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P Tempst

Publications and source records attributed to P Tempst.

At least 109 records · Page 6Linked to original sources
PubMed1995

Interleukin-3 signals through multiple isoforms of Stat5.

The interleukin (IL)-3 family of cytokines mediates its numerous effects on myeloid growth and maturation by binding a family of related receptors. It has been shown recently that IL-3 induces the activation of two distinct cytoplasmic signal transducing factors (STFs) that are likely to mediate the induction of immediate early genes. In immature myeloid cells, IL-3 activates STF-IL-3a, which comprises two tyrosine-phosphorylated DNA binding proteins of 77 and 80 kDa. In mature myeloid cells, IL-3 and granulocyte-macrophage colony-stimulating factor activate STF-IL-3b, which consists of a 94 and 96 kDa tyrosine-phosphorylated DNA binding protein. Peptide sequence data obtained from the purified 77 and 80 kDa proteins (p77 and p80) indicate that they are closely related but are encoded by distinct genes. Both peptide and nucleotide sequence data demonstrate that these two proteins are the murine homologs of ovine mammary gland factor (MGF)/Stat5. The peptide data also indicate that p77 and p80 are phosphorylated on tyrosine 699, a position analogous to the tyrosine that is phosphorylated in Stat1 and Stat2 in response to interferon. Additionally, antiserum raised against bacterially expressed p77/p80 recognizes the 94 and 96 kDa protein components of STF-IL-3b, suggesting that these may be additional isoforms of Stat5. These studies indicate that the IL-3 family of ligands is able to activate multiple isoforms of the signal transducing protein Stat5.

Amino Acid Sequence↗
PubMed1995

I kappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B.

We have cloned the cDNA encoding I kappa B-beta, one of the two major I kappa B isoforms in mammalian cells. The recombinant I kappa B- beta protein interacts with equal affinity to p65 and c-Rel and does not exhibit a preference between these Rel proteins. Instead the primary difference between I kapp B-alpha and I kappa B-beta is in their response to different inducers of NF-kappa B activity. One class of inducers causes rapid but transient activation of NF-kappa B by primarily affecting I kappa B-alpha complexes, whereas another class of inducers causes persistent activation of NF-kapa B by affecting both I kappa B-alpha and I kappa B-beta complexes. Therefore, the overall activation of NF-kappa B consists of two overlapping phases, a transient phase mediated through I kappa B-alpha and a persistent phase mediated through I kappa B-beta.

Amino Acid Sequence↗
PubMed1995

Binding of defined regions of a polypeptide to GroEL and its implications for chaperonin-mediated protein folding.

Unfolded rhodanese in a complex with the chaperonin GroEL was subjected to limited proteolysis. Sequence analysis indentified a GroEL-bound fragment of approximately 11,000 M(r) and a well defined fragment of approximately 7,000 M(r) from the two homologous domains of rhodanese. The shorter segment contains one hydrophobic and one amphiphilic alpha-helix mapping to the domain interface while the other fragment contains the homologous regions and an additional hydrophobic helix. Our results suggest a mechanism for the GroEL-mediated folding of rhodanese in which the domain-forming regions of the polypeptide are kept apart and are then released, perhaps sequentially, resulting in correct folding.

Amino Acid Sequence↗
PubMed1995

Porin activity and sequence analysis of a 31-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp1).

We have recently reported the isolation and purification of the Treponema pallidum outer membrane and the identification of its rare protein constituents, including a 31-kDa protein markedly enriched in the outer membrane preparation (D.R. Blanco, K. Reimann, J. Skare, C.I. Champion, D. Foley, M. M. Exner, R. E. W. Hancock, J. N. Miller, and M. A. Lovett, J. Bacteriol. 176:6088-6099, 1994). In this study, we report the cloning, sequencing, and expression of the structural gene which encodes the 31-kDa outer membrane protein, designated Tromp1. The deduced amino acid sequence from the tromp1 gene sequence encodes a 318-amino-acid polypeptide with a putative 40-amino-acid signal peptide. Processing of Tromp1 results in a mature protein with a predicted molecular mass of 30,415 Da and a calculated pI of 6.6. Secondary-structure predictions identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of Tromp1 containing 14 transmembrane segments is proposed. Specific antiserum against a recombinant Tromp1 fusion protein was generated and was used to identify native Tromp1 in cellular fractionation. Upon Triton X-114 extraction and phase separation of T. pallidum, the 31-kDa Tromp1 protein was detected in the detergent-phase fraction but not in the protoplasmic cylinder or aqueousphase fractions, consistent with a hydrophobic outer membrane protein. Anti-Tromp1 antiserum was also used to identify native Tromp1 purified from whole T. pallidum by Triton X-100 solubilization followed by nondenaturing isoelectric focusing. Reconstitution of purified Tromp1 into planar lipid bilayers showed porin activity based on the measured single channel conductanes of 0.15 and 0.7 nS in 1 M KCl. These findings demonstrate that Tromp1 is a transmembrane outer membrane porin protein of T. pallidum.

