Insulin-regulated aminopeptidase : analysis of peptide substrate and inhibitor binding to the catalytic domain

Peptide inhibitors of insulin-regulated aminopeptidase (IRAP) accelerate spatial learning and facilitate memory retention and retrieval by binding competitively to the catalytic site of the enzyme and inhibiting its catalytic activity. IRAP belongs to the M1 family of Zn2+-dependent aminopeptidases characterized by a catalytic domain that contains two conserved motifs, the HEXXH(X)18E Zn2+-binding motif and the GXMEN exopeptidase motif. To elucidate the role of GXMEN in binding peptide substrates and competitive inhibitors, site-directed mutagenesis was performed on the motif. Non-conserved mutations of residues G428, A429 and N432 resulted in mutant enzymes with altered catalytic activity, as well as divergent changes in kinetic properties towards the synthetic substrate leucine β-naphthalamide. The affinities of the IRAP inhibitors angiotensin IV, Nle1-angiotensin IV, and LVV-hemorphin-7 were selectively decreased. Substrate degradation studies using the in vitro substrates vasopressin and Leu-enkephalin showed that replacement of G428 by either D, E or Q selectively abolished the catalysis of Leu-enkephalin, while [A429G]IRAP and [N432A]IRAP mutants were incapable of cleaving both substrates. These mutational studies indicate that G428, A429 and N432 are important for binding of both peptide substrates and inhibitors, and confirm previous results demonstrating that peptide IRAP inhibitors competitively bind to its catalytic site.

Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein

The human ZC3H14 gene encodes an evolutionarily conserved Cys(3)His zinc finger protein that binds specifically to polyadenosine RNA and is thus postulated to modulate post-transcriptional gene expression. Expressed sequence tag (EST) data predicts multiple splice variants of both human and mouse ZC3H14. Analysis of ZC3H14 expression in both human cell lines and mouse tissues confirms the presence of multiple alternatively spliced transcripts. Although all of these transcripts encode protein isoforms that contain the conserved C-terminal zinc finger domain, suggesting that they could all bind to polyadenosine RNA, they differ in other functionally important domains. Most of the alternative transcripts encode closely related proteins (termed isoforms 1, 2. 3, and 3short) that differ primarily in the inclusion of three small exons, 9, 10, and 11, resulting in predicted protein isoforms ranging from 82 to 64 kDa. Each of these closely related isoforms contains predicted classical nuclear localization signals (cNLS) within exons 7 and 11. Consistent with the presence of these putative nuclear targeting signals, these ZC3H14 isoforms are all localized to the nucleus. In contrast, an additional transcript encodes a smaller protein (34 kDa) with an alternative first exon (isoform, 4). Consistent with the absence of the predicted cNLS motifs located in exons 7 and 11, ZC3H14 isoform 4 is localized to the cytoplasm. Both EST data and experimental data suggest that this variant is enriched in testes and brain. Using an antibody that detects endogenous ZC3H14 isoforms 1-3 reveals localization of these isoforms to nuclear speckles. These speckles co-localize with the splicing factor, SC35, suggesting a role for nuclear ZC3H14 in mRNA processing. Taken together, these results demonstrate that multiple transcripts encoding several ZC3H14 isoforms exist in vivo. Both nuclear and cytoplasmic ZC3H14 isoforms could have distinct effects on gene expression mediated by the common Cys(3)His zinc finger polyadenosine RNA binding domain. (C) 2009 Elsevier B.V. All rights reserved.

Deconstructing the DGAT1 enzyme: Binding sites and substrate interactions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Allergen motifs and the prediction of allergenicity

We have recently shown that the majority of allergens can be represented by allergen motifs. This observation prompted us to experimentally investigate the synthesized peptides corresponding to the in silico motifs with regard to potential IgE binding and cross-reactions with allergens. Two motifs were selected as examples to conduct in vitro studies. From the first motif, derived from allergenic MnSOD sequences, the motif stretch of the allergen Asp f 6 was selected and synthesized as a peptide (MnSOD Mot). The corresponding full-length MnSOD was also expressed in Escherichia coli and both were compared for IgE reactivity with sera of patients reacting to the MnSOD of Aspergillus fumigatus or Malassezia sympodialis. For the second motif, the invertebrate tropomyosin sequences were aligned and a motif consensus sequence was expressed as a recombinant protein (Trop Mot). The IgE reactivity of Trop Mot was analyzed in ELISA and compared to that of recombinant tropomyosin from the shrimp Penaeus aztecus (rPen a 1) in ImmunoCAP. MnSOD Mot was weakly recognized by some of the tested sera, suggesting that the IgE binding epitopes of a multimeric globular protein such as MnSOD cannot be fully represented by a motif peptide. In contrast, the motif Trop Mot showed the same IgE reactivity as shrimp full-length tropomyosin, indicating that the major allergenic reactivity of a repetitive structure such as tropomyosin can be covered by a motif peptide. Our results suggest that the motif-generating algorithm may be used for identifying major IgE binding structures of coiled-coil proteins.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Crystal structures of progressive Ca2+ binding states of the Ca2+ sensor Ca2+ binding domain 1 (CBD1) from the CALX Na+/Ca2+ exchanger reveal incremental conformational transitions.

Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.

Dynamics of a minimal model of interlocked positive and negative feedback loops of transcriptional regulation by cAMP-response element binding proteins.

cAMP-response element binding (CREB) proteins are involved in transcriptional regulation in a number of cellular processes (e.g., neural plasticity and circadian rhythms). The CREB family contains activators and repressors that may interact through positive and negative feedback loops. These loops can be generated by auto- and cross-regulation of expression of CREB proteins, via CRE elements in or near their genes. Experiments suggest that such feedback loops may operate in several systems (e.g., Aplysia and rat). To understand the functional implications of such feedback loops, which are interlocked via cross-regulation of transcription, a minimal model with a positive and negative loop was developed and investigated using bifurcation analysis. Bifurcation analysis revealed diverse nonlinear dynamics (e.g., bistability and oscillations). The stability of steady states or oscillations could be changed by time delays in the synthesis of the activator (CREB1) or the repressor (CREB2). Investigation of stochastic fluctuations due to small numbers of molecules of CREB1 and CREB2 revealed a bimodal distribution of CREB molecules in the bistability region. The robustness of the stable HIGH and LOW states of CREB expression to stochastic noise differs, and a critical number of molecules was required to sustain the HIGH state for days or longer. Increasing positive feedback or decreasing negative feedback also increased the lifetime of the HIGH state, and persistence of this state may correlate with long-term memory formation. A critical number of molecules was also required to sustain robust oscillations of CREB expression. If a steady state was near a deterministic Hopf bifurcation point, stochastic resonance could induce oscillations. This comparative analysis of deterministic and stochastic dynamics not only provides insights into the possible dynamics of CREB regulatory motifs, but also demonstrates a framework for understanding other regulatory processes with similar network architecture.

Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

A Z-DNA binding domain present in the human editing enzyme, double-stranded RNA adenosine deaminase

Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Zα) that binds specifically to the left-handed Z-DNA conformation with high affinity (KD = 4 nM). As formation of Z-DNA in vivo occurs 5′ to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Zα has allowed identification of motifs common to this class of nucleic acid binding domain.

Identification of the endophilins (SH3p4/p8/p13) as novel binding partners for the β1-adrenergic receptor

Several G-protein coupled receptors, such as the β1-adrenergic receptor (β1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein–protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the β1-AR either as a glutathione S-transferase fusion protein in biochemical “pull-down” assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4/p8/p13 (also referred to as endophilin 1/2/3), a SH3 domain-containing protein family, as binding partners for the β1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the β1-AR but not to that of the β2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of β1-ARs in HEK293 cells while having no effect on β2-ARs. Thus, our studies demonstrate a role of the SH3p4/p8/p13 protein family in β1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

Identification of a family of low-affinity insulin-like growth factor binding proteins (IGFBPs): Characterization of connective tissue growth factor as a member of the IGFBP superfamily

The insulin-like growth factor (IGF) binding proteins (IGFBPs) modulate the actions of the insulin-like growth factors in endocrine, paracrine, and autocrine settings. Additionally, some IGFBPs appear to exhibit biological effects that are IGF independent. The six high-affinity IGFBPs that have been characterized to date exhibit 40–60% amino acid sequence identity overall, with the most conserved sequences in their NH2 and COOH termini. We have recently demonstrated that the product of the mac25/IGFBP-7 gene, which shows significant conservation in the NH2 terminus, including an “IGFBP motif” (GCGCCXXC), exhibits low-affinity IGF binding. The closely related mammalian genes connective tissue growth factor (CTGF) gene, nov, and cyr61 encode secreted proteins that also contain the conserved sequences and IGFBP motifs in their NH2 termini. To ascertain if these genes, along with mac25/IGFBP-7, encode a family of low-affinity IGFBPs, we assessed the IGF binding characteristics of recombinant human CTGF (rhCTGF). The ability of baculovirus-synthesized rhCTGF to bind IGFs was demonstrated by Western ligand blotting, affinity cross-linking, and competitive affinity binding assays using 125I-labeled IGF-I or IGF-II and unlabeled IGFs. CTGF, like mac25/IGFBP-7, specifically binds IGFs, although with relatively low affinity. On the basis of these data, we propose that CTGF represents another member of the IGFBP family (IGFBP-8) and that the CTGF gene, mac25/IGFBP-7, nov, and cyr61 are members of a family of low-affinity IGFBP genes. These genes, along with those encoding the high-affinity IGFBPs 1–6, together constitute an IGFBP superfamily whose products function in IGF-dependent or IGF-independent modes to regulate normal and neoplastic cell growth.

