Obligate Heterodimer Research Articles

The function and allosteric control of the human sweet taste receptor.

Humans perceive sweet taste via activation of a specific taste receptor expressed at the surface of taste receptor cells located on the tongue and soft palate papillae. The sweet taste receptor functions as an obligate heterodimer, comprising two different class C GPCR subunits. This receptor is unique in that it is activated or modulated by a plethora of ligands from highly diverse chemical classes, from small molecules to peptides and proteins and interacting with topologically distinct sites on each of its subunits. Modulators acting at separate functional domains of the sweet taste receptor can behave as full agonists. However, contrary to observations made with other class C GPCRs such as the metabotropic glutamate receptors and the γ-aminobutyric acid type B receptor (GABAB) receptor, modulators interacting within the allosteric sites in the transmembrane domains of the sweet taste receptor only exert a relatively small effect on the affinity and efficacy of the agonist interacting at the orthosteric binding site located within the Venus fly trap domain (VFD). Newly identified potent and efficacious positive allosteric modulators (PAM)s of the sweet taste receptor rather interact at a site in close proximity to the agonist, within the VFD, display significant probe dependence, and markedly increase the affinity of the orthosteric ligand. Several sweet taste receptor inhibitors have also been characterized. Functional studies reveal a complex relationship between different ligands. Whether the antagonist will be surmountable or insurmountable and will act competitively or non-competitively largely depends on the agonist being studied and the location of its interaction site on the receptor.

Mechanisms and Regulation of Neuronal GABAB Receptor-Dependent Signaling.

γ-Aminobutyric acid B receptors (GABABRs) are broadly expressed throughout the central nervous system where they play an important role in regulating neuronal excitability and synaptic transmission. GABABRs are G protein-coupled receptors that mediate slow and sustained inhibitory actions via modulation of several downstream effector enzymes and ion channels. GABABRs are obligate heterodimers that associate with diverse arrays of proteins to form modular complexes that carry out distinct physiological functions. GABABR-dependent signaling is fine-tuned and regulated through a multitude of mechanisms that are relevant to physiological and pathophysiological states. This review summarizes the current knowledge on GABABR signal transduction and discusses key factors that influence the strength and sensitivity of GABABR-dependent signaling in neurons.

Evolution of vascular plants through redeployment of ancient developmental regulators

Vascular plants provide most of the biomass, food, and feed on earth, yet the molecular innovations that led to the evolution of their conductive tissues are unknown. Here, we reveal the evolutionary trajectory for the heterodimeric TMO5/LHW transcription factor complex, which is rate-limiting for vascular cell proliferation in Arabidopsis thaliana Both regulators have origins predating vascular tissue emergence, and even terrestrialization. We further show that TMO5 evolved its modern function, including dimerization with LHW, at the origin of land plants. A second innovation in LHW, coinciding with vascular plant emergence, conditioned obligate heterodimerization and generated the critical function in vascular development in Arabidopsis In summary, our results suggest that the division potential of vascular cells may have been an important factor contributing to the evolution of vascular plants.

ABCG subfamily (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

This family of 'half-transporters' act as homo- or heterodimers; particularly ABCG5 and ABCG8 are thought to be obligate heterodimers. The ABCG5/ABCG heterodimer sterol transporter structure has been determined [4], suggesting an extensive intracellular nucleotide binding domain linked to the transmembrane domains by a fold in the primary sequence. The functional ABCG2 transporter appears to be a homodimer with structural similarities to the ABCG5/ABCG8 heterodimer [7].

Abstract 3397: The mechanism of PI3K activation at the atomic level

Abstract PI3Ks are a family of lipid kinases in the PI3K/Akt/mTOR pathway that regulates cellular processes. PI3Kα harbors the frequent somatic oncogenic driver mutations conferring gain of function. It is an obligate heterodimer with the regulatory p85α subunit and catalytic p110α subunit. Here, we determine the PI3Kα activation mechanism by nSH2 release at the atomic resolution. The PIP2 substrate binding site is far from ATP and the membrane binding surface is buried in the inactive PI3Kα, raising the question of how catalysis is executed. Our results show that release of nSH2 domain from PI3Kα triggers significant conformational change in p110α, exposing kinase domain for membrane interaction. The C-lobe of kinase domain shows the structural rearrangement to reduce the distance between the ATP and the substrate binding site, offering an explanation to how phosphoryl transfer is executed. This explains how oncogenic mutations promote PI3Kα activation by facilitating nSH2 release and offers an innovative, PI3K isoform-specific drug discovery principle that prevents substrate phosphorylation by targeting isoform-specific residues in the substrate binding site. Citation Format: Mingzhen Zhang, Hyunbum Jang, Ruth Nussinov. The mechanism of PI3K activation at the atomic level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3397.

