C-terminal Position Research Articles

Polyglutamine tracts as modulators of transcriptional activation from yeast to mammals

Microsatellite repeats are genetically unstable and subject to expansion and shrinkage. A subset of them, triplet repeats, can occur within the coding region and specify homomeric tracts of amino acids. Polyglutamine (polyQ) tracts are enriched in eukaryotic regulatory proteins, notably transcription factors, and we had shown before that they can contribute to transcriptional activation in mammalian cells. Here we generalize this finding by also including evolutionarily divergent organisms, namely, Drosophila and baker's yeast. In all three systems, Gal4-based model transcription factors were more active if they harbored a polyQ tract, and the activity depended on the length of the tract. By contrast, a polyserine tract was inactive. PolyQs acted from either an internal or a C-terminal position, thus ruling out a merely structural 'linker' effect. Finally, a two-hybrid assay in mammalian cells showed that polyQ tracts can interact with each other, supporting the concept that a polyQ-containing transcription factor can recruit other factors with polyQ tracts or glutamine-rich activation domains. The widespread occurrence of polyQ repeats in regulatory proteins suggests a beneficial role; in addition to the contribution to transcriptional activity, their genetic instability might help a species to adapt to changing environmental conditions in a potentially reversible manner.

ChemInform Abstract: Aminoboronic Acids and Esters: From Synthetic Challenges to the Discovery of Unique Classes of Enzyme Inhibitors

AbstractReview: 156 refs.

A Systematic Study of Site-specific GalNAc-type O-Glycosylation Modulating Proprotein Convertase Processing

Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model for O-glycosylation especially of isolated sites, but serine and to a lesser extent threonine residues are frequently found adjacent to PC processing sites. In the present study we used in vitro enzyme assays and ex vivo cell models to systematically address the boundaries of the region within site-specific O-glycosylation affect PC processing. The results demonstrate that O-glycans within at least ±3 residues of the RXXR furin cleavage site may affect PC processing suggesting that site-specific O-glycosylation is a major co-regulator of PC processing.

A new fluorescence complementation biosensor for detection of estrogenic compounds

Estrogenic compounds are an important class of hormonal substances that can be found as environmental contaminants, with sources including pharmaceuticals, human and animal waste, the chemical industry, and microbial metabolism. Here we report the creation of a biosensor useful for monitoring such compounds, based on complementation of fluorescent protein fragments. A series of sensors were made consisting of fragments of a split mVenus fluorescent protein fused at several different N-terminal and C-terminal positions flanking the ligand binding domain of the estrogen receptor alpha. When expressed in HeLa cells, sensor 6 (ERα 312-595) showed a nine-fold increase in fluorescence in the presence of estrogen receptor agonists or antagonists. Sensor 2 (ERα 281-549) discriminated between agonists and antagonists by showing a decrease in fluorescence in the presence of agonists while being induced by antagonists. The fluorescent signal of sensor 6 increased over a period of 24 h, with a two-fold induction visible at 4 h and four-fold at 8 h of ligand incubation. Ligand titration showed a good correlation with the known relative binding affinities of the compound. The sensor could detect a number of compounds of interest that can act as environmental endocrine disruptors. The lack of a substrate requirement, the speed of signal development, the potential for high throughput assays, and the ability to distinguish agonists from antagonists make this an attractive sensor for widespread use.

Stability Screening of Arrays of Major Histocompatibility Complexes on Combinatorially Encoded Flow Cytometry Beads

The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.

Tandem termination signal in plant mRNAs

It was proposed that if some mRNA characteristics resulted in a low efficiency of termination signal, an additional closely located stop codon (tandem stop codons) could be used to prevent the harmful readthrough. However, the role of tandem terminators in higher eukaryotes was not verified and remains hypothetical. In this work the sequence features of Arabidopsis thaliana and Oryza sativa mRNAs were analyzed. It was found that plant mRNAs with UGA terminator were characterized by a higher frequency of nonsense codons in the first triplet position of 3′-UTR that could result from a weak natural selection for “reserve” stop signal. Interestingly, the presence of tandem stop codons positively correlated with a specific amino acid composition in the C-terminal position of the encoded proteins. In particular, C-terminal glycine positively correlated with significantly higher frequencies of reserve terminators at the beginning positions of 3′-UTR in UGA-containing mRNAs. This finding coincides with some earlier observations concerning the role of glycine and its codons in inefficient termination of translation and recoding (e.g., 2A oligopeptide).