Amino Acid Sequence↗
PubMed1994

Adipocyte-specific transcription factor ARF6 is a heterodimeric complex of two nuclear hormone receptors, PPAR gamma and RXR alpha.

Previously, we identified a novel transcription factor, ARF6, as a key regulator of the tissue-specific adipocyte P2 (aP2) enhancer. In order to identify the proteins which comprise the adipocyte ARF6 complex, we have purified this DNA binding activity from a cultured adipocyte cell line. We have developed a system for growth and differentiation of HIB-1B brown adipocytes in suspension culture that facilitates the production of large quantities of adipocyte nuclear extract. ARF6 was purified from HIB-1B nuclear extract by a combination of conventional and sequence-specific DNA affinity chromotography. Chemical sequencing and mass spectral analysis of tryptic peptides derived from the purified polypeptides identifies the ARF6 complex as a heterodimer of the retinoid X receptor alpha (RXR alpha) and the murine peroxisome proliferator activated receptor gamma (PPAR gamma). Of the known PPAR gamma isoforms, PPAR gamma is the predominant form expressed in adipose tissue. These results suggest that PPAR gamma 2 serves a unique function among PPAR family members as an important regulator of adipocyte-specific gene expression.

Adipocytes↗
PubMed1994

Acute transcriptional response of the honeybee peptide-antibiotics gene repertoire and required post-translational conversion of the precursor structures.

The cell-free immune repertoire of honeybees (Apis mellifera) consists of four polypeptides that are induced by bacterial infection and, through complementarity, provide broad-spectrum antibacterial defense. apidaecin is overproduced by a combination of low threshold transcriptional activation and a unique, genetically encoded amplification mechanism. In contrast, sizable experimental infections are required for induction of the normally silent hymenoptaecin, abaecin, and bee defensin genes; even so, bee defensin transcription is minimal and delayed, and only minute quantities of corresponding peptide are produced. The specific, temporal organization of the multi-component immune response in bees has therefore likely been selected to cope with infection of prevalent, plant-associated Gram-negative bacteria. Post-translational processing and modifications are substantially different for each of the four antibacterial peptides. While no similarities were observed among precursor structures of the various bee peptides, surprisingly, the signal sequences of abaecin (bee) and drosocin (Drosophila) shared unmistakable homology, possibly indicating common ancestral secretion/processing mechanisms. Finally, we report that bee defensin contains a typical disulfide-rich structure (40 amino acids) but also a unique, amphipathic, putatively amidated carboxyl-terminal tail (10 amino acids). We speculate that this structure is a "co-drug," assembled by fusing "disulfide-rich" and "alpha-helical" class peptide antibiotics, a novel concept in naturally occurring antibacterials.

Amino Acid Sequence↗
PubMed1994

Identification of CRKL as the constitutively phosphorylated 39-kD tyrosine phosphoprotein in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is characterized by the presence of the Philadelphia (Ph) chromosome in clonally derived hematopoietic precursors and their progeny. The Ph chromosome arises from a translocation that deregulates the c-ABL protein tyrosine kinase, giving it transforming potential and increased kinase activity. We observed a unique 39-kD tyrosine phosphoprotein (pp39), previously reported in blastic CML cell lines, in neutrophils from 50 cases of chronic phase CML. This protein was prominently and constitutively tyrosine-phosphorylated in CML neutrophils and was not phosphorylated in normal neutrophils. Stimulation of normal neutrophils with cytokines and agonists did not induce tyrosine phosphorylation of proteins migrating in the region of pp39, and the phosphorylation state of pp39 in CML neutrophils was not affected by kinase inhibitors known to downregulate the ABL kinase. The pp39 was not phosphorylated in hematopoietic cells from healthy donors or from patients with Ph chromosome-negative myeloproliferative disorders. Using micro amino acid sequencing of purified preparations of pp39, we identified pp39 as CRKL protein, which is consistent with recent immunologic studies in the blastic K562 cell line. Immunoblotting with anti-CRKL antibodies showed the presence of CRKL protein in CML cells and cell lines as well as in antiphosphotyrosine immunoprecipitates from CML cells. Our results suggest that pp39 CRKL in CML neutrophils may be stably tyrosine-phosphorylated by the BCR/ABL kinase at an early stage of myeloid differentiation when the ABL kinase is active. CRK, CRKL, and other SH2 (SRC homology domain)/SH3-containing proteins function as adaptor molecules in nonreceptor tyrosine kinase signalling pathways. Although the CRKL protein is present in normal neutrophils, it is not tyrosine-phosphorylated, and the inability to induce such phosphorylation in normal neutrophils suggests a special role of this phosphoprotein in the pathogenesis of CML. Constitutive phosphorylation of CRKL is unique to CML, indicating that it may be a useful target for therapeutic intervention.

