No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Misregulation of protein signaling pathways is the basis for many human diseases, and thus 95% of Food and Drug Administration approved drugs target proteins. Proteins are dynamic entities which can undergo transitions to reach different conformational states. The conformational state of a protein, or its three-dimensional shape, is intricately linked to functions, such as association with endogenous or exogenous binding partners, or catalysis. Thus, it is of interest to the pharmacological community to understand the mechanisms of protein conformational state transitions in order to better target and control protein functions. In two case studies, I show the importance of understanding protein dynamics in protein function and drug design. In the case of human immunodeficiency virus-1 (HIV-1) protease, a tremendous “open-and-closed” conformational transition is revealed by Molecular Dynamics Simulations (MDS). Through observing the dramatic difference in effectiveness of two Darunavir inhibitor derivatives differentiated by a single atom at locking the protease in the closed conformation, we discovered the residues and mechanism that lead to the protease’s conformational transition. This mechanism also explained the significant difference in the binding conformation and binding affinity of these two inhibitors. This study provides insight on how to improve the potency and anti-viral capacity of these compounds. In the second case study, MDS enabled us to observe the conformational transitions of a family of seven isoforms known as the 14-3-3 proteins. Many vital cellular processes involve all or select 14-3-3 isoforms, making this family very difficult to target. Through MDS, I discovered different conformational samplings among these 14-3-3 isoforms which were then validated by SAXS. Subsequently, a FRET-based ligand binding assay was developed which can screen for preferential 14-3-3 isoform binding of endogenous ligands, giving hope that using conformations unique to a 14-3-3 isoform of interest can provide a method for selective drug design. / 2022-03-09

Prostate tumor overexpressed 1 (PTOV1) is highly expressed in several forms of cancer. High expression of PTOV1 is associated with tumor aggressiveness in several tumor types, including ovarian and breast cancer. Currently, PTOV1 is known to act both as a translational and transcriptional regulator aiding in the expression of prosurvival genes. Although PTOV1 is known to pass in and out of the nucleus in a cell cycle-dependent manner, the regulation of PTOV1 activity is not well understood and here we identify 14-3-3 as a PTOV1 interactor and show that high levels of 14-3-3 expression, like PTOV1, correlate with prostate cancer progression. Further, we identify SGK2-mediated phosphorylation at S36 of PTOV1 that is required for 14-3-3 binding. Disruption of the PTOV1-14-3-3 interaction results in an accumulation of PTOV1 in the nucleus and a proteasome-dependent reduction in PTOV1 protein levels, which requires ubiquitination at K114 of PTOV1. We also observed HUWE1 as a PTOV1-interacting partner responsible for the degradation of PTOV1 through the proteasome. We show that loss of 14-3-3 binding leads to an increase in PTOV1-HUWE1 binding, suggesting that 14-3-3 stabilizes PTOV1 protein by sequestering PTOV1 in the cytosol and inhibiting its interaction with HUWE1. Finally, our data suggest that stabilization of the 14-3-3-bound form of PTOV1 promotes PTOV1-mediated expression of cJun. Together, these data support a model that explains how 14-3-3 and HUWE1 regulate the PTOV1 stability, localization, and function within the cell.