Expression of myogenin during embryogenesis is controlled by Six/sine oculis homeoproteins through a conserved MEF3 binding site

Myogenin, one of the MyoD family of proteins, is expressed early during somitogenesis and is required for myoblast fusion in vivo. Previous studies in transgenic mice have shown that a 184-bp myogenin promoter fragment is sufficient to correctly drive expression of a β-galactosidase transgene during embryogenesis. We show here that mutation of one of the DNA motifs present in this region, the MEF3 motif, abolished correct expression of this β-galactosidase transgene. We have found that the proteins that bind to the MEF3 site are homeoproteins of the Six/sine oculis family. Antibodies directed specifically against Six1 or Six4 proteins reveal that each of these proteins is present in the embryo when myogenin is activated and constitutes a muscle-specific MEF3-binding activity in adult muscle nuclear extracts. Both of these proteins accumulate in the nucleus of C2C12 myogenic cells, and transient transfection experiments confirm that Six1 and Six4 are able to transactivate a reporter gene containing MEF3 sites. Altogether these results establish Six homeoproteins as a family of transcription factors controlling muscle formation through activation of one of its key regulators, myogenin.

Karyopherin β2 mediates nuclear import of a mRNA binding protein

We have cloned and sequenced cDNA for human karyopherin β2, also known as transportin. In a solution binding assay, recombinant β2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1’s previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin β1, karyopherin β2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both β1 and β2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, β2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of β2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of β2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin β2 inhibited karyopherin α/β1-mediated import of a classical NLS containing substrate and, vice versa, β1 inhibited β2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of β1 and β2 to repeat nucleoporins.

Crystal structure of the ribosomal RNA domain essential for binding elongation factors

The structure of a 29-nucleotide RNA containing the sarcin/ricin loop (SRL) of rat 28 S rRNA has been determined at 2.1 Å resolution. Recognition of the SRL by elongation factors and by the ribotoxins, sarcin and ricin, requires a nearly universal dodecamer sequence that folds into a G-bulged cross-strand A stack and a GAGA tetraloop. The juxtaposition of these two motifs forms a distorted hairpin structure that allows direct recognition of bases in both grooves as well as recognition of nonhelical backbone geometry and two 5′-unstacked purines. Comparisons with other RNA crystal structures establish the cross-strand A stack and the GNRA tetraloop as defined and modular RNA structural elements. The conserved region at the top is connected to the base of the domain by a region presumed to be flexible because of the sparsity of stabilizing contacts. Although the conformation of the SRL RNA previously determined by NMR spectroscopy is similar to the structure determined by x-ray crystallography, significant differences are observed in the “flexible” region and to a lesser extent in the G-bulged cross-strand A stack.

Dystrobrevin and dystrophin: An interaction through coiled-coil motifs

Dystrobrevin, a dystrophin-related and -associated protein, has been proposed to be important in the formation and maintenance of the neuromuscular junction. Dystrobrevin coprecipitates with both the acetylcholine receptor complex as well as the dystrophin glycoprotein complex. Although the nature of dystrobrevin’s association with the dystrophin glycoprotein complex remains unclear, it is known that dystrobrevin binds directly to the syntrophins, a heterologous group of dystrophin-associated proteins. Using the yeast two-hybrid system to identify protein–protein interactions, we present evidence for the heterodimerization of dystrobrevin directly with dystrophin. The C terminus of dystrobrevin binds specifically to the C terminus of dystrophin. We further refined this site of interaction to these proteins’ homologous coiled-coil motifs that flank their respective syntrophin-binding sites. We also show that the interaction between the dystrobrevin and dystrophin coiled-coil domains is specific and is not due to a nonspecific coiled-coil domain interaction. From the accumulated evidence of protein–protein interactions presented here and elsewhere, we propose a partially revised model of the organization of the dystrophin-associated glycoprotein complex.