Adaptor Proteins, Signal Transducing↗
PubMed1994

Biodiversity of apidaecin-type peptide antibiotics. Prospects of manipulating the antibacterial spectrum and combating acquired resistance.

Insects have a unique repertoire of peptide antibiotics but, to date, prospects of clinical applications are not clear. Apidaecin, a small peptide isolated from honeybees, inhibits viability of Gram-negative bacteria; lethal activity is near immediate, independent of a conventional "lytic" mechanism, and involves stereoselective recognition of target molecules. Here we report structural analysis of 14 naturally occurring apidaecin-type peptides and the existence of evolutionarily conserved ("constant") regions. By detailed analysis of activities against clinically relevant bacteria, we demonstrate that the diversity of the intervening ("variable") regions confers specificity to the antibacterial spectrum of each homolog. As a result, apidaecin-homolog-based antibiograms (using 16 peptides) differ markedly between bacterial strains, contrasting the most between Yersinia enterocolitica and Campylobacter jejuni. Furthermore, in at least one instance, acquired resistance to apidaecin could be negated by minor substitutions in the variable regions. The delineation in a short peptide of constant and variable regions, responsible for, respectively, general antibacterial capacity and specificity of the antibacterial spectrum, is unprecedented. Taken together, we provide evidence that antibacterial spectra of apidaecin-type peptides can be manipulated, and that, in some cases, resistance can be countered and perhaps prevented. The current findings will guide rational design of second generation peptide antibiotics for clinical trials.

Amino Acid Sequence↗
PubMed1994

A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles.

Rab proteins are generally required for transport vesicle docking. We have exploited yeast secretion mutants to demonstrate that a rab protein is required for v-SNAREs and t-SNAREs to assemble. The absence of the rab protein in the docking complex suggests that, in a broad sense, rab proteins participate in a reaction catalyzing SNARE complex assembly. In so doing, rab proteins could help impart an additional layer of specificity to vesicle docking. This mechanism likely involves the Sec1 homolog Sly1, which we identified in isolated docking complexes. We also report the identification of a novel v-SNARE (Ykt6p) component of the yeast ER-Golgi docking complex that has a CAAX box and is predicted to be lipid anchored. The surprising finding that docking complexes can contain many distinct species of SNAREs (Sed5p, Bos1p, Sec22p, Ykt6p, and likely Bet1p, p28, and p14) suggests that multimeric interactions are features of the fusion machinery, and may also improve the fidelity of vesicle targeting.

Amino Acid Sequence↗
PubMed1994

Metal chelates as reversible stains for detection of electroblotted proteins: application to protein microsequencing and immunoblotting.

Coomassie brilliant blue and Ponceau red have traditionally been used to stain electroblotted proteins, since they are compatible with existing N-terminal and internal protein microsequencing as well as with immunoblotting procedures. With recent improvements in sequencing and immunoblotting technology, detection of significantly smaller amounts of protein has become necessary. Metal complexes were evaluated as alternatives to conventional stains. Electroblotted proteins were detected by blocking nonspecific sites with polyvinylpyrrolidone-40 followed by incubation in metal chelate solutions at acidic pH values. Two of the most promising metal chelate stains were the Ferrozine/ferrous complex and the ferrocyanide/ferric complex. Both stained a wide variety of proteins and peptides quantitatively. Dot blots and 1D and 2D electroblots were successfully stained using iron chelates. When these two stains were utilized in combination, they were of equivalent sensitivity to colloidal gold stain. The reversibility of the metal chelate stains was substantiated by incubating stained membranes at neutral to basic pH in the presence of 20 mM ethylenediaminetetraacetic acid to rapidly elute the complexes from the bound proteins. The chelate stains were determined to be fully compatible with immunoblotting, N-terminal, and in situ internal protein microsequencing.

