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The folding of proteins in living cells may start during their synthesis when the poiypeptides emerge gradually at the ribosomal exit tunnel. However, our current understanding of cotranslational folding processes at the atomic level is limited. We employed NMR spectroscopy to monitor the conformation of the SH3 domain from [alpha]-spectrin at sequential stages of elongation via in vivo ribosome-arrested [sup.15]N,[sup.13]C-labeled nascent polypeptides. These nascent chains exposed either the entire SH3 domain or C-terminally truncated segments thereof, thus providing snapshots of the translation process. We show that nascent SH3 polypeptides remain unstructured during elongation but fold into a compact, native-like [beta]-sheet assembly when the entire sequence information is available. Moreover, the ribosome neither imposes major conformational constraints nor significantly interacts with exposed unfolded nascent SH3 domain moieties. Our data provide evidence for a domainwise folding of the SH3 domain on ribosomes without significant population of folding intermediates. The domain follows a thermodynamically favorable pathway in which sequential folding units are stabilized, thus avoiding kinetic traps during the process of cotranslational folding. nascent chains | protein folding | ribosome doi/ 10.1073/pnas.0914300107

Two small polypeptides, PorA and PorH, are known to form cell wall channels in Corynebacterium glutamicum and in Corynebacterium efficiens. The genes coding for both polypeptides are localized in close proximity to one another between the genes coding for GroEl2 and a polyphosphate kinase (PKK2). In this study, we investigated the relationship of PorA and PorH to one another. The results suggested that the major cell wall channels of Corynebacterium glutamicum, Corynebacterium efficiens, and Corynebacterium diphtheriae need the obligatory presence of two distinct polypeptides, one of class PorA and one of class PorH, to form an active cell wall channel. Identification of genes coding for homologous proteins in the chromosome of Corynebacterium callunae suggested a similar result for this strain. Contrary to our previous reports on channel-forming proteins in these strains, a heterooligomeric structure composed of PorA and PorH is needed in all of them to form the major cell wall channel. This was concluded from complementation experiments using a porH- and porA-deficient C. glutamicum strain. The stringent necessity of proteins of either class to recover the wild-type channels was demonstrated by black lipid bilayer experiments using detergent or organic solvent extracts of the complemented porH- and porA-deficient C. glutamicum strain. The channel-forming capability of recombinant expressed, affinity-purified PorA and PorH proteins of C. glutamicum revealed that the channels consisted solely of these two components. This agreed with results obtained from a transcript coding for both channel-forming components identified in C. glutamicum by Northern blot analysis and reverse transcription-PCR analysis. The transcription start point of the genes was determined by the rapid amplification of cDNA ends approach, allowing the prediction of the -35 and -10 regions of the promoter. The results demonstrate that the cell wall channels within the genus Corynebacterium may be formed by two-component oligomers. doi: 10.1128/JB.0 1142-09

Musson MC, Jepeal LI, Mabray PD, Zhdanova IV, Cardoso WV, Wolfe MM. Expression of glucose-dependent insulinotropic polypeptide in the zebrafish. Am J Physiol Regul Integr Comp Physiol 297: R1803-R1812, 2009. First published September 30, 2009; doi: 10.1152/ajpregu.00288.2009.--In mammals, glucose-dependent insulinotropic polypeptide (GIP) is synthesized predominately in the small intestine and functions in conjunction with insulin to promote nutrient deposition. However, little is known regarding GIP expression and function in early vertebrates like the zebrafish, a model organism representing an early stage in the evolutionary development of the compound vertebrate pancreas. Analysis of GIP and insulin (insa) expression in zebrafish larvae by RT-PCR demonstrated that although insa was detected as early as 24 h postfertilization (hpf), GIP expression was not demonstrated until 72 hpf, shortly after the completion of endocrine pancreatic development but prior to the commencement of independent feeding. Furthermore, whole mount in situ hybridization of zebrafish larvae showed expression of GIP and insa in the same tissues, and in adult zebrafish, RT-PCR and immunohistochemistry demonstrated GIP expression in both the intestine and the pancreas. Receptor activation studies showed that zebrafish GIP was capable of activating the rat GIP receptor. Although previous studies have identified four receptors with glucagon receptor-like sequences in the zebrafish, one of which possesses the capacity to bind GIP, a functional analysis of these receptors has not been performed. This study demonstrates interactions between the latter receptor and zebrafish GIP, identifying it as a potential in vivo target for the ligand. Finally, food deprivation studies in larvae demonstrated an increase in GIP and proglucagon II mRNA levels in response to fasting. In conclusion, the results of these studies suggest that although the zebrafish appears to he a model of an early stage of evolutionary development of GIP expression, the peptide may not possess incretin properties in this species. incretin hormones; enteroinsular axis; endocrine pancreas doi: 10.1152/ajpregu.00288.2009

