2230 on the B. subtilis genetic map.The heat shock response is a homeostatic mechanism that enables cells to survive a variety of environmental stresses. It is characterized by the increased synthesis of a group of evolutionarily conserved proteins, heat shock proteins (HSPs), and is a universal feature of both prokaryotic and eukaryotic cells (35). When Eschertichia coli cells are shifted to a high temperature, the synthesis of a set of about 20 HSPs transiently increases and then declines rapidly to steady-state levels characteristic of the new ambient temperature (44).The highly conserved HSPs perform similar functions in all organisms. One of these functions is the well-established regulation of protein-protein interactions by the chaperonins (69). One of the most abundant HSPs, HSP70, is highly conserved in evolution. It is found in such diverse organisms as E. coli, Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens (14, 35).The dnaK gene of E. coli was originally discovered because mutations in it blocked bacteriophage lambda DNA replication at all temperatures (22,23). Subsequently, the dnaK gene product was shown to be essential for E. coli viability at high and low temperatures (22,30,46,51,52), and genetic evid...

Kininogens, the precursor proteins of the vasoactive kinins, bind specifically, reversibly, and saturably to platelets, neutrophils, and endothelial cells. Two domains of the kininogens expose major cell binding sites: domain D3 that is shared by H- and L-kininogen and domain D5H that is exclusively present in H-kininogen. Previously we have mapped the kininogen cell binding sites to 27 residues of D3 ("LDC27") and 20 residues of D5H ("HKH20"", respectively (Herwald, H., Hasan, A. A. K., Godovac-Zimmermann, J., Schmaier, A. H., and Müller-Esterl, W. (1995) J. Biol. Chem. 270, 14634-14642; Hasan, A. A. K., Cines, D. B., Herwald, H., Schmaier, A. H., and Müller-Esterl, W. (1995) J. Biol. Chem. 270, 19256-19261). The corresponding kininogen acceptor site(s) exposed by the cell surfaces are still poorly defined. Using a non-ionic detergent, Nonidet P-40, we have been able to solubilize kininogen binding sites from an endothelial cell line, EA.hy926, in their functionally active form. Affinity chromatography of the solubilized kininogen binding sites on HKH20, a synthetic peptide representing the D5H cell binding site, allowed us to isolate a 33-kDa protein ("p33") that binds specifically and reversibly to H-kininogen with a KD (apparent dissociation constant) of 9 +/- 2 nM. Preparative SDS electrophoresis followed by NH2-terminal amino acid sequence analysis identified the kininogen-binding protein p33 as the gC1q receptor ("gC1qR"), an extrinsic membrane protein that interacts with the globular domains of the complement component C1q. The purified p33 binds C1q with moderate affinity, KD = 240 +/- 10 nM. Recombinant expression of the corresponding cDNA in Escherichia coli demonstrated that p33 binds H-kininogen, but not L-kininogen. Peptide HKH20 but not peptide LDC27 inhibited binding of H-kininogen to the recombinant p33 in a concentration-dependent manner, indicating that H-kininogen binds to p33 via domain D5H. Recombinant p33 efficiently inhibited the binding of H-kininogen to EA.hy926 cells. Factor XII, but not prekallikrein, competed with H-kininogen binding to p33. These findings suggest that an endothelial binding protein mediates the assembly of critical components of the kinin-generating pathway on the surface of endothelial cells, thereby linking the early events of kinin formation and complement activation.

Obesity is a major risk factor in the development of chronic renal failure. Rimonabant, a cannabinoid CB1 receptor antagonist, improves body weight and metabolic disorders; however, its effect on mortality and chronic renal failure associated with obesity is unknown. Obese Zucker rats received either rimonabant or vehicle for 12 months and were compared to a pair-fed but untreated group of obese rats. Mortality in the obese rats was significantly reduced by rimonabant along with a sustained decrease in body weight, transient reduction in food intake, and an increase in plasma adiponectin. This was associated with significant reduction in plasma total cholesterol, low-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio, triglycerides, glucose, norepinephrine, plasminogen activator inhibitor 1, and preservation of pancreatic weight and beta-cell mass index. The cannabinoid antagonist attenuated the increase in proteinuria, urinary N-acetylglucosaminidase excretion, plasma creatinine, and urea nitrogen levels while improving creatinine clearance. Renal hypertrophy along with glomerular and tubulointerstitial lesions were reduced by rimonabant. Although the drug did not modify hemodynamics, it normalized the pressor response to angiotensin II. Our study suggests that in a rat model of chronic renal failure due to obesity, rimonabant preserves renal function and increases survival.

Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+-calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme; however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two-hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting protein), which avidly binds to the carboxyl-terminal region of the eNOS oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of eNOS and NOSIP in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the caveolin-1 scaffolding domain. NO production was significantly reduced in eNOS-expressing CHO cells (CHO-eNOS) that transiently overexpressed NOSIP. Stimulation with the calcium ionophore A23187 induced the reversible translocation of eNOS from the detergent-insoluble to the detergent-soluble fractions of CHO-eNOS, and this translocation was completely prevented by transient coexpression of NOSIP in CHO-eNOS. Immunofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO-eNOS that was abolished in the presence of NOSIP. Subcellular fractionation studies identified eNOS in the caveolin-rich membrane fractions of CHO-eNOS, and coexpression of NOSIP caused a shift of eNOS to intracellular compartments. We conclude that NOSIP is a novel type of modulator that promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.

