Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis

DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.     

Maternal care and development of stress responses in baboons

The ability to mount a successful response to threats is critical for an organism's survival. A key element of the stress response is its nonspecificity toward the stress source, with similar endocrine and behavioral changes expected under a variety of stressors. In this project we utilized an experimental design that accounts for multiple sources of variation to further understand the nature of stress responsivity and its relationship to the early rearing environment. A sample of baboons (n=73) was observed during the early phase of life in their social group, and then tested as juveniles in a challenging situation. Maternal cortisol levels were measured during the peripartum period. The challenging situation (individuals were isolated for a few minutes in a single cage) was designed to be a moderate source of psychological stress. Patterns in individual differences during the stress test were "mapped" by means of multidimensional scaling (MDS). After the observation was made, the subject was sedated and a blood sample was taken to measure cortisol levels. Our results indicate that when juvenile baboons are confronted with a source of psychological stress, they show a multidimensional behavioral response, probably mediated by the activation and synergic interaction among different neurohormonal systems that, ultimately, act on the hypothalamus-pituitary-adrenal (HPA) axis. Different components of the multidimensional, or nonspecific, behavioral response are associated with the quality and quantity of interactions with their mothers during early life. Juveniles whose mothers displayed higher levels of positive interaction were characterized by vigilant but less active reactions to the stress test, whereas juveniles of mothers that displayed high levels of stress-related behaviors had higher cortisol and locomotion levels.     

Crystal structure of the E. coli dipeptidyl carboxypeptidase Dcp: further indication of a ligand-dependent hinge movement mechanism

Dcp from Escherichia coli is a 680 residue cytoplasmic peptidase, which shows a strict dipeptidyl carboxypeptidase activity. Although Dcp had been assigned to the angiotensin I-converting enzymes (ACE) due to blockage by typical ACE inhibitors, it is currently grouped into the M3 family of mono zinc peptidases, which also contains the endopeptidases neurolysin and thimet oligopeptidase (TOP). We have cloned, expressed, purified, and crystallized Dcp in the presence of an octapeptide "inhibitor", and have determined its 2.0A crystal structure using MAD methods. The analysis revealed that Dcp consists of two half shell-like subdomains, which enclose an almost closed two-chamber cavity. In this cavity, two dipeptide products presumably generated by Dcp cleavage of the octapeptide bind to the thermolysin-like active site fixed to side-chains, which are provided by both subdomains. In particular, an Arg side-chain backed by a Glu residue, together with two Tyr phenolic groups provide a charged anchor for fixing the C-terminal carboxylate group of the P2' residue of a bound substrate, explaining the strict dipeptidyl carboxypeptidase specificity of Dcp. Tetrapeptidic substrates are fixed only via their main-chain functions from P2 to P2', suggesting a broad residue specificity for Dcp. Both subdomains exhibit very similar chain folds as the equivalent but abducted subdomains of neurolysin and TOP. Therefore, this "product-bound" Dcp structure seems to represent the inhibitor/substrate-bound "closed" form of the M3 peptidases, generated from the free "open" substrate-accessible form by a hinge-bending mechanism. A similar mechanism has recently been demonstrated experimentally for ACE2.     

S100A9 deficiency alters adenosine-5'-triphosphate induced calcium signalling but does not generally interfere with calcium and zinc homeostasis in murine neutrophils

The two calcium- and zinc-binding proteins, S100A9 and S100 A8, abundant in myeloid cells are considered to play important roles in both calcium signalling and zinc homeostasis. Polymorphonuclear neutrophils from S100A9 ko mice are also devoid of S100A8. Therefore, S100A9-deficient neutrophils were used as a model to study the role of the two S100 proteins in the neutrophils's calcium and zinc metabolism. Analysis of the intracellular zinc level upon pyrithione and (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide (NOR-1) treatment revealed no differences between S100A9-deficient and wildtype neutrophils. Similar, the calcium signals were not distinguishable from S100A9-deficient and wildtype neutrophils upon stimulation with platelet activating factor (PAF), thapsigargin or macrophage inflammatory protein 1 alpha (MIP-1 alpha), indicating despite their massive expression S100A8/A9 do neither serve as calcium nor as zinc buffering proteins in granulocytes. In contrast, stimulation with adenosine-5'-triphosphate (ATP) induces a significant stronger increase of the intracellular free calcium level in S100A9-deficient cells compared to wildtype cells. Moreover, the ATP-induced calcium signal was still different when the cells were incubated in calcium free buffer suggesting that pirinergic receptors of the P(2Y) class could be involved in this signalling pathway.     

Formation of the head organizer in hydra involves the canonical Wnt pathway

Stabilization of beta-catenin by inhibiting the activity of glycogen synthase kinase-3beta has been shown to initiate axis formation or axial patterning processes in many bilaterians. In hydra, the head organizer is located in the hypostome, the apical portion of the head. Treatment of hydra with alsterpaullone, a specific inhibitor of glycogen synthase kinase-3beta, results in the body column acquiring characteristics of the head organizer, as measured by transplantation experiments, and by the expression of genes associated with the head organizer. Hence, the role of the canonical Wnt pathway for the initiation of axis formation was established early in metazoan evolution.     