Moriya R, Shirakura T, Ito J, Mashiko S, Seo T. Activation of sodium-glucose cotransporter 1 ameliorates hyperglycemia by mediating incretin secretion in mice. Am J Physiol Endocrinol Metab 297: EI358-E1365, 2009. First published October 6, 2009; doi: 10.1152/ajpendo.00412.2009.--Glucose ingestion stimulates the secretion of the incretin hormones, glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1). Despite the critical role of incretins in glucose homeostasis, the mechanism of glucose-induced incretin secretion has not been established. We investigated the underlying mechanism of glucose-induced incretin secretion in vivo in mice. Injection of glucose at 1 g/kg in the upper intestine significantly increased plasma GIP and GLP-1 levels, whereas injection of glucose in the colon did not increase GIP or GLP-1 levels. This finding indicates that the glucose sensor for glucose-induced incretin secretion is in the upper intestine. Coadministration of a sodium-glucose cotransporter- 1 (SGLT 1) inhibitor, phloridzin, with glucose in the upper intestine blocked glucose absorption and glucose-induced incretin secretion. [alpha]-methyl-D-glucopyranoside (MDG), an SGLT1 substrate that is a nonmetabolizable sugar, significantly increased plasma GIP and GLP-1 levels, whereas phloridzin blocked these increases, indicating that concomitant transport of sodium ions and glucose (substrate) via SGLT1 itself triggers incretin secretion without the need for subsequent glucose metabolism. Interestingly, oral administration of MDG significantly increased plasma GIP, GLP-1, and insulin levels and reduced blood glucose levels during an intraperitoneal glucose tolerance test. Furthermore, chronic MDG treatment in drinking water (3%) for 13 days reduced blood glucose levels after a 2-h fast and in an oral glucose tolerance test in diabetic db/db mice. Our findings indicate that SGLT1 serves as the intestinal glucose sensor for glucose-induced incretin secretion and that a noncalorigenic SGLT1 substrate ameliorates hyperglycemia by stimulating incretin secretion. glucagon-like peptide-l; glucose-dependent insulinotropic peptide; [alpha]-methyl-D-glucopyranoside; insulin resistance doi: 10.1152/ajpendo.00412.2009

The primary sigma factor of Staphylococcus aureus, [[sigma].sup.SA], regulates the transcription of many genes, including several essential genes, in this bacterium via specific recognition of exponential growth phase promoters. In this study, we report the existence of a novel staphylococcal phage G1-derived growth inhibitory polypeptide, referred to as G1ORF67, that interacts with [[sigma].sup.SA] both in vivo and in vitro and regulates its activity. Delineation of the minimal domain of [[sigma].sup.SA] that is required for its interaction with G1ORF67 as amino acids 294 to 360 near the carboxy terminus suggests that the G1 phage-encoded anti-[sigma] factor may occlude the -35 element recognition domain of [[sigma].sup.SA]. AS would be predicted by this hypothesis, the G1ORF67 polypeptide abolished both RNA polymerase core-dependent binding of [[sigma].sup.SA] to DNA and [[sigma].sup.SA]-dependent transcription in vitro. While G1ORF67 profoundly inhibits transcription when expressed in S. aureus cells in mode of action studies, our finding that G1ORF67 was unable to inhibit transcription when expressed in Escherichia coli concurs with its inability to inhibit transcription by the E. coli holoenzyme in vitro. These features demonstrate the selectivity of G1ORF67 for S. aureus RNA polymerase. We predict that G1ORF67 is one of the central polypeptides in the phage G1 strategy to appropriate host RNA polymerase and redirect it to phage reproduction.

The extent to which polypeptide conformation depends on side-chain composition and sequence has been widely studied, but less is known about the importance of maintaining an [alpha]-amino acid backbone. Here, we examine a series of peptides with backbones that feature different repeating patterns of [alpha]- and [beta]-amino acid residues but an invariant side-chain sequence. In the pure [alpha]-backbone, this sequence corresponds to the previously studied peptide GCN4-pLI, which forms a very stable four-helix bundle quaternary structure. Physical characterization in solution and crystallographic structure determination show that a variety of [alpha]/[beta]-peptide backbones can adopt sequence-encoded quaternary structures similar to that of the a prototype. There is a loss in helix bundle stability upon [beta]-residue incorporation; however, stability of the quaternary structure is not a simple function of [beta]-residue content. We find that cyclically constrained [beta]-amino acid residues can stabilize the folds of [alpha]/[beta]-peptide GCN4-pLI analogues and restore quaternary structure formation to backbones that are predominantly unfolded in the absence of cyclic residues. Our results show a surprising degree of plasticity in terms of the backbone compositions that can manifest the structural information encoded in a sequence of amino acid side chains. These findings offer a framework for the design of nonnatural oligomers that mimic the structural and functional properties of proteins. [alpha]/[beta]-peptides | foldamers | protein folding

A system is described for the synthesis of polypeptides with novel folds in which existing sequence and structural data from bacterial proteins are used to assemble a pool of secondary structural elements. The results have shown that the protein evolution strategy has generated novel polypeptide sequences containing secondary structure.