14-3-3 proteins are among a family of phospho-binding proteins that are known to regulate many essential cellular mechanisms. By binding to sites of phosphorylation, 14-3-3s are integrated into multiple signaling pathways that govern critical processes, such as apoptosis, cell cycle progression, autophagy, glucose metabolism, and cell motility. These processes are crucial for tumorigenesis and 14-3-3 proteins are known to play a central role in facilitating cancer progression. In this study, my colleagues and I discover two novel 14-3-3 interacting proteins, ATG9A and PTOV1, that are both vital to essential cellular functions and describe various mechanisms that these two proteins regulate. ATG9A is a multi-pass transmembrane lipid scramblase that is found primarily as a homotrimer in the ER or small ATG9A vesicles. It is essential in the cellular recycling process called autophagy and is believed to act at the earliest stages of autophagy by providing the seed for the growth of the double membrane vesicle called an autophagosome. Previous work in our lab demonstrated that upon hypoxic stress, AMPK, the master nutrient-sensing kinase, phosphorylates S761 on the C-terminus of ATG9A. This triggers the binding of 14-3-3ζ to contribute to ATG9A function in hypoxia induced autophagy. Despite this revelation, the exact function of ATG9A is still poorly understood, especially in unstimulated conditions where autophagy functions at a basal level and AMPK is inactive. In this study, we sought to understand ATG9A function more broadly by identifying novel interactors of ATG9A and the role ATG9A plays in basal autophagy. To do this, we employed BioID mass spectrometry and various biochemical approaches to identify LRBA as a bona fide ATG9A interactor and autophagy regulator. Furthermore, using deuterium labeling and quantitative whole proteome mass spectrometry, and various other biochemical techniques, we show that ATG9A regulates the basal degradation of p62 and is recruited to sites of basal autophagy by active poly-ubiquitination to initiate basal autophagy. PTOV1 is an oncogenic protein that is poorly understood. Our current understanding of PTOV1 is limited to a few studies, which demonstrate that PTOV1 is highly expressed in primary prostate tumor samples and is correlated with metastasis, drug resistance, and poor clinical outcomes. In this study, we identify a mechanism by which SGK2, a poorly understood kinase, phosphorylates PTOV1 at S36 to trigger 14-3-3 binding at that site to increase PTOV1 stability in the cytosol and increase c-Jun expression. Upon SGK2 inhibition, 14-3-3 releases PTOV1 and PTOV1 is shuttled into the nucleus where HUWE1, an E3 ubiquitin ligase, ubiquitinates PTOV1 and initiates PTOV1 degradation by the proteasome. This is the first detailed mechanism of regulation identified for the poorly understood oncogene, PTOV1, and sheds light on potential therapeutic targets for cancer treatments.

No description available.

Decades of research have begun to identify oncogenic mut-drivers that are responsible for driving a large percentage of cancers. These high frequency mut-drivers have therapeutics in the clinic for patient treatment. However, there is another group of low frequency mut-drivers that fail to rise above the noise of the high frequently drivers. These low frequency drivers represent a group of genes with untapped potential for new targeted therapies. However, identifying these drivers can be difficult. This study focuses on identifying new functional phosphorylations using the phospho-docking protein 14-3-3. The family of 14-3-3 proteins have been linked to many oncogenic pathways due to the diversity in their client protein interactions. One critical problem in studying 14-3-3 interactors is uncovering the docking site on the phospho-binding partner. Our work indicates that intrinsic disorder and unbiased mass spectrometry identification rate of a given phosphorylation are important for improving the selection of a 14-3-3 docking site. Using a machine learning model, we developed a tool that combines current available 14-3-3 prediction data and our observations about disorder and phosphorylation observation to predict 14-3-3 binding sites. Our publicly available tool "14-3-3-site-finder" produces a rank order list of potential 14-3-3 docking sites that could help overcome the time-consuming process of identifying the correct site. In our efforts of identifying functional phosphorylations with 14-3-3, we have observed that 14-3-3 interacts with a non-receptor tyrosine kinase, TNK1. TNK1 is a poorly characterized kinase that has essentially nothing known about its substrates, function or regulation. TNK1 has been implicated in both tumor suppressor and oncogenic roles. Particularly, a Hodgkin Lymphoma cell line is dependent on a truncated form of TNK1 for growth. Our work uncovers the first mechanism of regulation for this kinase. We found that MARK kinase phosphorylates TNK1 within the proline rich domain allowing 14-3-3 to dock on this phosphorylation. 14-3-3 binding restrains TNK1 in the cytosol and holds TNK1 in an inactive state. Upon the release of 14-3-3, TNK1 moves to a membrane fraction where it is active. One unique feature of TNK1 is that it has a c-terminal ubiquitin association domain (UBA). In vitro ubiquitin pulldowns indicate that the TNK1 UBA has no preference for linkage type or length of ubiquitin. Further, biolayer interferometry indicates that the UBA binds ubiquitin tightly. Mutation of residues within the ubiquitin:TNK1 interface prevent ubiquitin binding and decrease TNK1 activity, preventing downstream oncogenic signaling, suggesting a UBA-centric mechanism of regulation for TNK1. Finally, we developed a small molecule inhibitor, TP-5801, that selectively targets TNK1. TP-5801 prevents downstream TNK1 phosphorylation of STAT3. Further, TP-5801 prolonged the life of mice injected with TNK1 driven Ba/F3 cells. Taken together, our data reveal the first mechanism of kinase regulation for TNK1 involving 14-3-3 binding and ubiquitin association as well as the development of a TNK1 specific therapeutic