Amino Acid Sequence↗
PubMed1994

Microbore reversed-phase high-performance liquid chromatographic purification of peptides for combined chemical sequencing-laser-desorption mass spectrometric analysis.

An optimized microbore RP-HPLC system (1.0 mm I.D. columns) for the purification of low picomole amounts (< 5 pmol) of peptides is described. It is comprised of commercially available columns, instrument components and parts. These were selected on the basis of a comparative evaluation and to yield the highest resolution and most efficient peak collection. The sensitivity of this system equals, probably surpasses, that of advanced chemical microsequencing for which 2-4 pmol of peptide are minimally required. As an automated sequencer cannot be "on-line" connected with a micro-preparative HPLC system, fractions must be collected and transferred. With a typical flow of 30 microliters, efficient manual collection is possible and fractions (about 20 microliters in volume) can still be handled without unacceptable losses, albeit with great precaution. Furthermore, major difficulties were encountered to efficiently and quantitatively load low- or sub-picomole amounts of peptide mixtures onto the RP-HPLC column for separation. Discipline and rigorous adherence to sample handling protocols are thus on order when working at those levels of sensitivity. With adequate instrumentation and handling procedures in place, we demonstrate that low picomole amounts of peptides can now be routinely prepared for analysis by combined Edman-chemical sequencing-matrix-assisted laser-desorption mass spectrometry (MALDI-MS). The integrated method was applied to covalent structural characterization of minute quantities of a gel-purified protein of known biological function but unknown identity. The results allowed unambiguous identification and illustrated the power of MALDI-MS-aided interpretation of chemical sequencing data: accurate peptide masses were crucial for (i) confirmation of the results, (ii) deconvolution of mixed sequences, (iii) proposal of complete structures on the basis of partial sequences, and (iv) confirmation of protein identification (obtained by database search with a single, small stretch of peptide sequence) by "mass matching" of several more peptides with predicted proteolytic fragments.

Amino Acid Sequence↗
PubMed1994

RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs.

The immunosuppressants rapamycin and FK506 bind to the same intracellular protein, the immunophilin FKBP12. The FKB12-FK506 complex interacts with and inhibits the Ca(2+)-activated protein phosphatase calcineurin. The target of the FKBP12-rapamycin complex has not yet been identified. We report that a protein complex containing 245 kDa and 35 kDa components, designated rapamycin and FKBP12 targets 1 and 2 (RAFT1 and RAFT2), interacts with FKBP12 in a rapamycin-dependent manner. Sequences (330 amino acids total) of tryptic peptides derived from the 245 kDa RAFT1 reveal striking homologies to the yeast TOR gene products, which were originally identified by mutations that confer rapamycin resistance in yeast. A RAFT1 cDNA was obtained and found to encode a 289 kDa protein (2549 amino acids) that is 43% and 39% identical to TOR2 and TOR1, respectively. We propose that RAFT1 is the direct target of FKBP12-rapamycin and a mammalian homolog of the TOR proteins.

Amino Acid Sequence↗
PubMed1994

Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals.

We cloned p27Kip1, a cyclin-dependent kinase inhibitor implicated in G1 phase arrest by TGF beta and cell-cell contact. p27Kip1 associates with cyclin E-Cdk2 complexes in vivo and in vitro, prevents their activation, and inhibits previously activated complexes, and p27Kip1 overexpression obstructs cell entry into S phase. p27Kip1 potently inhibits Rb phosphorylation by cyclin E-Cdk2, cyclin A-Cdk2, and cyclin D2-Cdk4. p27Kip1 is highly conserved and broadly expressed in human tissues, and its mRNA levels are similar in proliferating and quiescent cells. p27Kip1 has a region of sequence similarity to p21Cip1/WAF1, the Cdk inhibitor whose transcription is stimulated by p53. A p27Kip1 peptide corresponding to this region retains Cdk inhibitory activity. We suggest that cell contact, TGF beta, and p53 all restrain cell proliferation through related Cdk inhibitors.