Type I diabetes affects skeletal muscle glutamine uptake in a fiber-specific manner

Skeletal muscle serves as the body's major glutamine repository, and releases glutamine at enhanced rates during diabetes, but whether all muscles are equally affected is unknown. System N(m) activity mediates most trans-sarcolemmal glutamine movement, and although two System N (SN) isoforms have been identified (SN1/sodium-coupled neutral amino acid transporter or System N and A transporters [SNAT]-3; and SN2/SNAT5), their expression in skeletal muscle remains controversial. Here, the impact of Type I diabetes on glutamine uptake and System N transporter expression were examined in fast- and slow-twitch skeletal muscle from spontaneously diabetic (BB/Wor-DP) rats. Net glutamine uptake in fast-twitch fibers was decreased 75%-95%, but enhanced more than 2-fold in slow-twitch muscle from diabetic animals relative to nondiabetic controls. Both SNAT3 and SNAT5 mRNA were expressed in both muscle fiber types and their abundance was unaffected by diabetes. This represents the first report of differential fiber-specific effects of diabetes on skeletal muscle glutamine transport and the co-expression of distinct System N transporters in skeletal muscle.     

The intermediate filament protein vimentin is a new target for epigallocatechin gallate

Epigallocatechin gallate (EGCG) is the major active polyphenol in green tea. Protein interaction with EGCG is a critical step in the effects of EGCG on the regulation of various key proteins involved in signal transduction. We have identified a novel molecular target of EGCG using affinity chromatography, two-dimensional electrophoresis, and mass spectrometry for protein identification. Spots of interest were identified as the intermediate filament, vimentin. The identification was confirmed by Western blot analysis using an anti-vimentin antibody. Experiments using a pull-down assay with [3H]EGCG demonstrate binding of EGCG to vimentin with a Kd of 3.3 nm. EGCG inhibited phosphorylation of vimentin at serines 50 and 55 and phosphorylation of vimentin by cyclin-dependent kinase 2 and cAMP-dependent protein kinase. EGCG specifically inhibits cell proliferation by binding to vimentin. Because vimentin is important for maintaining cellular functions and is essential in maintaining the structure and mechanical integration of the cellular space, the inhibitory effect of EGCG on vimentin may further explain its anti-tumor-promoting effect.     

Mutations of the PC2 substrate binding pocket alter enzyme specificity

By taking advantage of the recently published furin structure, whose catalytic domain shares high homology with other proprotein convertases, we designed mutations in the catalytic domain of PC2, altering residues Ser206, Thr271, Asp278, ArgGlu282, AlaSer323, Leu341, Asn365, and Ser380, which are both conserved and specific to this convertase, and substituting residues specific to PC1 and/or furin. In order to investigate the determinants of PC2 specificity, we have tested the mutated enzymes against a set of proenkephalin-derived substrates, as well as substrates representing Arg, Ala, Leu, Phe, and Glu positional scanning variants of a peptide B-derived substrate. We found that the exchange of the Ser206 residue with Arg or Lys led to a total loss of activity. Increased positive charge of the substrate generally resulted in an increased specificity constant. Most intriguingly, the RE281GR mutation, corresponding to a residue placed distantly in the S6 pocket, evoked the largest changes in the specificity pattern. The D278E and N356S mutations resulted in distinct alterations in PC2 substrate preferences. However, when other residues that distinguish PC2 from other convertases were substituted with PC1-like or furin-like equivalents, there was no significant alteration of the PC2 specificity pattern, suggesting that the overall structure of the substrate binding cleft rather than individual residues specifies substrate binding.     

Dual function of interleukin-1beta for the regulation of interleukin-6-induced suppressor of cytokine signaling 3 expression

Interleukin-6 (IL-6) exerts pro- as well as anti-inflammatory activities in response to infection, injury, or other stimuli that affect the homeostasis of the organism. IL-6-induced expression of acute-phase protein genes in the liver is tightly regulated through both IL-6-induced feedback inhibitors and the activity of pro-inflammatory cytokines such as tumor necrosis factor alpha and interleukin-1beta. In previous studies mechanisms for how IL-1beta counteracts IL-6-dependent acute-phase protein gene induction have been proposed. Herein we analyzed IL-1beta-mediated regulation of IL-6-induced expression of the feedback inhibitor SOCS3. In hepatocytes IL-1beta alone does not induce SOCS3 expression, but it counteracts SOCS3-promoter activation in long term studies. Surprisingly, short term stimulation revealed IL-1beta to be a potent enhancer of SOCS3 expression in concert with IL-6. This activity of IL-1beta does not depend on IL-1beta-dependent STAT1-serine phosphorylation but on NF-kappaB-dependent gene induction. Such a regulatory network allows IL-1beta to counteract IL-6-dependent expression of acute-phase protein genes without inhibiting IL-6-induced SOCS3 expression and provides a reasonable mechanism for the IL-1beta-dependent inhibition of acute-phase gene induction, because reduced SOCS3 expression would lead to enhanced IL-6 activity.