Activity and localization of endothelial nitric oxide synthase (eNOS) is regulated in a remarkably complex fashion, yet the complex molecular machinery mastering stimulus-induced eNOS translocation and trafficking is poorly understood. In a search by the yeast two-hybrid system using the eNOS oxygenase domain as bait, we have identified a previously uncharacterized eNOS-interacting protein, dubbed NOSTRIN (for eNOS traffic inducer). NOSTRIN contains a single polypeptide chain of 506-aa residues of 58 kDa with an N-terminal cdc15 domain and a Cterminal SH3 domain. NOSTRIN mRNA is abundant in highly vascularized tissues such as placenta, kidney, lung, and heart, and NOSTRIN protein is expressed in vascular endothelial cells. Coimmunoprecipitation experiments demonstrated the eNOS-NOSTRIN interaction in vitro and in vivo, and NOSTRIN's SH3 domain was essential and sufficient for eNOS binding. NOSTRIN colocalized extensively with eNOS at the plasma membrane of confluent human umbilical venous endothelial cells and in punctate cytosolic structures of CHO-eNOS cells. NOSTRIN overexpression induced a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern and at the same time led to a significant inhibition of NO release. We conclude that NOSTRIN contributes to the intricate protein network controlling activity, trafficking, and targeting of eNOS. N itric oxide (NO) is a potent mediator in biological processes such as neurotransmission, inflammatory response, and vascular homeostasis (1). The prime source of NO in the cardiovascular system is endothelial NO synthase (eNOS), which is tightly regulated with respect to activity and localization. For example, coordinated phosphorylation contributes to activity control of eNOS because of activating and inhibiting phosphorylation at S1179 and T495, respectively (2-6). Myristoylation and dual palmitoylation at its extreme N terminus target eNOS to the cytoplasmic face of the Golgi complex and to the plasma membrane (7), where eNOS is thought to be fully capable of activation (8, 9). Misrouting of acylation-deficient eNOS impairs NO production (10, 11), indicating that correct subcellular targeting is critical for stimulus-dependent activation of the enzyme (8). Posttranslational modifications are efficiently complemented by multiple proteinprotein interactions that help regulate eNOS activity with respect to time and space. For instance, chaperone hsp90 bound to eNOS may mediate vascular endothelial growth factor-induced eNOS phosphorylation by promoting the interaction between eNOS and Akt (12, 13). At the plasma membrane, eNOS is complexed to and inhibited by the master components of caveolae, i.e., caveolin-1 in endothelial cells (9, 14) and caveolin-3 in cardiac myocytes (15). After stimulus-induced [Ca 2ϩ ] i increase, the Ca 2ϩ -calmodulin complex displaces eNOS from caveolin (16), stimulates eNOS to produce NO, and subsequently leads to the redistribution of eNOS from plasma membrane caveolae (17). The complexity...

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We identified the multifunctional chaperon protein p32 as a protein kinase C (PKC)-binding protein interacting with PKC␣, PKC, PKC␦, and PKC. We have analyzed the interaction of PKC with p32 in detail, and we show here in vivo association of PKC, as revealed from yeast two-hybrid analysis, precipitation assays using glutathione S-transferase fusion proteins, and reciprocal coimmunoprecipitation. In SKW 6.4 cells, PKC is constitutively associated with p32 at mitochondrial membranes, evident from colocalization with cytochrome c. p32 interacts with PKC in a compartmentspecific manner, as it can be coimmunoprecipitated mainly from the particulate and not from the soluble fraction, despite the presence of p32 in both fractions. Although p32 binds to the kinase domain of PKC, it does not serve as a substrate. Interestingly, PKC-p32 immunocomplexes precipitated from the particulate fraction of two distinct cell lines, SKW 6.4 and 293T, show no detectable substrate phosphorylation. In support of a kinase regulatory function of p32, addition of p32 to in vitro kinase assays blocked, in a dose-dependent manner, aldolase but not autophosphorylation of PKC, suggesting a steric hindrance of substrate within the kinase domain. Together, these findings identify p32 as a novel, compartment-specific regulator of PKC kinase activity. The protein kinases C (PKC)1 comprise a family of intracellular serine/threonine-specific kinases, which are implicated in signal transduction of a wide range of biological responses including changes in cell morphology, proliferation, and differentiation (1-3). The currently defined 11 members of the PKC family can be grouped into the three major classes of Ca 2ϩ -dependent classical PKCs, Ca 2ϩ -independent, novel PKCs, and Ca 2ϩ -and lipid-independent atypical PKCs as well as PKC and its mouse homologue PKD (4, 5), which do not conform to either one of these major classes and may thus define a new subgroup (6). PKC/PKD differ from the three major groups of PKC isozymes by the presence of an amino-terminal hydrophobic domain, an acidic domain (7), a pleckstrin homology domain within the regulatory region (8), and lack of a typical pseudosubstrate site. PKC is ubiquitously expressed, and evidence for the involvement of PKC in diverse cellular functions stems from reports showing enhancement of constitutive transport processes in PKC-overexpressing epithelial cells (9), G protein-mediated regulation of Golgi organization (10), and involvement in protection from apoptosis (11). Interestingly, PKC shows particularly high expression in thymus and hematopoietic cells suggesting a potential role in immune functions (12). In accordance with this is the finding that, upon B cell receptor stimulation, PKC is recruited together with the tyrosine kinase Syk and phospholipase C␥ to the B cell receptor complex and negatively regulates phospholipase C␥ activity (13).In addition to lipid second messengers as regulators of PKC translocation and activation, there is increasing evidence for a role of regulatory prot...

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