Abstract 3042: Splicing repressor HNRNPC is an indispensable and 'druggable' target in acute myeloid leukemia

Abstract Refractory and relapsed disease is the greatest challenge in acute myeloid leukemia (AML), and we have shown that blood vessels serve as sanctuary sites for AML. Using a high throughput screening assay mimicking AML in the vascular niche, we screened 31 million compounds and identified a hit compound 2470-51 that selectively killed AML cells and CD34+CD38-CD123+ AML stem cells, while sparing bone marrow-derived endothelial cells, normal hematopoietic stem and progenitor cells (HSPC) as well as CD4+ T lymphocytes from healthy volunteers. In vivo AML patient-derived xenograft modeling further validated the efficacy of 2470-51 as a selective anti-leukemic agent compared to cytarabine (conventional control). We performed quantitative proteomics combining ITRAQ differential protein expression analysis, label-free shotgun analysis and SPR imaging to identify heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC) as the target of 2470-51. HNRNPC depletion in AML cell lines THP1, K562, MV411 and HL-60 significantly decreased cell proliferation, viability and clonogenic capacity of the AML cells. In contrast, normal mesenchymal/fibroblastic cells, endothelial cells and normal HSPCs from human umbilical cord blood specimens were unaffected by HNRNPC depletion, indicating the selective dependence of AML cells on hnRNPC. Furthermore, clinical studies using TCGA AML datasets showed significant survival advantage associated with lower HNRNPC expression. RNA-seq analyses revealed a distinct gene expression pattern suggesting widespread inhibition of Myc transcriptional targets. Using differential alternative splicing and differential transcript expression analyses, we discovered significant alternative splicing of MAX after HNRNPC depletion, resulting in MAX transcript isoforms that lacked Myc-interacting domains (“inactive” MAX). These Myc-interacting domains are necessary for the obligate heterodimerization of Myc and Max and are critical for Myc transcriptional activation. We further analyzed splicing landscapes of 578 AML patients and 33 healthy controls and identified substantial mis-splicing of mRNA in AML patients, despite the absence of somatic gene mutations of splicing factors, when compared to the healthy controls. Moreover, we found significant overexpression of “inactive” MAX isoforms in the healthy controls, while AML patients overexpressed fully functional MAX isoforms, suggesting that MAX splicing may have an important functional role in AML. Collectively, our studies show significant RNA splicing changes in AML and an essential role of HNRNPC in AML in contrast to normal hematopoietic and stromal cells where HNRNPC is dispensable. We present a pharmacologic agent for targeting HNRNPC and Myc-Max as a molecular mechanism of action. Our data indicate that hnRNPC is a critical factor in AML and inhibiting this splicing repressor may represent a new therapeutic strategy. Citation Format: Vindhya Vijay, Amy Meacham, Lauren Katzell, Aaron Winer, Jesse Terrell, Vincent Archibald, Jodi Bubenik, Alberto Riva, Jon Boatwright, Cristina Tognon, Jeffrey Tyner, Brian Druker, Christopher Cogle. Splicing repressor HNRNPC is an indispensable and 'druggable' target in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3042.

KMT9 monomethylates histone H4 lysine 12 and controls proliferation of prostate cancer cells.

Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-β-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.

Structural basis for auxiliary subunit KCTD16 regulation of the GABAB receptor

Metabotropic GABAB receptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABAB receptor complexes contain the principal subunits GABAB1 and GABAB2, which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABAB receptors and modify the kinetics of GABAB receptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABAB receptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABAB2 receptor. A single GABAB2 C-terminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABAB2-KCTD16 interface disrupted both the biochemical association and functional modulation of GABAB receptors and G protein-activated inwardly rectifying K+ channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABAB receptors. Defining the binding interface of GABAB receptor and KCTD reveals a potential regulatory site for modulating GABAB-receptor function in the brain.

Floral MADS-box protein interactions in the early diverging angiosperm Aristolochia fimbriata Cham. (Aristolochiaceae: Piperales).