Amylin infusion reduces food intake and slows body weight gain in rodents. In obese male rats, amylin (but not pair feeding) caused a preferential reduction of fat mass with protein preservation despite equal body weight loss in amylin-treated (fed ad libitum) and pair-fed rats. In the present study, the effect of prior or concurrent food restriction on the ability of amylin to cause weight loss was evaluated. Retired female breeder rats were maintained on a high-fat diet (40% fat) for 9 wk. Prior to drug treatment, rats were either fed ad libitum or food restricted for 10 days to lose 5% of their starting body weight. They were then subdivided into treatment groups that received either vehicle or amylin (100 [micro]g x [kg.sup.-1] x [day.sup.-1] via subcutaneous mini-pump) and placed under either a restricted or ad libitum feeding schedule (for a total of 8 treatment arms). Amylin 1) significantly reduced body weight compared with vehicle under all treatment conditions, except in always restricted animals, 2) significantly decreased percent body fat in all groups, and 3) preserved lean mass in all groups. These results indicate that amylin's anorexigenic and fat-specific weight loss properties can be extended to a variety of nutritive states in female rats. amylin; diet-induced obese rats; food restriction; weight; body composition

The molecular dynamics simulation studies of HNP defensins sequences that are human antimicrobial peptides produced in response to microbial invasion are reported. The results provide insight into their relationship, structural dissimilarities, and different microbial actions.

A new method, 2D circularly polarized IR photon echo spectroscopy was employed to study various polypeptide systems. It was shown that this novel method can provide additional information on the angles between the transition magnetic dipole and the transition electric dipole of two different vibrationally excited states of polypeptide.

This study examines the actions of the novel enzyme-resistant, N[H.sub.2]-terminally modified GIP analog ([Hyp.sup.3])GIP and its fatty acid-derivatized analog ([Hyp.sup.3])[GIPLys.sup.16]PAL. Acute effects are compared with the established GIP receptor antagonist ([Pro.sup.3])GIP. All three peptides exhibited DPP IV resistance, and significantly inhibited GIP stimulated cAMP formation and insulin secretion in GIP receptor-transfected fibroblasts and in clonal pancreatic BRIN-BD11 cells, respectively. Likewise, in obese diabetic ob/ob mice, intraperitoneal administration of GIP analogs significantly inhibited the acute antihyperglycemic and insulin-releasing effects of native GIP. Administration of once daily injections of ([Hyp.sup.3])GIP or ([Hyp.sup.3])[GIPLys.sup.16]PAL for 14 days resulted in significantly lower plasma glucose levels (P < 0.05) after ([Hyp.sup.3])GIP on days 12 and 14 and enhanced glucose tolerance (P < 0.05) and insulin sensitivity (P < 0.05 to P < 0.001) in both groups by clay 14. Both ([Hyp.sup.3])GIP and ([Hyp.sup.3])[GIPLys.sup.16]PAL treatment also reduced pancreatic insulin (P < 0.05 to P < 0.01) without affecting islet number. These data indicate that ([Hyp.sup.3])GIP and ([Hyp.sup.3])[GIPLys.sup.16]PAL function as GIP receptor antagonists with potential for ameliorating obesity-related diabetes. Acylation of ([Hyp.sup.3])GIP to extend bioactivity does not appear to be of any additional benefit. glucose-dependent insulinotropic polypeptide receptor antagonists; insulin secretion; antihyperglycemic activity; dipeptidyl peptidase IV; fatty acid conjugation doi: 10.1152/ajpendo.00391.2006

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent growth hormone (GH)-releasing factor in lower vertebrates. However, its functional interactions with other GH regulators have not been fully characterized. In fish models, norepinephrine (NE) inhibits GH release at the pituitary cell level, but its effects on GH synthesis have yet to be determined. We examined adrenergic inhibition of PACAP-induced GH secretion and GH gene expression using grass carp pituitary cells as a cell model. Through activation of pituitary [[alpha].sub.2]-adrenoreceptors, NE or the [[alpha].sub.2]-agonist clonidine reduced both basal and PACAP-induced GH release and GH mRNA expression. In carp pituitary cells, clonidine also suppressed cAMP production and intracellular [Ca.sup.2+] levels and blocked PACAP induction of these two second messenger signals. In G[H.sub.3] cells transfected with a reporter carrying the grass carp GH promoter, PACAP stimulation increased GH promoter activity, and this stimulatory effect could be abolished by NE treatment. In parallel experiments, clonidine reduced GH primary transcript and GH promoter activity without affecting GH mRNA stability, and these inhibitory actions were mimicked by inhibiting adenylate cyclase (AC), blocking protein kinase A (PKA), removing extracellular [Ca.sup.2+] in the culture medium, or inactivating L-type voltage-sensitive [Ca.sup.2+] channels (VSCC). Since our recent studies have shown that PACAP can induce GH secretion in carp pituitary cells through cAMP/PKA- and [Ca.sup.2+]/calmodulin-dependent mechanisms, these results, taken together, suggest that [[alpha].sub.2]-adrenergic stimulation in the carp pituitary may inhibit PACAP-induced GH release and GH gene transcription by blocking the AC/cAMP/PKA pathway and [Ca.sup.2+] entry through L-type VSCC. pituitary adenylate cyclase-activating polypeptide; growth hormone; signal transduction; norepinephrine doi:10.1152/ajpendo.00001.2007.