Abstract 5584: Green tea polyphenols inhibit histone deacetylases and modulate p53 transcriptional activities in human prostate cancer cells

Abstract Inhibitors of histone deacetylases (HDACs) are emerging as a promising class of anticancer drugs. HDAC inhibitors have been demonstrated to induce cellular activities similar to p53-mediated responses such as induction of cell cycle arrest and apoptosis in various malignant cells. Green tea polyphenols (GTP) have shown protective effects against development and progression of various human cancers. GTP-mediated cellular responses include p53 activation, cell cycle arrest and induction of apoptosis in cancer cells. In the present study, we sought to determine whether GTP-mediated cellular responses are similar to that elicited by HDAC inhibitors. For these studies, we used human prostate cancer LNCaP cells, possessing wild-type p53, infected with short hairpin (sh) RNA vector and sh-p53 RNA vector to generate isogenic cell lines. Knockdown of p53 in LNCaP cells caused an increase in the activity of class I HDACs with specific increase in the protein expression of HDAC viz. 1 and 8, respectively. Treatment of both cell lines with 20, 40 and 80 mcg/mL doses of GTP for 24 h resulted in dose-dependent decrease in the total HDAC activity and protein expression of HDAC 2, 3 and 8 and HDAC 1, albeit at highest GTP dose. Similar dose-dependent responses were achieved by trichostatin A (TSA), a well known pharmacological inhibitor of class I and II HDACs in these cells. Interestingly, chemosensitization by GTP was more pronounced in the cells expressing p53 than the isogenic cell lines lacking p53 as observed by cell cycle analysis and annexin V flow cytometric analysis. Furthermore, GTP treatment resulted in marked increase in acetylation of p53 at its C-terminal amino acid positions K373 and K382, other than K305 along with increase in the acetylated forms of histone H3 and H4 which correlated with increased p53 expression, cell cycle arrest and apoptosis. Taken together, these results suggest that interaction of HDACs with p53 probably results in p53 deacetylation, thereby reducing the transcriptional activity; whereas GTP has the ability to cause deacetylation of p53 by reducing the levels of class I HDACs. This seems likely to be, in part, the mechanism for GTP-mediated p53 activation, a similar response elicited by HDAC inhibitors, which are developed as a promising class of drugs for the treatment of various human malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5584. doi:10.1158/1538-7445.AM2011-5584

Determination of the Membrane Topology of the Small EF-Hand Ca2+-Sensing Proteins CaBP7 and CaBP8

The CaBPs represent a subfamily of small EF-hand containing calcium (Ca2+)-sensing proteins related to calmodulin that regulate key ion channels in the mammalian nervous system. In a recent bioinformatic analyses we determined that CaBP7 and CaBP8 form an evolutionarily distinct branch within the CaBPs (also known as the calneurons) a finding that is consistent with earlier observations characterising a putative C-terminal transmembrane (TM) spanning helix in each of these proteins which is essential for their sub-cellular targeting to the Golgi apparatus and constitutive secretory vesicles. The C-terminal position of the predicted TM-helix suggests that CaBP7 and CaBP8 could be processed in a manner analogous to tail-anchored integral membrane proteins which exhibit the ability to insert across membranes post-translationally. In this study we have investigated the topology of CaBP7 and CaBP8 within cellular membranes through a combination of trypsin protection and epitope accessibility analyses. Our results indicate that the TM-helices of CaBP7 and CaBP8 insert fully across membranes such that their extreme C-termini are luminal. The observed type-II membrane topology is consistent with processing of CaBP7 and CaBP8 as true tail-anchored proteins. This targeting mechanism is distinct from any other calmodulin related Ca2+-sensor and conceivably underpins unique physiological functions of these proteins.

Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

Aminoboronic acids and esters: from synthetic challenges to the discovery of unique classes of enzyme inhibitors

Although boronic acids have attracted considerable interest as versatile intermediates in organic synthesis, their contributions in chemical biology and drug discovery programs have long been underestimated. This situation is changing since the beginning of the 2000s, mainly due to significant advances in modern organoborane chemistry and the recent FDA approval of Velcade®, a boropeptide used for multiple myeloma treatment. There is now a significant renewed interest in the design and synthesis of new boron-containing compounds. Due to their close analogy to their carbon counterparts, aminoboronic acids, alone or incorporated at the C-terminal position of a peptide, represent one of the major classes of organoboranes evaluated as potential drug candidates. This critical review aims to provide an overview of the current state of the art in their synthesis and their most relevant biological properties (156 references).