Floral identity MADS-box A, B, C, D, E, and AGL6 class genes are predominantly single copy in Magnoliids, and predate the whole genome duplication (WGD) events in monocots and eudicots. By comparison with the model species Arabidopsis thaliana, the expression patterns of B-, C-, and D-class genes in stamen, carpel, and ovules are conserved in Aristolochia fimbriata, whereas A-, E-class, and AGL6 genes have different expression patterns. Nevertheless, the interactions of these proteins that act through multimeric complexes remain poorly known in early divergent angiosperms. This study evaluates protein interactions among all floral MADS-box A. fimbriata proteins using the Yeast Two Hybrid System (Y2H). We found no homodimers and less heterodimers formed by AfimFUL when compared to AfimAGL6, which allowed us to suggest AGL6 homodimers in combination with AfimSEP2 as the most likely tetramer in sepal identity. We found AfimAP3-AfimPI obligate heterodimers and AfimAG-AfimSEP2 protein interactions intact suggesting conserved stamen and carpel tetrameric complexes in A. fimbriata. We observed a broader interaction partner set for AfimSEP2 than for its paralog AfimSEP1. We show conserved and exclusive MADS-box protein interactions in A. fimbriata in comparison with other eudicot and monocot model species in order to establish plesiomorphic MADS-box protein floral networks in angiosperms.

Abstract B037: Tunable thermal bioswitches for control of cell-based therapeutics

Abstract Temperature is a unique input signal that could be used by therapeutic agents, including engineered proteins and cells, to sense and respond to patient conditions or spatially targeted external triggers such as focused ultrasound. A critical capability for many envisioned applications is the ability to control the function of such therapies in situ to enable spatially and temporally specific activation at tumors and surrounding local tissue. However, among existing control methods, systemic chemical administration typically lacks the spatial precision needed to modulate activity at specific anatomic locations, while optical approaches suffer from poor light penetration into tissues. On the other hand, temperature can be controlled both globally and locally—at depth—using technologies such as focused ultrasound, infrared light and magnetic particle hyperthermia. In addition, body temperature can serve as an indicator of the patient’s condition. We have previously described the development of temperature-dependent protein actuators for control of gene expression in prokaryotic hosT-cells (1). Here, we generalize this technology to control protein-protein interactions with the aim of modulating arbitrary cell signaling pathways, including those of engineered therapeutic mammalian cells.To develop temperature-responsive protein-protein interaction domains, we turned to the thermo-sensitive bacterial transcriptional repressor TlpA. We demonstrated that this protein exhibits a tunable activation threshold which can be modulated within the biomedically relevant range of 32–46 °C, and that genes controlled by this repressor activate upon exposure to focused ultrasound hyperthermia applied noninvasively to a murine host (1). We then utilized a structure-based rational design strategy to convert the homodimeric TlpA protein into a pair of obligate heterodimers. We subsequently demonstrated that these heterodimers can be fused to sub-cellular anchoring moieties and cargo domains to conditionally sequester their payload into various compartments of human cells. Release of the cargo can be triggered via a mild thermal elevation, and the release temperature can be tuned via mutagenesis. In this study, we demonstrate that the temperature-sensing components of a bacterial transcriptional repressor can be isolated and repurposed into a generalized strategy to exert direct and rapid control over protein function in the context of both bacterial and mammalian cells. We aim to harness this technology to regulate therapeutically relevant processes in mammalian cells such as transcriptional activation and enzymatic signaling cascades. This class of bioswitches can be utilized to engineer a broad range of research tools and biological therapies with actuation driven by spatiotemporally precise noninvasively applied stimuli or by real-time sensing of host conditions.Reference: 1. Piraner DI, Abedi MH, Moser BA, Lee-Gosselin A, Shapiro MG. Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nat Chem Biol 2016;13(1):75–80. Citation Format: Dan Ilya Piraner, Mikhail G. Shapiro. Tunable thermal bioswitches for control of cell-based therapeutics [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B037.

Diversity of structure and function of GABAB receptors: a complexity of GABAB-mediated signaling.

