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At least 127 records · Page 7Linked to original sources
PubMed2003

Protein-protein, protein-RNA and protein-lipid interactions of signal-recognition particle components in the hyperthermoacidophilic archaeon Acidianus ambivalens.

The signal-recognition particle (SRP) of one of the most acidophilic and hyperthermophilic archaeal cells, Acidianus ambivalens, and its putative receptor component, FtsY (prokaryotic SRP receptor), were investigated in detail. A. ambivalens Ffh (fifty-four-homologous protein) was shown to be a soluble protein with strong affinity to membranes. In its membrane-residing form, Ffh was extracted from plasma membranes with chaotropic agents like urea, but not with agents diminishing electrostatic interactions. Using unilamellar tetraether phospholipid vesicles, both Ffh and FtsY associate independently from each other in the absence of other factors, suggesting an equilibrium of soluble and membrane-bound protein forms under in vivo conditions. The Ffh protein precipitated from cytosolic cell supernatants with anti-Ffh antibodies, together with an 7 S-alike SRP-RNA, suggesting a stable core ribonucleoprotein composed of both components under native conditions. The SRP RNA of A. ambivalens depicted a size of about 309 nucleotides like the SRP RNA of the related organism Sulfolobus acidocaldarius. A stable heterodimeric complex composed of Ffh and FtsY was absent in cytosolic supernatants, indicating a transiently formed complex during archaeal SRP targeting. The FtsY protein precipitated in cytosolic supernatants with anti-FtsY antisera as a homomeric protein lacking accessory protein components. However, under in vitro conditions, recombinantly generated Ffh and FtsY associate in a nucleotide-independent manner, supporting a structural receptor model with two interacting apoproteins.

Archaeal Proteins↗
PubMed2004

Hyperpolarization-activated, cyclic nucleotide-gated HCN2 cation channel forms a protein assembly with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction.

Hyperpolarization-activated cation currents, termed Ih, are non-uniformly distributed along dendritic arbors with current density increasing with increasing distance from the soma. The non-uniform distribution of Ih currents contributes to normalization of location-dependent variability in temporal integration of synaptic input, but the molecular basis for the graded HCN distribution remains to be investigated. The hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCNs) underlie Ih currents and consist of four members (HCN1-HCN4) of the gene family in mammals. In this investigation, we report that HCN2 forms a protein assembly with tamalin, S-SCAM and Mint2 scaffold proteins, using several different approaches including immunoprecipitation of rat brain and heterologously expressing cell extracts and glutathione S-transferase pull-down assays. The PDZ domain of tamalin interacts with HCN2 at both the PDZ-binding motif and the internal carboxy-terminal tail of HCN2, whereas binding of the PDZ domain of S-SCAM occurs at the cyclic nucleotide-binding domain (CNBD) and the CNBD-downstream sequence of the carboxy-terminal tail of HCN2. A protein assembly between HCN2 and Mint2 is formed by the interaction of the munc18-interacting domain of Mint2 with the CNBD-downstream sequence of HCN2. The results demonstrate that HCN2 forms a protein complex with multiple neuronal scaffold proteins in distinct modes of protein-protein interaction.

Animals↗
PubMed2003

Direct interactions between the heterotrimeric G protein subunit G beta 5 and the G protein gamma subunit-like domain-containing regulator of G protein signaling 11: gain of function of cyan fluorescent protein-tagged G gamma 3.

We used fluorescence resonance energy transfer imaging of enhanced cyan fluorescent protein (CFP)-tagged and enhanced yellow fluorescent protein (YFP)-tagged protein pairs to examine the hypothesis that G protein gamma subunit-like (GGL) domain-containing regulators of G protein signaling (RGS) can directly bind to the Gbeta5 subunit of heterotrimeric G proteins in vivo. We observed that Gbeta5 could interact with Ggamma2 and Ggamma13, after their expression in human embryonic kidney 293 cells. Interestingly, although untagged Ggamma3 did not interact with Gbeta5, CFP-tagged Ggamma3 strongly interacted with YFP-tagged Gbeta5 in FRET studies. Moreover, CFP-Ggamma3 supported Ca(2+) channel inhibition when paired with Gbeta5 or YFP-Gbeta5, indicating a "gain of function" for CFP-Ggamma3. Gbeta5 could also interact with RGS11 and its N-terminal, but not its C-terminal domain. On the other hand, RGS11 did not interact with Gbeta1. These studies demonstrate that the GGL domain-containing N terminus of RGS 11 can directly interact with Gbeta5 in vivo and supports the hypothesis that this interaction may contribute to the specificity of Gbeta5 interactions with cellular effector molecules.