Amino Acid Sequence↗
PubMed1994

A glutathione S-transferase (GST) isozyme from broccoli with significant sequence homology to the mammalian theta-class of GSTs.

A novel glutathione S-transferase (GST) was purified from broccoli (Brassica oleracea var. italica). Partial amino-acid sequencing indicated that the protein shared significant homology with several different plant GSTs from maize, silene, Dianthus, Nicotiana and Triticum, but little homology to yeast (Issatchenkia) GST. One region of the polypeptide near the N-terminal also shared significant homology to a region of rat 5-5, rat 12-12 and human theta-GST (collectively referred to as the theta-GST-class) but little structural homology to the common mammalian cytosolic GSTs (alpha-, mu- or pi-classes). The broccoli GST was retained on a novel membrane based glutathione affinity matrix and displayed activity towards 1-chloro-2,4-dinitro-benzene (CDNB), a general GST substrate, as well as 4-nitrophenethyl bromide, a marker substrate for the theta-class of GSTs. The characteristics of the broccoli GST potentially define it as a member of the theta-class. This is consistent with the view that the theta-class may have arisen prior to the divergence of animals and plants while the mammalian mu-, pi- and alpha-classes evolved after the two kingdoms were established.

Amino Acid Sequence↗
PubMed1994

Apidaecin-type peptide antibiotics function through a non-poreforming mechanism involving stereospecificity.

Insect resistance to bacterial infections is dependent on the production of specialized defense peptides. We report here that lethal activities of apidaecin, a small peptide from honeybees, cannot possibly be the result of a conventional 'lytic' mechanism. Evidence includes the complete lack of membrane permeabilization, at concentrations that exceed lethal doses by four orders of magnitude, and undiminished sensitivity of apidaecin-resistant mutants to 'poreforming' peptides. In addition, the D-enantiomer of apidaecin is completely devoid of antibacterial activities. We propose therefore, that the antagonistic effects of apidaecin involve stereoselective recognition of a chiral cellular target, establishing this peptide as functionally unique among insect antibacterials. Identification of the apidaecin target may provide the scientific basis for rational drug design.

Amino Acid Sequence↗
PubMed1994

High-sensitivity sequencing of large proteins: partial structure of the rapamycin-FKBP12 target.

We report on studies leading to refinements of various steps of the protein internal sequencing process. Specifically, the developments comprise (1) higher-sensitivity chemical sequencing through background reduction; (2) improved peptide recovery from rapid in situ digests of nanogram amount, nitrocellulose-bound proteins; and (3) accurate UV spectroscopic identification of Trp- and Cys-containing peptides. In addition, we describe strategies for 2-dimensional liquid chromatographic peptide isolation from complex mixtures and a multi-analytical approach to peptide sequence analysis (Edman sequencing, matrix-assisted laser desorption mass spectrometry, and UV spectroscopy). Both strategies were applied in tandem to the primary structural analysis of a gel-purified, 250-kDa protein (mammalian target of rapamycin-FKBP12 complex), available in low picomolar quantities only. More than 300-amino acids worth of sequence was obtained in mostly uninterrupted stretches, several containing Trp, Cys, His, and Ser. That information has allowed the matching of a biological function of a mammalian protein to a yeast gene product with a well-characterized mutant phenotype. The results also demonstrate that extended chemical sequencing analysis (e.g., 26 successive amino acids) is now feasible, starting with initial yields well below 1 pmol.

Amino Acid Sequence↗
PubMed1994

The recombination signal sequence-binding protein RBP-2N functions as a transcriptional repressor.

We have identified a cellular protein, RBP-2N, a presumed recombinase, as a repressor of transcription. Inhibition of transcription by RBP-2N was dependent on its DNA recognition site and was demonstrated in vitro and in vivo. This repression appears to be general, as transcription mediated by SP1 and Gal4/VP16 was inhibited by RBP-2N. The protein was purified to near homogeneity from human cells on the basis of its binding to a site present in the promoter of the adenovirus pIX gene. The DNA recognition sequence is 5'-TGGGAAAGAA, which is markedly different from the recombination signal sequence originally identified as the target site for this protein. The sequence of the purified protein is 97% identical with that published for the mouse RBP-2N protein. The reported homolog in Drosophila is Suppressor of Hairless. RBP-2N binding sites are present in a number of cellular and viral promoters, so RBP-2N may have a general role in transcriptional repression.

Amino Acid Sequence↗