γ-aminobutyric acid type B (GABAB) receptors are broadly expressed in the nervous system and play an important role in neuronal excitability. GABAB receptors are G protein-coupled receptors that mediate slow and prolonged inhibitory action, via activation of Gαi/o-type proteins. GABAB receptors mediate their inhibitory action through activating inwardly rectifying K+ channels, inactivating voltage-gated Ca2+ channels, and inhibiting adenylate cyclase. Functional GABAB receptors are obligate heterodimers formed by the co-assembly of R1 and R2 subunits. It is well established that GABAB receptors interact not only with G proteins and effectors but also with various proteins. This review summarizes the structure, subunit isoforms, and function of GABAB receptors, and discusses the complexity of GABAB receptors, including how receptors are localized in specific subcellular compartments, the mechanism regulating cell surface expression and mobility of the receptors, and the diversity of receptor signaling through receptor crosstalk and interacting proteins.

Abstract 182: Superior targeting of tumor-stromal interactions and endothelial migration with a bispecific antibody to α5 and αv integrins

Abstract Introduction: The integrin α5β1 has been implicated in the adhesive and migratory response of prostate cancer cells induced by fibronectin fragments secreted by human bone-marrow derived mesenchymal stromal cells (hBM-MSCs) [Joshi, Cell Adh and Migr 2016). Genetic inactivation of integrin α5 in PC-3 cells downregulated the bcl-2 family of proteins and induced programmed cell death (Ren, Mol Cell Res 2017) - linking hBM-MSCs to a putative bone marrow niche survival program for prostate cancer cells. We hypothesized that the fibronectin-binding integrins α5β1 and integrin αv collaborate in broader tumor-stromal interactions and sought to examine the impact of combinatorial inactivation of these integrins. Methods: Monospecific antibodies to α5 (or its obligate α5β1 heterodimer) and to integrin alpha v (or the αvβ3 heterodimer) were utilized to assess the efficacy of a combinatorial neutralization approach over either single agent in α5/αv co-expressing cells from diverse tumor types including prostate, breast, glioma, cervix and uterine cancer. Assays included adhesion, migration, cell survival and the induction of endothelial migration in co-culture with stromal cells. To address the hypothesis that a bispecific integrin targeting antibody would be superior to a combinatorial approach with monospecific integrin antibodies, a bispecific antibody prototype that simultaneously targets the α5β1 and αv integrins [Bsα5β1/αvAb] was constructed and deployed in comparative assays. Results: Combined α5 and av neutralization with dual monospecific antibodies was superior to individual single agents in blocking adhesion, migration and the induction of endothelial chemotaxis across diverse tumor types that co-expressed α5 and αv integrins. However, a significant superiority of the Bsα5β1/αvAb was noted over the combinatorial approach with monospecific antibodies in these assays. In addition, a significant reduction in cell survival was noted in selected tumor types with the Bsα5β1/αvAb. Strikingly, the Bsα5β1/αvAb was significantly more potent than bevacizumab in the inhibition of endothelial migration induced by tumor-stromal cell interactions Conclusions: Combinatorial α5 and av integrin targeting in tumor cells that co-express α5 and av integrins effectively abrogates tumor-stromal interactions and the resultant endothelial migratory response. A Bsα5β1/αvAb demonstrates a significant improvement over combinatorial monospecific antibodies likely through diverse mechanisms including cross-priming. Targeting tumor, stromal and endothelial cells simultaneously with a Bsα5β1/αvAb approach represents a potentially effective therapeutic strategy for targeting diverse mechanisms of progressive disease in the tumor microenvironment. Citation Format: Raghav Joshi, Wenying Ren, Paul Mathew. Superior targeting of tumor-stromal interactions and endothelial migration with a bispecific antibody to α5 and αv integrins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 182.

Abstract 1367: BRCA1 and BARD1 protein interactions that are required for DNA repair function

Abstract Breast and ovarian cancers are prevalent among women, and hereditary breast and ovarian cancers (HBOCs) have been associated with germline mutations in genes such as BRCA1 and BARD1. BRCA1 and BARD1 form an obligate heterodimer, and the BRCA1/BARD1 complex is required for tumor suppression functions. Our lab has tested hundreds of BARD1 and BRCA1 variants and identified many that are deficient in homologous recombination, which we have shown to accurately predict cancer predisposition in the clinic. Several of these functionally defective BRCA1 mutants map to a pocket of amino acids on the surface of the protein that does not have any known binding partners. Repair-deficient mutations are also present on the surface of the BARD1 protein in domains that are not known to be associated with DNA repair. We hypothesize that the DNA repair deficiencies mediated by these BRCA1 and BARD1 mutants are due to differences in protein binding when compared to wild-type protein. We have found that BRCA1 mutants in this protein pocket do not phosphorylate and do not localize to the nucleus following DNA damage, both of which are characteristic of the DNA damage response. However, BARD1 repair-deficient mutants still bind phosphorylated BRCA1, indicating that their deficiencies are not due to loss of BRCA1 function. To investigate protein interaction differences we are creating fusion proteins of wild-type and mutant BRCA1 with BioID2, which will biotinylate proteins that bind to BRCA1. We will then be able to identify, via avidin purification and mass spectrometry, protein interactions that are present in the wild-type but absent in the mutant BRCA1. Novel proteins that we identify will be tested for DNA repair and tumor suppressor function. This information will allow us to better understand both BARD1 and BRCA1 function and the mechanism of homologous recombination. Citation Format: Aleksandra Adamovich, Margaret Wingo, Tapahsama Banerjee, Miranda Gardner, Michael Freitas, Jeffrey Parvin. BRCA1 and BARD1 protein interactions that are required for DNA repair function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1367.