Calcium Channel Blockers↗
PubMed1994

Interleukin-1-induced intracellular signaling pathways converge in the activation of mitogen-activated protein kinase and mitogen-activated protein kinase-activated protein kinase 2 and the subsequent phosphorylation of the 27-kilodalton heat shock protein in monocytic cells.

Interleukin (IL)-1 plays a central role in human host defense. Binding of IL-1 to its receptor is associated with phosphorylation of various cellular target proteins, most of which are unidentified. The kinases responsible for target protein phosphorylation after IL-1 stimulation are also still not completely understood. We report here that IL-1 induced activation of mitogen-activated protein (MAP) kinase in primary monocytes and in the human monocytic leukemia cell line U-937. Activation of MAP kinase was followed by activation of MAP kinase-activated protein (MAPKAP) kinase 2, a serine/threonine kinase, leading to subsequent phosphorylation of the small heat shock protein [27-kDa heat shock protein (Hsp27)]. Phosphorylation of Hsp27 triggered by IL-1 was both dose and time dependent. IL-1 failed to phosphorylate Hsp27 when cells had been previously deactivated with tyrosine kinase inhibitors such as genistein. In those cells, however, Hsp27 phosphorylation could be reconstituted when activated immunoprecipitated MAP kinase or purified MAPKAP kinase 2 was added. Phosphorylation of Hsp27 could also be inhibited when NaF, a serine/threonine phosphatase inhibitor, was omitted. Taken together, our findings indicate that IL-1-induced intracellular signaling pathways converge in the activation of MAP kinase and MAPKAP kinase 2 and the subsequent phosphorylation of Hsp27.

Cell Line↗
PubMed2003

Amyloid beta protein precursor (AbetaPP), but not AbetaPP-like protein 2, is bridged to the kinesin light chain by the scaffold protein JNK-interacting protein 1.

Proteolytic processing of amyloid beta protein precursor (AbetaPP) generates peptides that regulate normal cell signaling and are implicated in Alzheimer's disease pathogenesis. AbetaPP processing also occurs in nerve processes where AbetaPP is transported from the cell body by kinesin-I, a microtubule motor composed of two kinesin heavy chain and two kinesin light chain (Klc) subunits. AbetaPP transport is supposedly mediated by the direct AbetaPP-Klc1 interaction. Here we demonstrate that the AbetaPP-Klc1 interaction is not direct but is mediated by JNK-interacting protein 1 (JIP1). The phosphotyrosine binding domain of JIP1 binds the cytoplasmic tail of AbetaPP, whereas the JIP1 C-terminal region interacts with the tetratrico-peptide repeats of Klc1. We also show that JIP1 does not bridge the AbetaPP gene family member AbetaPP-like protein 2, APLP2, to Klc1. These results support a model where JIP1 mediates the interaction of AbetaPP to the motor protein kinesin-I and that this JIP1 function is unique for AbetaPP relative to its family member APLP2. Our data suggest that kinesin-I-dependent neuronal AbetaPP transport, which controls AbetaPP processing, may be regulated by JIP1.

Adaptor Proteins, Signal Transducing↗
PubMed1989

The protein requirement of adult marmosets: nitrogen balances and net protein utilization of milk proteins, soy protein, and amino acid mixtures.

Nitrogen balance studies were conducted in adult male marmosets (Callithrix jacchus) using purified and semipurified diets with protein levels between 0% and 7%. Daily nitrogen loss in a state of prolonged protein free nutrition was 131 +/- 16 mg/kg body weight0.75. Zero nitrogen balance resulted from mean daily intake of 261 mg nitrogen/kg0.75 when high quality protein sources were used. Very low protein intake or the lack of arginine and histidine in an amino acid mixture induced coprophagy. It is concluded that the protein requirement of adult marmosets is very similar to the protein requirement of adult humans (based on metabolic body weight). About 6-7% high quality protein, based on dry matter, suffice to avoid a negative nitrogen balance in all individuals.