Intracellular Cysteine 346 Is Essentially Involved in Regulating Panx1 Channel Activity

Pannexins constitute a family of proteins exhibiting predominantly hemichannel activity. Pannexin channels have been suggested to participate in a wide spectrum of biological functions such as propagation of calcium waves, release of IL-1β, and responses to ischemic conditions. At present, the molecular mechanisms regulating pannexin hemichannel activity are essentially unknown. Because cysteines have been shown to constitute key elements in regulating hemichannel properties of the connexin-type we performed site-directed mutagenesis of intracellular cysteine residues of Panx1. Cysteine to serine exchange (Cys → Ser) at the C-terminal position amino acid 346 led to a constitutively leaky hemichannel and subsequently to cell death. Increased channel activity was demonstrated by dye uptake and electrophysiological profiling in injected Xenopus laevis oocytes and transfected N2A cells. Mutations of the remaining intracellular cysteines did not result in major changes of Panx1 channel properties. From these data we conclude that the Cys-346 residue is important for proper functioning of the Panx1 channel.

Conformations of peptides containing a chiral cyclic α, α‐disubstituted α‐amino acid within the sequence of Aib residues

A single chiral cyclic α,α-disubstituted amino acid, (3S,4S)-1-amino-(3,4-dimethoxy)cyclopentanecarboxylic acid [(S,S)-Ac(5)c(dOM)], was placed at the N-terminal or C-terminal positions of achiral α-aminoisobutyric acid (Aib) peptide segments. The IR and (1)H NMR spectra indicated that the dominant conformations of two peptides Cbz-[(S,S)-Ac(5)c(dOM)]-(Aib)(4)-OEt (1) and Cbz-(Aib)(4)-[(S,S)-Ac(5)c(dOM)]-OMe (2) in solution were helical structures. X-ray crystallographic analysis of 1 and 2 revealed that a left-handed (M) 3(10)-helical structure was present in 1 and that a right-handed (P) 3(10)-helical structure was present in 2 in their crystalline states.

Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection

BackgroundMolecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products.ResultsOur vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expression of target proteins in E.coli cells. Variable tags are available both in N-terminal or C-terminal position. A newly developed β-lactamase (ΔW290) selection cassette contains a segment inside the β-lactamase open reading frame encoding a stretch of hydrophilic amino acids that result in a T7 promoter when back-translated. This position of the promoter permits positive selection and attenuated expression of fusion proteins with C-terminal tags. We have tested eight vectors by inserting six target sequences of variable length, provenience and function. The target proteins were cloned, expressed and detected using an automated Tecan Freedom Evo II liquid handling work station. Only two colonies had to be picked to score with 85% correct inserts while 80% of those were positive in expression tests.ConclusionsOur results establish co-transformation and positive/negative selection cloning in conjunction with the provided vectors and selection cassettes as an automatable alternative to commercialized high-throughput cloning systems like Gateway® or ligase-independent cloning (LIC) .

Characterization of the Substrate Specificity of Human Carboxypeptidase A4 and Implications for a Role in Extracellular Peptide Processing

CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment.

Exploration of structure–activity relationships at the two C-terminal residues of potent 11mer Glucagon-Like Peptide-1 receptor agonist peptides via parallel synthesis

We report the identification of potent agonists of the Glucagon-Like Peptide-1 receptor (GLP-1R) via evaluation of two positional scanning libraries and a two-dimensional focused library, synthesized in part on SynPhase™ Lanterns. These compounds are 11 amino acid peptides containing several unnatural amino acids, including (in particular) analogs of biphenylalanine (Bip) at the two C-terminal positions. Typical activities of the most potent peptides in this class are in the picomolar range in an in vitro functional assay using human GLP-1 receptor.