Allosteric interactions between GABAB1 subunits control orthosteric binding sites occupancy within GABAB oligomers

The GABAB receptor was the first G protein-coupled receptor identified as an obligate heterodimer. It is composed of two subunits, GABAB1 containing the agonist binding site and GABAB2 responsible for G protein activation. The GABAB receptor was found to associate into larger complexes through GABAB1-GABAB1 interactions, both in transfected cells and in brain membranes. Here we assessed the possible allosteric interactions between GABAB heterodimers by analyzing the effect of mutations located at the putative interface between the extracellular binding domains. These mutations decrease, but do not suppress, the Förster resonance energy transfer (FRET) signal measured between GABAB1 subunits. Further analysis of one of these mutations revealed an increase in G protein-coupling efficacy and in the maximal antagonist binding by approximately two-fold. Hypothesizing that a tetramer is an elementary unit within oligomers, additional FRET data using fluorescent ligands and tagged subunits suggest that adjacent binding sites within the GABAB oligomers are not simultaneously occupied. Our data show a strong negative effect between GABAB1 binding sites within GABAB oligomers. Accordingly, GABAB receptor assembly appears to limit receptor signaling to G proteins, a property that may offer novel regulatory mechanism for this important neuronal receptor.This article is part of the “Special Issue Dedicated to Norman G. Bowery”.

Intersubunit Crosstalk in the Rag GTPase Heterodimer Enables mTORC1 to Respond Rapidly to Amino Acid Availability

mTOR complex I (mTORC1) is a central growth regulator that senses amino acids through a pathway that converges on the Rag GTPases, an obligate heterodimer of two related GTPases. Despite their central role in amino acid sensing, it is unknown why the Rag GTPases are heterodimeric and whether their subunits communicate with each other. Here, we find that the binding of guanosine triphosphate (GTP) to one subunit inhibits the binding and induces the hydrolysis of GTP by the other. This intersubunit communication pushes the Rag GTPases into either of two stable configurations, which represent active "on" or "off" states that interconvert via transient intermediates. Subunit coupling confers on the mTORC1 pathway its capacity to respond rapidly to the amino acid level. Thus, the dynamic response of mTORC1 requires intersubunit communication by the Rag GTPases, providing a rationale for why they exist as a dimer and revealing a distinct mode of control for a GTP-binding protein.

Beta-catenin represses protein kinase D1 gene expression by non-canonical pathway through MYC/MAX transcription complex in prostate cancer.

Down regulation of Protein Kinase D1 (PrKD1), a novel serine threonine kinase, in prostate, gastric, breast and colon cancers in humans leads to disease progression. While the down regulation of PrKD1 by DNA methylation in gastric cancer and by nuclear beta-catenin in colon cancer has been shown, the regulatory mechanisms in other cancers are unknown. Because we had demonstrated that PrKD1 is the only known kinase to phosphorylate threonine 120 (T120) of beta-catenin in prostate cancer resulting in increased nuclear beta-catenin, we explored the role of beta-catenin in gene regulation of PrKD1. An initial CHIP assay identified potential binding sites for beta-catenin in and downstream of PrKD1 promoter and sequencing confirmed recruitment of beta-catenin to a 166 base pairs sequence upstream of exon 2. Co-transfection studies with PrKD1-promoter-reporter suggested that beta-catenin represses PrKD1 promoter. Efforts to identify transcription factors that mediate the co-repressor effects of beta-catenin identified recruitment of both MYC and its obligate heterodimer MAX to the same binding site as beta-catenin on the PrKD1 promoter site. Moreover, treatment with MYC inhibitor rescued the co-repressor effect of beta-catenin on PrKD1 gene expression. Prostate specific knock out of PrKD1 in transgenic mice demonstrated increased nuclear expression of beta-catenin validating the in vitro studies. Functional studies showed that nuclear translocation of beta-catenin as a consequence of PrKD1 down regulation, increases AR transcriptional activity with attendant downstream effects on androgen responsive genes. In silico human gene expression analysis confirmed the down regulation of PrKD1 in metastatic prostate cancer correlated inversely with the expression of MAX, but not MYC, and positively with MXD1, a competing heterodimer of MAX, suggesting that the dimerization of MAX with either MYC or MXD1 regulates PrKD1 gene expression. The study has identified a novel auto-repressive loop that perpetuates PrKD1 down regulation through beta-catenin/MYC/MAX protein complex.