Amino Acids↗
PubMed1986

[Protein and amino acid metabolism in the intestinal tract of growing young bulls. 7. Protein estimation and evaluation of protein supply in growing ruminants by the apparently digestible total protein in the duodenum based on rations].

The following values were derived from experimental studies of the crude protein (CP) and amino acid (AA) metabolism of growing fattening bulls, the detailed results of which were described in installments 1-6, for the ascertainment of the content of apparently digestible total protein at the duodenum (ADTPD): a) partial method: Degradation quotaCP = NPN + 0.69 X g pure protein/CP; g AD feed PD = g CP (1 -decomposition quotaCP) X 0.72 or g AD feed PD = 0.31 X g pure protein X 0.72; g ADBPDE = 0.149 X g AD org. matter X 0.80 X 0.72 (ADBPDE = apparently digestible pure bacteria protein at the duodenum, dependent on energy release in the reticulo-rumen; g ADBPDN = g CP X (degradation quotaCP) X 0.80 X 0.72 (ADBPDN = apparently digestible pure bacteria protein at the duodenum, dependent on CP supply of rumen microbes); g AD total PD = g AD feed PD + ADBPDE g AD total PD = g AD feed PD + ADBPDN; b) summarizing method: AD total PD = 0.429 + 10.9 X 0.80/CP in % AD org. matter X (CP intake) X 0.72; AD total PD = 0.196 + 16.6 X 0.80/pure protein % AD org. m. X pure pr. intake X 0.72. The requirement of AD total PD was calculated under consideration of the endogenous urine N, skin N and hair N losses as well as protein retention per kg live weight assuming a utilization of 0.70 for this partial performance. The practical application of this balance method is demonstrated in comparison to the digestible CP-system.

Amino Acids↗
PubMed1984

Fine-structure analyses of lipid-protein and protein-protein interactions of gag protein p19 of the avian sarcoma and leukemia viruses by cyanogen bromide mapping.

In avian sarcoma and leukemia viruses, the gag protein p19 functions structurally as a matrix protein, connecting internal components with the viral envelope. We have used a combination of in situ cross-linking and peptide mapping to localize within p19 the regions responsible for two major interactions in this complex, p19 with lipid and p19 with p19. Lipid-protein cross-links were localized near the amino terminus within the first 35 amino acids of the polypeptide. Homotypic protein-protein disulfide bridges were found to originate from near the carboxy terminus of p19, from cysteine residues at amino acids 111 and 153. These results suggest that p19 is divided into domains with distinct functions. The peptide maps constructed for p19, and for the related proteins p23 in avian sarcoma and leukemia viruses and p19 beta in recombinant avian sarcoma viruses, should serve as useful tools for other types of studies involving these proteins.

Amino Acids↗
PubMed1976

Protein-protein interaction in transport: periplasmic histidine-binding protein J interacts with P protein.

A component of the high-affinity histidine transport system in Salmonella typhimurium, the periplasmic histidine-binding protein J, interacts with another transport component, the P protein. A mutant J protein, with a defective interaction site but intact histidine-binding site, can function in histidine transport if an appropriate compensating mutation is introduced in the P protein. The interaction between the J and P proteins is an obligatory step in transport. The significance of this interaction and of the involvement of the P protein in multiple transport functions is discussed.

Bacterial Proteins↗
PubMed1994

PP1 gamma 2, a testis-specific protein-serine/threonine-phosphatase type 1 catalytic subunit, is associated with a protein having high sequence homology with the 78-kDa glucose-regulated protein, a member of the 70-kDa heat shock protein family.