Paroxysmal atrial fibrillation associated with focal ectopy originating from the pulmonary vein (PV) can be preceded by variations in autonomic tone; however, the underlying cellular mechanisms are not clear. To determine the mechanisms of autonomically mediated PV ectopy, high-resolution optical mapping techniques were used to measure action potentials and [Ca.sup.2] [+ or -]transients from the PV and the ligament of Marshall area in the arterially perfused canine left atrium. Rapid pacing was used to initiate ectopic activity during pituitary adenylate cyclase-activating polypeptide (PACAP) injection (1 nmol), as a surrogate for autonomic imbalance, before (n = 9) and after (n = 6) verapamil (10 nmol) administration. In all preparations, spontaneous activity was absent before rapid pacing. During PACAP injection, rapid pacing induced ectopic activity in eight of nine preparations. In contrast, before PACAP injection, rapid pacing did not induce ectopic activity. Activation maps of each episode of ectopic activity indicated that the site of origin occurred more frequently in the PV (70%) than in the ligament of Marshall (30%) area. As rapid pacing cycle length increased, so did the ectopic beat coupling interval. In addition, PACAP-induced ectopic activity was associated with large [Ca.sup.2] [+ or -]transient amplitudes and was always suppressed by verapamil, a [Ca.sup.2] [+ or -] channel blocker (P < 0.05). Finally, during PACAP injection in the absence of an ectopic beat, spontaneous [Ca.sup.2] [+ or -] release and delayed after depolarizations were observed simultaneously after termination of rapid pacing. In conclusion, these data suggest that autonomically mediated PV ectopy may be due to [Ca.sup.2+] -mediated triggered activity arising from delayed afterdepolarizations. delayed afterdepolarizations; pituitary adenylate cyclase-activating polypeptide; ligament of Marshall; high-resolution optical mapping

The role played by the aromatic residues in the formation of amyloid by islet amyloid polypeptide (IAPP) is examined. The results have shown that aromatic residues play a role in the fibril assembly process, although it is not an absolute requirement for amyloid formation by IAPP.

Two models of the origin of novel polypeptide domains are proposed, based on 'exonization' of intron sequences and insertion-based polymorphisms between orthologous genes. These processes are discussed, also analyzing how each might participate in the evolutionary emergence of novel polypeptide domains.

Polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytoplasmic, integral membrane, or exported proteins. In Escherichia coli, the chaperones SecB, Trigger Factor (TF), and DnaK are key players in this process. Here, we report that, as with dnaK or dnaJ mutants, a secB null strain exhibits a strong cold-sensitive (Cs) phenotype. Through suppressor analyses, we found that inactivating mutations in the tig gene encoding TF fully relieve both the Cs phenotype and protein aggregation observed in the absence of SecB. This antagonistic effect of TF depends on its ribosome-binding and chaperone activities but unrelated to its peptidyl-prolyl cis/trans isomerase (PPlase) activity. Furthermore, in contrast to the previously known synergistic action of TF and DnaK/DnaJ above 30[degrees]C, a tig null mutation partially suppresses the Cs phenotype exhibited by a compromised DnaK/DnaJ chaperone machine. The antagonistic role of TF is further exemplified by the fact that the secB dnaJ double mutant is viable only in the absence of TF. Finally, we show that, in the absence of TF, more SecA and ribosomes are associated with the inner membrane, suggesting that the presence of TF directly or indirectly interferes with the process of cotranslational protein targeting to the Sec translocon. cold sensitivity | genetic suppression | nascent polypeptides | protein aggregation | protein export

Intrachain loop formation allows unfolded polypeptide chains to search for favorable interactions during protein folding. We applied triplet-triplet energy transfer between a xanthone moiety and naphthylalanine to directly measure loop formation in various unfolded polypeptide chains with loop regions consisting of polyserine, poly(glycine--serine) or polyproline. By combination of femtosecond and nanosecond laserflash experiments loop formation could be studied over many orders of magnitude in time from picoseconds to microseconds. The results reveal processes on different time scales indicating motions on different hierarchical levels of the free energy surface. A minor ( conformational substates | femtoseconds spectroscopy | peptide dynamics | protein folding | triplet-triplet energy transfer

J-proteins and Hsp70 chaperones function together in diverse cellular processes. We identified a cytosolic J-protein, Jjj1, of Saccharomyces cerevisiae that is associated with 60S ribosomal particles. Unlike Zuo1, a 60S subunit-associated J-protein that is a component of the chaperone machinery that binds nascent polypeptide chains upon their exit from the ribosome, Jjj1 plays a role in ribosome biogenesis. Cells lacking Jjj1 have phenotypes very similar to those lacking Rei1, a ribosome biogenesis factor associated with pre-60S ribosomal particles in the cytosol. Jjj1 stimulated the ATPase activity of the general cytosolic Hsp70 Ssa, but not Ssb, Zuo1's ribosome-associated Hsp70 partner. Overexpression of Jjj1, which is normally [approximately equal to] 40-fold less abundant than Zuo1, can partially rescue the phenotypes of cells lacking Zuo1 as well as cells lacking Ssb. Together, these results are consistent with the idea that Jjj1 normally functions with Ssa in a late, cytosolic step of the biogenesis of 60S ribosomal subunits. In addition, because of its ability to bind 60S subunits, we hypothesize that Jjj1, when overexpressed, is able to partially substitute for the Zuo1:Ssb chaperone machinery by recruiting Ssa to the ribosome, facilitating its interaction with nascent polypeptide chains. Hsp70 | Reil | ribosome biogenesis | Hsp40