Dans le système nerveux central, le récepteur inhibiteur GABAB est un archétype des RCPGs hétérodimériques. Il est composé en effet de deux sous-unités, la sous-unité GABAB1 (B1a ou B1b) qui lie l’agoniste et la sous-unité GABAB2 couplée aux protéines G. L’activation de ce récepteur a un effet antinociceptif bien établi concernant les douleurs aiguës mais son effet reste cependant très limité en cas de douleurs neuropathiques. Notre hypothèse est que son activation et sa signalisation peuvent être altérées par des protéines partenaires, aboutissant à des processus de désinhibition dans la moelle épinière en conditions de neuropathie. Nos résultats mettent en évidence le rôle de deux protéines partenaires qui sont surexprimées en conditions douloureuses et qui diminuent l’activation du récepteur GABAB via deux mécanismes différents. D’un part, la protéine cytosolique 14-3-3? induit la dissociation de l’hétérodimère B1b/B2. Cette action a lieu principalement dans les compartiments post-synaptiques. D’autre part, la fibuline-2, protéine de la matrice extracellulaire diminue l’activation de l’hétérodimère B1a/B2. Il s’agit cette fois préférentiellement d’une action dans les compartiments pré-synaptiques. Des stratégies anti-sens (siRNA anti-14-3-3? ou anti-fibuline-2) ou des peptides de compétition sélectifs de l’interaction B1b/14-3-3? permettent de potentialiser les effets antinociceptifs d’un agoniste du récepteur sur un modèle animal de neuropathie. L’ensemble de ces résultats suggèrent que l’état d’oligomérisation des RCPGs peut être modulé in vivo par des protéines partenaires endogènes impliquées dans le développement ou le maintien d’états pathologiques de sensibilisation à la douleur. / In the central nervous system, the inhibitory GABAB receptor is an obligate heterodimeric GPCR that requires the association between GABAB1 (B1a or B1b) and GABAB2 subunits. The heterodimeric GABAB receptor activation has a well-known antinociceptive action in acute pain but its effect appears limited in pathological states. Our hypothesis is that the GABAB activation and signaling could be altered by partner proteins, thus resulting in desinhibition processes in the spinal cord. In the present study, we investigated the role of two partner proteins overexpressed in neuropathic states which decrease GABAB activation through two different mechanisms. On the one hand, the cytosolic 14-3-3? protein induces the dissociation of the heterodimer B1b/B2. This effect occurs in post-synaptic compartments. On the other hand, fibulin-2, an extracellular matrix protein, which decreases the activation of the heterodimer B1a/B2 localized preferentially in presynaptic compartments. Anti-sens strategies (anti-14-3-3? or anti-fibulin-2 siRNA) or competing peptides specific of 14-3-3?/B1b interaction, potentiate the antinociceptive effects of GABAB agonist in an animal model of neuropathic pain. Taken together, our data suggest that GPCR oligomeric state can be modulated in vivo by endogenous partners proteins that are involved in the development and the maintenance of pain sensitization.