Protein phosphatase 1 gamma 2 (PP1 gamma 2) is a testis-specific isotype of the protein-serine/threonine-phosphatase type 1 catalytic subunit. Three native forms of PP1 gamma 2 were detected in a crude fraction of rat testis by electrophoresis in a nondenaturing polyacrylamide gel. We purified a major native form of PP1 gamma 2 to homogeneity by successive column chromatography on Mono Q-Sepharose, EAH-agarose, protamine-agarose, and G3000SW and by electrophoresis in a nondenaturing polyacrylamide gel. The G3000SW-purified PP1 gamma 2 native form had an apparent molecular mass of 170 kDa. The purified holoenzyme from nondenaturing polyacrylamide gel was composed of the catalytic subunit and two noncatalytic subunits, of 78 kDa and 55 kDa. Partial amino acid sequence analysis of the 78-kDa protein suggested that it is the 78-kDa glucose-regulated protein, a member of the 70-kDa heat shock protein family. The 78-kDa protein may possibly function as a chaperone or by confining substrate specificity of PP1 gamma 2.

Amino Acid Sequence↗
PubMed1986

Proteins from the prokaryotic nucleoid. A protein-protein cross-linking study on the quaternary structure of Escherichia coli DNA-binding protein NS (HU).

Escherichia coli DNA-binding proteins NS1, NS2 and NS (NS1 + NS2) react with the protein-protein bifunctional cross-linking reagents dimethylsuberimidate and dimethyladipimidate to yield oligomers up to hexamers. The former reagent, with the longer arm, is more efficient than the other shorter one. Both one- and two-dimensional gel electrophoreses show that the cross-linked trimers are homogeneous, while the dimers appear heterogeneous, suggesting that at least two types of dimers but geometrically equivalent trimers are formed. In the presence of DNA, the cross-linking reaction with either reagent yields fewer dimers and more of the larger products. The yield of cross-linked products of various sizes was determined for NS1, NS2 and NS as a function of the protein concentration (0.03-3000 microM). From the results obtained in these experiments, we derived a model of quaternary structure in which dimers and tetramers are predominant in very solutions of the proteins. Above a critical concentration (10-50 microM), interactions among tetramers become increasingly important, yielding octamers and perhaps larger products. Our data do not support a recently proposed model in which the DNA is packaged around a protein disc consisting of 8-10 NS dimers.

Bacterial Proteins↗
PubMed2006

Protein-protein interactions and nuclear trafficking of coat protein and betaC1 protein associated with Bhendi yellow vein mosaic disease.

Bhendi yellow vein mosaic disease (BYVMD) is caused by a complex consisting of a monopartite begomovirus BYVMV and a satellite DNA beta component. BYVMV represents a new member of the emerging group of monopartite begomoviruses requiring a satellite component for symptom induction. Here we report the results of the transient expression of green fluorescent protein (GFP) fused with the betaC1 and coat protein (CP) coding regions, in the epidermal cells of Nicotiana benthamiana. GFPCP was found to be targeted into the nucleus whereas GFPbetaC1 was localized towards the periphery of the cell. The sub-cellular localization of the betaC1 protein has been compared with that of the CP in yeast cells using a genetic system for detection of protein nuclear import and export. Expression of betaC1 ORF in transgenic N. benthamiana under the control of the Cauliflower mosaic virus 35S promoter produced severe developmental abnormalities in the plant, like distorted stem, leaves and stunting of the plant. We also present the results on the interaction of CP and betaC1 proteins using yeast two hybrid analysis, suggesting a collaborative role in the inter- and intracellular dynamics of BYVMD.

Active Transport, Cell Nucleus↗
PubMed1999

Endogenous substrates of sphingosine-dependent kinases (SDKs) are chaperone proteins: heat shock proteins, glucose-regulated proteins, protein disulfide isomerase, and calreticulin.