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction, whereas VIP induces relaxation. Here, we have attempted to identify key residues responsible for their interactions with PACAP ([PAC.sub.1]) and VIP (VPAC) receptors in the guinea pig gallbladder. We synthesized PACAP-27/VIP hybrid peptides and compared their actions on isolated guinea pig gallbladder smooth muscle strips using isotonic transducers. [[Ala.sup.4]]- and [[Val.sup.5]]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [[Ala.sup.4], [Val.sup.5]]- and [[Ala.sup.4], [Val.sup.5], [Asn.sup.9]]PACAP-27 induced relaxation similarly to VIP. [[Asn.sup.9]]-, [[Thr.sup.11]]-, or [[Leu.sup.13]]PACAP-27 had 20-70% contractile activity of PACAP-27, whereas [[Asn.sup.24], [Ser.sup.25], [Ile.sup.26]]PACAP-27 showed no change in the activity. All VIP analogs, including [[Gly.sup.4], [Ile.sup.5], [Ser.sup.9]] VIP, induced relaxation. In the presence of a [PAC.sub.1] receptor antagonist, PACAP(6-38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of [PAC.sub.1] receptor, 'hop' splice variant, and [VPAC.sub.1] and [VPAC.sub.2] receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via [PAC.sub.1]/hop receptors. [Gly.sup.4] and [Ile.sup.5] are the key N[H.sub.2]-terminal residues of PACAP-27 that distinguish [PAC.sub.1]/hop receptors from [VPAC.sub.1]/[VPAC.sub.2] receptors. However, both the N[H.sub.2]-terminal and [alpha]-helical regions of PACAP-27 are required for initiating gallbladder contraction. [PAC.sub.1] receptor; [VPAC.sub.1] receptor; [VPAC.sub.2] receptor; splice variant

Monte Carlo simulations were performed within a model based on a set of distance-dependent effective potentials, which are used to describe the interactions between a pair of distinct amino acids. The model has a reduced number of variables and its main is the use of effective pair potentials (EPPs) extracted from experimental correlation functions that were obtained from the crystallographic data of the Protein Data Bank (PDB).

Although transmembrane proteins generally require membrane-embedded machinery for integration, a few can insert spontaneously into liposomes. Previously, we established that the tail-anchored (TA) protein cytochrome b(5) (b5) can post-translationally translocate 28 residues downstream to its transmembrane domain (TMD) across protein-free bilayers (Brambillasca, S., M. Yabal, P. Soffientini, S. Stefanovic, M. Makarow, R.S. Hegde, and N. Borgese. 2005. EMBO J. 24:2533-2542). In the present study, we investigated the limits of this unassisted translocation and report that surprisingly long (85 residues) domains of different sequence and charge placed downstream of b5's TMD can posttranslationally translocate into mammalian microsomes and liposomes at nanomolar nucleotide concentrations. Furthermore, integration of these constructs occurred in vivo in translocon-defective yeast strains. Unassisted translocation was not unique to b5 but was also observed for another TA protein (protein tyrosine phosphatase 1B) whose TMD, like the one of b5, is only moderately hydrophobic. In contrast, more hydrophobic TMDs, like synaptobrevin's, were incapable of supporting unassisted integration, possibly because of their tendency to aggregate in aqueous solution. Our data resolve long-standing discrepancies on TA protein insertion and are relevant to membrane evolution, biogenesis, and physiology.

Gastric cancer in humans arises in the setting of oxyntic atrophy (parietal cell loss) and attendant hyperplastic and metaplastic lineage changes within the gastric mucosa. Helicobacter infection in mice and humans leads to spasmolytic polypeptide-expressing metaplasia (SPEM). In a number of mouse models, SPEM arises after oxyntic atrophy. In mice treated with the parietal cell toxic protonophore DMP-777, SPEM appears to arise from the transdifferentiation of chief cells. These results support the concept that intrinsic mucosal influences regulate and modulate the appearance of gastric metaplasia even in the absence of significant inflammation, whereas chronic inflammation is required for the further neoplastic transition. gastric adenocareinoma; spasmolytic polypeptide-expressing metaplasia; trefoil factor 2; intestinal metaplasia

Although protein structures are primarily encoded by their sequences, they are also critically dependent on environmental factors such as solvents and interactions with other molecules. Here we investigate how the folding-energy landscape of a short peptide is altered by interactions with another peptide, by performing atomistic replica-exchange molecular dynamics simulations of polyalanines in various environments. We analyzed the free-energy landscapes of [Ala.sup.7] and [Ala.sup.8] in isolation, near an [alpha]-helix template, and near a [beta]-strand template. The isolated [Ala.sup.7] and [Ala.sup.8] at 270 K were mainly in polyproline II helix conformations and in equilibrium between the [alpha]-helix and polyproline II helix, respectively, in harmony with the experiment. Interestingly, we found remarkably strong secondary-structure 'templating'; namely, the [alpha]-helix template enhanced [alpha]-helix conformation and the [beta]-strand template induced [beta]-strand conformation in the simulated [Ala.sup.8]. The [alpha]-helix template lowered the nearby dielectric constant, which strengthened hydrogen bonds in the simulated [Ala.sup.8], leading to [alpha]-helix stabilization. The [beta]-strand template provided hydrogen bond positions to the simulated [Ala.sup.8], sharply inducing [beta]-strand structure. With or without templates, the energy landscape of [Ala.sup.8] is always funnel-like and centered at the [alpha]-helix conformation, whereas entropic contribution disfavors the [alpha]-helix, leading to subtle competition. Secondary-structure templating may play a critical role in protein conformation dynamics in the cellular environment. energy landscape | generalized Born | polyproline II | protein folding | replica exchange

The larvicidal activity of Bacillus thuringiensis subsp, israelensis against dipteran larvae is determined by four major polypeptides of the parasporal crystalline body produced during sporulation. Cyt1Aa shows the lowest toxicity when used alone but is the most synergistic with any of the other proteins. The sequence of the plasmid pBtoxis, which contains all the toxin genes in this subspecies, revealed a new cyt-like coding sequence named cyt1Ca. In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be a [beta]-trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression in Escherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of Aedes aegypti of Cyt1Ca in recombinant E. coli cells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.