Abstract 3801: p53 acetylation by green tea polyphenols leads to upregulation of GSTP1 activity

Abstract Epigenetic modifications including DNA methylation and post-translational changes in histone proteins cause alteration in gene regulation. Expression of glutathione-S-transferase Pi (GSTP1), an enzyme of phase II cellular metabolism involved in detoxification of carcinogens and other cellular functions found to be silenced in 80-90% prostate cancers due to hypermethylation of its promoter. We have recently demonstrated that green tea polyphenols (GTPs) cause re-expression of GSTP1 in human prostate cancer LNCaP cells [Int J Cancer. 2009; PMID: 19856314]. However, the mechanism of GSTP1 re-expression is not fully elucidated. Similar to GSTP1 gene, p53 tumor suppressor is also involved in a number of important cellular events including cell cycle, DNA repair, and apoptosis which is reported to be mutated or has defective signaling in most human cancers including prostate cancer. Human GSTP1 gene has a p53-binding motif located in intron 4 and is highly homologous to consensus p53-binding motifs in other p53-responsive genes having the ability to transcriptionally activate GSTP1. Whether p53 participates in GTP-mediated re-expression of GSTP1 is unknown. In the present study we attempted to elucidate the role of tumor suppressor p53 in GSTP1 re-expression after treatment with GTPs. Human prostate cancer LNCaP cells which possess epigenetic signatures of GSTP1 silencing and functional p53 were treated with physiologically achievable doses of GTPs (5μg/mL) or its major constituent epigallocatechin-3-gallate (20μM) up to 7 days. Exposure of LNCaP cells to green tea polyphenols caused acetylation of p53 at its C-terminal amino acid positions K373 and K382, other than K305, which correlated with increased GSTP1 expression. Discontinuation of treatment with GTPs resulted in the loss of p53 acetylation with progressive decrease in GSTP1 expression in these cells. Furthermore, GSTP1 levels were significantly reduced after GTP treatment in cells with the knockdown of p53 expression achieved using shRNA in lentiviral vector. Taken together, our results demonstrate that expression of GSTP1, in part, could be enhanced through p53 acetylation after treatment with green tea polyphenols and thus protect the cells from undergoing epigenetic alterations which may contribute to prostate tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3801.

Prokaryotic Ubiquitin-like Protein (Pup) Is Coupled to Substrates via the Side Chain of Its C-Terminal Glutamate

A prokaryotic protein tagging system called pupylation that is analogous to ubiquitylation in eukaryotes has recently been described. In this process, prokaryotic ubiquitin-like protein (Pup) is coupled to substrate proteins via an isopeptide bond in order to target them for degradation by the proteasome. The ligation occurs via a condensation reaction involving a carboxylate of the C-terminal glutamate of Pup (located in a conserved terminal Gly-Gly-Glu motif) and the side-chain amino group of a substrate lysine. Here we have used a combination of NMR and biochemical experiments with free lysine and a physiological protein substrate (PanB) to show that the coupling occurs through the side-chain carboxylate of the glutamate in the GGE motif rather than the carboxy terminus but that the glutamate must be located at the C-terminal position to be coupled.

Development and photo-responsive peptide bond cleavage reaction of two-photon near-infrared excitation-responsive peptide

Two-photon near-infrared excitation-responsive amino acid was developed. It was incorporated into a peptide, and focused near-infrared pulsed laser irradiation-induced peptide bond cleavage reaction at the C-terminal position of the photo-responsive amino acid was observed.

Identification of potent 11mer Glucagon-Like Peptide-1 Receptor agonist peptides with novel C-terminal amino acids: Homohomophenylalanine analogs

We report the identification of potent agonists of the Glucagon-Like Peptide-1 Receptor (GLP-1R). These compounds are short, 11 amino acid peptides containing several unnatural amino acids, including (in particular) analogs of homohomophenylalanine (hhPhe) at the C-terminal position. Typically the functional activity of the more potent peptides in this class is in the low picomolar range in an in vitro cAMP assay, with one example demonstrating excellent in vivo activity in an ob/ ob mouse model of diabetes.

Controlling the helical screw sense of peptides with C‐terminal L‐valine

One chiral L-valine (L-Val) was inserted into the C-terminal position of achiral peptide segments constructed from alpha-aminoisobutyric acid (Aib) and alpha,beta-dehydrophenylalanine (Delta(Z)Phe) residues. The IR, (1)H NMR and CD spectra indicated that the dominant conformations of the pentapeptide Boc-Aib-DeltaPhe-(Aib)(2)-L-Val-NH-Bn (3) and the hexapeptide Boc-Aib-DeltaPhe-(Aib)(3)-L-Val-NH-Bn (4) in solution were both right-handed (P) 3(10)-helical structures. X-ray crystallographic analyses of 3 and 4 revealed that only a right-handed (P) 3(10)-helical structure was present in their crystalline states. The conformation of 4 was also studied by molecular-mechanics calculations.