Protein kinases whose activity is detectable only in the presence of sphingosine (Sph) or N,N'-dimethyl-Sph (DMS), but not in the presence of 15 other sphingolipids, phospholipids, and glycerolipids tested (Megidish, T., et al. (1995) Biochem. Biophys. Res. Commun. 216, 739-747), have been termed "sphingosine-dependent kinases" (SDKs). We showed previously that a purified SDK (termed "SDK1") phosphorylates a specific Ser position of adapter/chaperone protein 14-3-3 isoforms beta, eta, and zeta but not tau or sigma (Megidish, T., et al. (1998) J. Biol. Chem. 273, 21834-45). In this study we found the following: (i) other SDKs with different substrate specificities are present in cytosolic and membrane extracts of mouse Balb/c 3T3 (A31) fibroblasts. (ii) The activation of these SDKs is specific to D-erythro-Sph and its N-methyl derivatives, the effect of L-threo-Sph or its N-methyl derivatives is minimal, and nonspecific cationic amphiphiles have no effect at all. An SDK separated as fractions "TN31-33" phosphorylated a 50 kDa substrate which was identified as calreticulin, as well as two endogenous substrates with molecular mass 58 and 55 kDa, both identified as protein disulfide isomerase (PDI). This SDK, which specifically phosphorylates calreticulin and PDI, both molecular chaperones found at high levels in endoplasmic reticulum, is tentatively termed "SDK2". Another SDK activity was copurified with glucose-regulated protein (GRP) and heat shock proteins (HSP). One GRP substrate had the same amino acid sequence as GRP94 (synonym: endoplasmin); another HSP substrate had the same amino acid sequence as mouse HSP86 or HSP84, the analogues of human HSP90. An SDK activity separated and present in "fraction 42" from Q-Sepharose chromatography specifically phosphorylated GRP105 (or GRP94) and HSP68 but did not phosphorylate PDI or 14-3-3. This SDK is clearly different from other SDKs in its substrate specificity and is tentatively termed "SDK3". Interestingly, substrates of all these SDKs so far identified are molecular chaperones or adapters capable of binding to enzymes and key molecules involved in signal transduction, maintaining tertiary structure of bioactive molecules, or maintaining cellular homeostasis in response to environmental stress. Thus, the essential role of Sph and DMS is to activate molecular chaperones, thereby providing a link to the mechanism by which SDK activity regulates cellular homeostasis and signal transduction.

3T3 Cells↗
PubMed2002

Regulation of uncoupling protein-2 mRNA in L6 myotubules: I: Thiazolidinediones stimulate uncoupling protein-2 gene expression by a mechanism requiring ongoing protein synthesis and an active mitogen-activated protein kinase.

The mitochondrial uncoupling protein-2 (UCP2) can uncouple phosphorylation to subserve several functions. It has been reported that the insulin sensitizers, thiazolidinediones (TZDs), increase UCP2 mRNA levels and, more recently, that TZDs stimulate UCP2 reporter genes but that the sequences involved do not bind peroxisome proliferator-activated receptor gamma (PPARgamma). We report here that TZDs stimulated UCP2 gene (ucp2) transcription in L6 myotubules involving an indirect mechanism. L6 cells contained comparatively small amounts of PPARgamma mRNA but clearly detectable amounts of PPARgamma2 protein. UCP2 mRNA levels were increased in a time- and concentration-dependent manner by TZDs. UCP2 mRNA had slow turnover (t 1/2 approximately 38 h), and this was not affected by TZDs. Bisphenol A diglycidyl ether, a PPARy antagonist, concentration dependently inhibited the TZD-induced increase in UCP2 mRNA. Blockade of protein synthesis with cycloheximide as well as abrogation of mitogen-activated protein kinase (MAPK) activity with PD98059 or U0126 also prevented the TZD-induced increase in UCP2 mRNA. As with autologous UCP2 mRNA, TZDs stimulated reporter gene expression directed by ucp2 sequences in transiently transfected L6 cells. The effect was enhanced by cotransfection of PPARgamma + retinoid X receptor gamma and prevented by MEK blockade. TZDs, however, did not increase the activation of MAPK, nor did its activation by other means (change of medium, insulin-like growth factor-1, insulin) increase UCP2 mRNA, indicating that phosphorylation is not limiting. These results suggest that TZDs indirectly stimulate ucp2 transcription by inducing-via PPARgamma-limiting amounts of a protein, which must be phosphorylated by MAPK to stimulate the gene.

Animals↗
PubMed1975

Protein-protein interactions of light-harvesting pigment protein from spinach chloroplasts. I.Ca-2+ binding and its relation to protein association.