Homo- and heteropolymer self-assembly of recombinant trichocytic keratins.

In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues. However, during extraction from natural sources, such as hair and wool fibers, natural keratins are subject to extensive processing conditions that lead to formation of unwanted by-products. Additionally, natural keratins suffer from limited sequence tunability. Recombinant keratin proteins can overcome these drawbacks while maintaining the desired chemical and physical characteristics of natural keratins. Herein, we present the bacterial expression, purification, and solution characterization of human hair keratins K31 and K81. The obligate heterodimerization of the K31/K81 pair that results in formation of intermediate filaments is maintained in the recombinant proteins. Surprisingly, we have for the first time observed new zero- and one-dimensional nanostructures from homooligomerization of K81 and K31, respectively. Further analysis of the self-assembly mechanism highlights the importance of disulfide crosslinking in keratin self-assembly.

The BRCA1 Ubiquitin ligase function sets a new trend for remodelling in DNA repair

ABSTRACTThe protein product of the breast and ovarian cancer gene, BRCA1, is part of an obligate heterodimer with BARD1. Together these RING bearing proteins act as an E3 ubiquitin ligase. Several functions have been attributed to BRCA1 that contribute to genome integrity but which of these, if any, require this enzymatic function was unclear. Here we review recent studies clarifying the role of BRCA1 E3 ubiquitin ligase in DNA repair. Perhaps the most surprising finding is the narrow range of BRCA1 functions this activity relates to. Remarkably ligase activity promotes chromatin remodelling and 53BP1 positioning through the remodeller SMARCAD1, but the activity is dispensable for the cellular survival in response to cisplatin or replication stressing agents. Implications for therapy response and tumor susceptibility are discussed.

Duplication and Whorl-Specific Down-Regulation of the Obligate AP3-PI Heterodimer Genes Explain the Origin of Paeonia lactiflora Plants with Spontaneous Corolla Mutation.

Herbaceous peony (Paeonia lactiflora) is a globally important ornamental plant. Spontaneous floral mutations occur frequently during cultivation, and are selected as a way to release new cultivars, but the underlying evolutionary developmental genetics remain largely elusive. Here, we investigated a collection of spontaneous corolla mutational plants (SCMPs) whose other floral organs were virtually unaffected. Unlike the corolla in normal plants (NPs) that withered soon after fertilization, the transformed corolla (petals) in SCMPs was greenish and persistent similar to the calyx (sepals). Epidermal cellular morphology of the SCMP corolla was also similar to that of calyx cells, further suggesting a sepaloid corolla in SCMPs. Ten floral MADS-box genes from these Paeonia plants were comparatively characterized with respect to sequence and expression. Codogenic sequence variation of these MADS-box genes was not linked to corolla changes in SCMPs. However, we found that both APETALA3 (AP3) and PISTILLATA (PI) lineages of B-class MADS-box genes were duplicated, and subsequent selective expression alterations of these genes were closely associated with the origin of SCMPs. AP3-PI obligate heterodimerization, essential for organ identity of corolla and stamens, was robustly detected. However, selective down-regulation of these duplicated genes might result in a reduction of this obligate heterodimer concentration in a corolla-specific manner, leading to the sepaloid corolla in SCMPs, thus representing a new sepaloid corolla model taking advantage of gene duplication. Our work suggests that modifying floral MADS-box genes could facilitate the breeding of novel cultivars with distinct floral morphology in ornamental plants, and also provides new insights into the functional evolution of the MADS-box genes in plants.

Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.