Glucose-dependent insulinotropic polypeptide [GIP-(1-42)] is degraded by dipeptidyl peptidase IV (DPP IV), forming GIP-(3-42). In mice, high concentrations of synthetic GIP-(3-42) may function as a GIP receptor antagonist, but it is unclear whether this occurs at physiological concentrations. In COS-7 cells transiently transfected with the human GIP receptor, GIP-(1-42) and -(3-42) bind with affinities (I[C.sub.50]) of 5.2 and 22 nM, respectively. GIP-(1-42) was a potent agonist, stimulating cAMP accumulation (E[C.sub.50], 13.5 pM); GIP-(3-42) alone had no effect. When incubated together with native GIP, GIP-(3-42) behaved as a weak antagonist (I[C.sub.50], 92 and 731 nM for inhibition of cAMP accumulation elicited by 10 pM and 1 nM native GIP, respectively). In the isolated perfused rat pancreas, GIP-(3-42) alone had no effect on insulin output and only reduced the response to GIP (1 nM) when coinfused in >50-fold molar excess (I[C.sub.50], 138 nM). The ability of GIP-(3-42) to affect the antihyperglycemic or insulinotropic actions of GIP-(1-42) was examined in chloralose-anesthetized pigs given intravenous glucose. Endogenous DPP IV activity was inhibited to reduce degradation of the infused GIP-(1-42), which was infused alone and together with GIP-(3-42), at rates sufficient to mimic postprandial concentrations of each peptide. Glucose, insulin, and glucagon responses were identical irrespective of whether GIP-(1-42) was infused alone or together with GIP-(3-42). We conclude that, although GIP-(3-42) can weakly antagonize cAMP accumulation and insulin output in vitro, it does not behave as a physiological antagonist in vivo. glucose homeostasis; dipeptidyl peptidase IV; inhibitor; valine-pyrrolidide doi: 10.1152/ajpendo.00577.2005

Autotransporters are single polypeptides consisting of an outer membrane translocation domain mediating the translocation of a passenger domain. The periplasmic folding state of the passenger domain is controversial. By comparisons of passenger domains differing in their folding properties, our results suggest that periplasmic folding of passenger domains interferes with translocation.

The physiological effects of glucose-dependent insulinotropic polypeptide (GIP) are mediated through specific receptors expressed on target cells. Because aberrant GIP receptor (GIPR) expression has been implicated in abnormal GIP responses associated with type 2 diabetes mellitus and food-induced Cushing's syndrome, we sought to identify factors that regulate the GIPR. We previously demonstrated that sequences between -1 and -100 of the GIPR gene were sufficient to direct transcription in a rat insulinoma cell line (RIN38). In the present study, we compared the 5'-flanking regions of the rat and human GIPR gene and demonstrated 88% identity within the first 92 bp. Subsequent serial deletion analyses showed that the region between -85 and -40 is essential for maximal promoter activity. Within this region, we identified three putative Sp1 binding motifs, located at positions -77, -60, and -50, that can specifically bind both Sp1 and Sp3. Whereas mutation of the Sp1 sites at -50 and -60 led to 36 and 40% reduction in promoter activity, respectively, mutation of the Sp1 motif at -70 did not affect promoter activity. Cotransfection of S2 Schneider cells with GIPR-luciferase chimeric constructs and either Sp1 or Sp3 expression vectors indicated that both Sp1 and the long form of Sp3 activate transcription through binding to the Sp1 sites located between -100 and -40. Lastly, chromatin immunoprecipitation analyses revealed that both Sp1 and Sp3 bind to the GIPR promoter region in RIN38 cells. These results indicate that cell-specific expression of GIPR is associated with the binding of the transcription factors Sp1 and Sp3 to the GIPR promoter. gastric inhibitory polypeptide; transcription factors; RIN38 cells; insulinoma; rat-2 cells; chromatin immunoprecipitation assay