The role of divalent cations in the regulation of the distribution of excitation energy between the two photosystems involved in green plants photosynthesis has led us to search for a better understanding of how such phenomena might occur at the molecular level. Since small changes in orientation of and distance between pigment molecules could greatly affect the distribution of excitation energy, we have decided to study the effects of ions on the light-harvesting pigment protein from spinach chloroplasts. The light-harvesting pigment protein is shown to have two types of binding sites for Ca-2+. Binding studies and analytical ultracentrifugation indicate that site I (K-d equals 2.5 mu-M, n equals 1.5-4.0 mu-mol Ca-2+ bound/mg chlorophyll) is lost as the protein associates. Site II (K-d equals 32 mu-M, n equals 9,5 mu-mol Ca-2+/mg chlorophyll) is not affected by the association of the protein. This site is responsible, however, for a further divalent cation-dependent association of the protein. The possible role of this protein in grana stacking and control of spillover is discussed.

Calcium↗
PubMed2003

Expression of multidrug resistance proteins P-glycoprotein, multidrug resistance protein 1, breast cancer resistance protein and lung resistance related protein in locally advanced bladder cancer treated with neoadjuvant chemotherapy: biological and clinical implications.

PURPOSE: Resistance to chemotherapy is a major obstacle to overcome in the conservative treatment of patients with locally advanced bladder cancer (LABC). We investigated the predictive value of the response to neoadjuvant chemotherapy (NACT) and prognosis of the expression of multidrug resistance (MDR) related proteins, P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), breast cancer resistance protein (BCRP) and lung resistance related protein/major vault protein (LRP/MVP) in LABC. MATERIALS AND METHODS: Using immunohistochemistry we studied the expression of MDR proteins in tumors from 83 patients with LABC treated with NACT using a bladder sparing approach. Expression was related to the response to NACT, bladder preservation and prognosis. RESULTS: P-gp, MRP1, BCRP and LRP/MVP were expressed at high levels in 53%, 59%, 28% and 70% of cases, respectively. P-gp expression correlated with shorter progression-free survival (p = 0.04) but not with overall survival. Surprisingly MRP1 expression correlated with a higher response (p = 0.005) and a higher probability of bladder preservation following NACT (p = 0.001). BCRP did not show any prognostic impact. High LRP/MVP expression was significantly associated with a worse response to NACT and a decreased probability of bladder preservation (p = 0.035). CONCLUSIONS: Our data suggest that MRP1 and LRP/MVP may be useful in combination with other clinicopathological prognostic factors for selecting patients with LABC to be candidates for bladder preservation after NACT. A large prospective study is warranted to confirm the prognostic value of these MDR proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Tumor necrosis factor signaling to stress-activated protein kinase (SAPK)/Jun NH2-terminal kinase (JNK) and p38. Germinal center kinase couples TRAF2 to mitogen-activated protein kinase/ERK kinase kinase 1 and SAPK while receptor interacting protein associates with a mitogen-activated protein kinase kinase kinase upstream of MKK6 and p38.

Tumor necrosis factor (TNF) elicits a diverse array of inflammatory responses through engagement of its type-1 receptor (TNFR1). Many of these responses require de novo gene expression mediated by the activator protein-1 (AP-1) transcription factor. We investigated the mechanism by which TNFR1 recruits the stress-activated protein kinases (SAPKs) and the p38s, two mitogen-activated protein kinase (MAPK) families that together regulate AP-1. We show that the human SPS1 homologue germinal center kinase (GCK) can interact in vivo with the TNFR1 signal transducer TNFR-associated factor-2 (TRAF2) and with MAPK/ERK kinase kinase 1 (MEKK1), a MAPK kinase kinase (MAPKKK) upstream of the SAPKs, thereby coupling TRAF2 to the SAPKs. Receptor interacting protein (RIP) is a second TNFR signal transducer which can bind TRAF2. We show that RIP activates both p38 and SAPK; and that TRAF2 activation of p38 requires RIP. We also demonstrate that the RIP noncatalytic intermediate domain associates in vivo with an endogenous MAPKKK that can activate the p38 pathway in vitro. Thus, TRAF2 initiates SAPK and p38 activation by binding two proximal protein kinases: GCK and RIP. GCK and RIP, in turn, signal by binding MAPKKKs upstream of the SAPKs and p38s.

Binding Sites↗