Development and normal function of the reproductive axis requires a precise degree of body energy stores. Polypeptide YY-(3-36) [PYY-(3-36)] is a gastrointestinal secreted molecule recently shown to be involved in the control of food intake with agonistic activity on neuropeptide Y (NPY) receptor subtypes [Y.sup.2] and [Y.sub.5]. Notably, PYY-(3-36) has been recently demonstrated as putative regulator of gonadotropin secretion in the rat. However, the 'reproductive' facet of this factor remains to be fully elucidated, In this context, we report herein our analyses of the influence of the nutritional status on the effects of PYY-(3-36) upon GnRH and gonadotropin secretion. The major findings of our study are 1) the stimulatory effect of central administration of PYY-(3-36) on LH secretion was significantly enhanced after fasting and blocked by a GnRH antagonist: 2) besides central effects, PYY-(3-36) elicited LH and FSH secretion directly at the pituitary level, a response that is also augmented by fasting; 3) PYY-(3-36) inhibited GnRH secretion by hypothalamic fragments from male rats fed ad libitum, whereas a significant stimulatory effect was observed after fasting; and 4) the increase in the gonadotropin responsiveness to PYY-(3-36) in fasting was not associated with changes in the expression of [Y.sub.2] and [Y.sub.5] receptor genes at hypothalamus and/or pituitary. In conclusion, our study extends our previous observations suggesting a relevant, mostly stimulatory, role of PYY-(3-36) in the control of gonadotropin secretion. Strikingly, such an effect was significantly enhanced by fasting. Considering the proposed decrease in PYY-(3-36) levels after fasting, the possibility that reduced PYY-(3-36) secretion might contribute to defective function of the gonadotropic axis after food deprivation merits further investigation. polypeptide YY-(3-36); gonadotropin-releasing hormone; luteinizing hormone; follicle-stimulating hormone; fasting; pituitary

Cytosolic chaperonin containing t-complex polypeptide 1 (CCT)/ TRiC is a group II chaperonin that assists in the folding of newly synthesized proteins. It is a eukaryotic homologue of the bacterial group I chaperonin GroEL. In contrast to the well studied functions of GroEL, the substrate recognition mechanism of CCT/TRiC is poorly understood. Here, we established a system for analyzing CCT/TRiC functions by using a reconstituted protein synthesis by using recombinant elements system and show that CCT/TRiC strongly recognizes WD40 proteins particularly at hydrophobic [beta]-strands. Using the G protein [beta] subunit (G[beta]), a WD40 protein that is very rich in [beta]-sheets, as a model substrate, we found that CCT/TRiC prevents aggregation and assists in folding of G[beta], whereas GroEL does not. G[beta] has a seven-bladed [beta]-propeller structure; each blade is formed from a WD40 repeat sequence encoding four [beta]-strands. Detailed mutational analysis of G[beta] indicated that CCT/TRiC, but not GroEL, preferentially recognizes hydrophobic residues aligned on surfaces of [beta]-strands in the second WD40 repeat of G[beta]. These findings indicate that one of the CCT/TRiC-specific targets is hydrophobic [beta]-strands, which are highly prone to aggregation. molecular chaperone | protein aggregation | protein folding | substrate recognition | WD40 repeat

Elaborate quality control strategies have evolved to counter inevitable mishaps in living cells where newly made and preexisting polypeptide chains are at risk for misfolding and aggregation. Studies describe the removal of aggregates from the cytosol, reveal mechanisms for protein quality control in the endoplasmic reticulum and provide new insight into two classes of molecular chaperones, the Hsp70 system and the AAA+ (Hsp 100) unfoldases.

The respiratory NADH:quinone oxidoreductase (complex I) (NDH-1) is a multisubunit enzyme that translocates protons (or in some cases [Na.sup.+]) across energy-conserving membranes from bacteria or mitochondria. We studied the reaction of the [Na.sup.+]-translocating complex I from the enterobacterium Klebsiella pneumoniae with N,N'-dicyclohexylcarbodiimide (DCCD), with the aim of identifying a subunit critical for [Na.sup.+] binding. At low [Na.sup.+] concentrations (0.6 mM), DCCD inhibited both quinone reduction and [Na.sup.+] transport by NDH-1 concurrent with the covalent modification of a 30-kDa polypeptide. In the presence of 50 mM [Na.sup.+], NDH-1 was protected from inhibition by DCCD, and the modification of the 30-kDa polypeptide with [[.sup.14]C]DCCD was prevented, indicating that [Na.sup.+] and DCCD competed for the binding to a critical carboxyl group in NDH-1. The 30-kDa polypeptide was assigned to NuoH, the homologue nf the ND1 subunit from mitochondrial complex I. It is proposed that [Na.sup.+] binds to the NuoH subunit during NADH-driven [Na.sup.+] transport by NDH-1.

The hydrophilic domain of the proton-translocating NADH:quinone oxidoreductase (complex I) from the thermophilic organism Thermus thermophilus HB8 was purified and characterized. The hydrophilic domain was found to be extremely stable and a new polypeptide was found to associate strongly and crystallize within the sub complex, indicating that it was a novel subunit and was named as Nqo15.

Lamina-associated polypeptide (LAP) 2[alpha] is a non-membrane-bound LAP2 isoform that forms complexes with nucleoplasmic A-type lamins. In this study, we show that the overexpression of LAP20[alpha] in fibroblasts reduced proliferation and delayed entry into the cell cycle from a GO arrest. In contrast, stable down-regulation of LAP20[alpha] by RNA interference accelerated proliferation and interfered with cell cycle exit upon serum starvation. The LAP2[alpha]-linked cell cycle phenotype is mediated by the retinoblastoma (Rb) protein because the LAP2[alpha] COOH terminus directly bound Rb, and overexpressed LAP2e[alpha] inhibited E2F/Rb-dependent reporter gene activity in G1 phase in an Rb-dependent manner. Furthermore, LAP2e[alpha] associated with promoter sequences in endogenous E2F/Rb-dependent target genes in vivo and negatively affected their expression. In addition, the expression of LAP2[alpha] in proliferating preadipocytes caused the accumulation of hypophosphorylated Rb, which is reminiscent of noncycling cells, and initiated partial differentiation into adipocytes. The effects of LAP20[alpha] on cell cycle progression and differentiation may be highly relevant for the cell-and tissue-specific phenotypes observed in laminopathic diseases.

The polypeptide growth factor, hepatocyte growth factor/scatter factor (HGF/SF), shares the multidomain structure and proteolytic mechanism of activation of plasminogen and other complex serine proteinases. HGF/SF, however, has no enzymatic activity. Instead, it controls the growth, morphogenesis, or migration of epithelial, endothelial, and muscle progenitor cells through the receptor tyrosine kinase MET. Using small-angle x-ray scattering and cryo-electron microscopy, we show that conversion of pro(single-chain)HGF/SF into the active two-chain form is associated with a major structural transition from a compact, closed conformation to an elongated, open one. We also report the structure of a complex between two-chain HGF/SF and the MET ectodomain (MET928) with 1:1 stoichiometry in which the N-terminal and first kringle domain of HGF/SF contact the face of the seven-blade [beta]-propeller domain of MET harboring the loops connecting the [beta]-strands b-c and d-a, whereas the C-terminal serine proteinase homology domain binds the opposite 'b' face. Finally, we describe a complex with 2:2 stoichiometry between two-chain HGF/SF and a truncated form of the MET ectodomain (MET567), which is assembled around the dimerization interface seen in the crystal structure of the NK1 fragment of HGF/SF and displays the features of a functional, signaling unit. The study shows how the proteolytic mechanism of activation of the complex proteinases has been adapted to cell signaling in vertebrate organisms, offers a description of monomeric and dimeric ligand-receptor complexes, and provides a foundation to the structural basis of HGF/SF-MET signaling. cell signaling | plasminogen | serine proteinases | kringle | x-ray scattering

In the plant-pathogenic mollicute Spiroplasma citri, spiralin is the major lipoprotein at the cell surface and is thought to be one of the components involved in the interactions of the spiroplasma with its insect vector. With the aim of identifying surface proteins other than spiralin, monoclonal antibodies (mAbs) were produced by immunization of mice with the spiralin-defective S. citri mutant GII3-9a2. mAb 10G3 was found to react with several polypeptides of 43-47 and 80-95 kDa, all of which were detected in the detergent phase after Triton X-114 partitioning of proteins. Mass spectrometry (MALDI-TOF) analyses of the two major polypeptides P47 and P80 of GII3-9a2, reacting with mAb 10G3, revealed that P47 was a processed product and represented the C-terminal moiety of P80. Search for sequence homologies revealed that P80 shared strong similarities with the S. citri adhesion-related protein P89 (Sarpl) of S. citri BR3, and is one (named Scarp4a) of the eight Scarps encoded by the S. citri GII-3 genome. The eight scarp genes are carried by plasmids pSci1-5. Western immunoblotting of proteins with mAb 10G3 revealed that, in contrast to the insect-transmissible S. citri strain GII-3, the non-insect-transmissible strains ASP-l, R8A2 and 44 did not express Scarps. Southern blot hybridization experiments indicated that these strains possessed no scarp genes, and did not carry plasmids pScil-5. However, S. citri strain GII3-5, lacking pSci5, was still efficiently transmitted, showing that, in the genetic background of S. citri GII-3, the pSci5-encoded genes, and in particular scarp2b, 3b and 5a, are not essential for insect transmission. Whether plasmid-encoded genes are involved in transmission of S. citri by its leafhopper vector remains to be determined.

Mammalian red blood cells, unlike those of other vertebrates, must withstand the rigors of circulation in the absence of new protein synthesis. Key to this is plasma membrane elasticity deriving from the protein spectrin, which forms a network on the cytoplasmic face. Spectrin is a tetramer [([alpha][beta]).sub.2], made up of [alpha][beta] dimers linked head to head. We show here that one component of erythrocyte spectrin, [alpha]I, is encoded by a gene unique to mammals. Phylogenetic analysis suggests that the other [alpha]-spectrin gene ([alpha]II) common to all vertebrates was duplicated after the emergence of amphibia, and that the resulting [alpha]I gene was preserved only in mammals. The activities of al and [alpha]II spectrins differ in the context of the human red cell membrane. An [alpha]I-spectrin fragment containing the site of head-to-head interaction with the [beta]-chain binds more weakly than the corresponding [alpha]II fragment to this site. The latter competes so strongly with endogenous [alpha]I as to cause destabilization of membranes at 100-fold lower concentration than the [alpha]I fragment. The efficacies of [alpha]I/[alpha]II chimeras indicate that the partial structural repeat, which binds to the complementary [beta]-spectrin element, and the adjacent complete repeat together determine the strength of the dimer--dimer interaction on the membrane. Alignment of all available [alpha]-spectrin N-terminal sequences reveals three blocks of sequence unique to [alpha]I. Furthermore, human [alpha]II-spectrin is closer to fruitfly [alpha]-spectrin than to human [alpha]I-spectrin, consistent with adaptation of [alpha]I to new functions. We conclude that [alpha]I-spectrin represents a neofunctionalized spectrin adapted to the rapid make and break of tetramers. cytoskeleton | membrane | red blood cell | triple-helix