Nos eucariotos, a evolução dos sistemas de transporte molecular foi essencial pois seu alto grau de compartimentalização requer mecanismos com maior especificidade para a localização de proteínas. Com o estabelecimento das mitocôndrias e plastídeos como organelas da célula eucariota, grande parte dos genes específicos para sua atividade e manutenção foram transferidos ao núcleo. Após a transferência gênica, a maioria das proteínas passaram a ser codificadas pelo núcleo, sintetizadas no citosol e direcionadas às organelas por uma maquinaria complexa que envolve receptores nas membranas das organelas, sequências de direcionamento nas proteínas e proteínas citossólicas que auxiliam o transporte. A importação depende em grande parte de uma sequência na região N-terminal das proteínas que contém sinais reconhecidos pelas membranas organelares. No entanto, muito ainda não é compreendido sobre o transporte de proteínas organelares e fatores ainda desconhecidos podem influenciar o direcionamento sub-celular. O objetivo deste trabalho foi a caracterização da General Regulatory Factor 9 (GRF9), uma proteína da família 14-3-3 de Arabidopsis thaliana potencialmente envolvida no direcionamento de proteínas organelares, e a geração de um genótipo para ser utilizado na obtenção de uma população mutante para genes que afetam o direcionamento da proteína Tiamina Monofosfato Sintetase (TH-1). Após experimentos in vivo e in planta, foi observado que GRF9 interage com as proteínas duplo-direcionadas Mercaptopyruvate Sulfurtransferase1 (MST1) e a Thiazole Biosynthetic Enzyme (THI1), e com a proteína direcionada aos cloroplastos TH-1. Experimentos de deleção e interação in vivo mostraram que a região Box1 de GRF9 é essencial para a interação com THI1 e MST1. Com a finalidade de dar continuidade a caracterização da GRF9 e para realização de testes com relação a sua função no direcionamento de proteínas organelares foi gerada uma linhagem homozigota que superexpressa GRF9. Plantas expressando o transgene TH-1 fusionado a Green Fluorescent Protein (GFP) em genótipo deficiente na TH-1 (CS3469/TH-1-GFP) foram obtidas para a geração de população mutante que possibilitará a descoberta de componentes genéticos ainda desconhecidos e responsáveis pelo direcionamento de proteínas aos cloroplastos. / In Eukaryotes, the evolution of molecular transport in the cell was essential due to their increase in compartmentalization, which requires more specific mechanisms for the correct localization of proteins. With the establishment of mitochondria and plastids as organelles, a great number of their genes, either specific for their metabolic functions or maintenance of their own transcription/translation processes, were transferred to the nucleus of the cell. These transfers caused most of the organellar proteins to be coded by the nucleus, then synthesized in the cytosol and targeted to the organelles by a complex machinery which involves membrane receptors in the organelles, targeting sequences in the proteins, and cytosolic proteins which assist them with the transport. Protein import depends greatly on an N-terminal sequence in proteins which has recognizable signals for the organellar membrane receptors. However, much is still not understood about the transport of organellar proteins, and unknown factors may still influence subcellular targeting. The goal of this work was the characterization of General Regulatory Factor 9 (GRF9), a protein of the 14-3-3 family in Arabidopsis thaliana potentially involved in the targeting of organellar proteins, and generating a genotype to be used in obtaining a mutant population for genes affecting the targeting of the protein Thiamine Requiring 1 (TH-1). After in vivo and in planta experiments it was observed that GRF9 interacts with the dual-targeted proteins Mercaptopyruvate Sulfurtransferase1 (MST1) and Thiazole Biosynthetic Enzyme (THI1), and with the chloroplast targeted protein TH-1. Deletion experiments followed by in vivo interaction assays showed that Box 1 region of GRF9 is essential for the interaction with THI1 and MST1. For the continuing characterization of GRF9 and for following tests of its function in the targeting of organellar proteins, a homozygous line was generated overexpressing GRF9. Plants expressing the transgene TH-1 fused to the Green Fluorescent Protein (GFP) in a TH-1 deficient genotype (CS3469/TH-1-GFP) were obtained for the generation of a mutant population which will allow the discovery of genetic components still unknown responsible for targeting proteins to the chloroplasts.

No description available.

Nos eucariotos, a evolução dos sistemas de transporte molecular foi essencial pois seu alto grau de compartimentalização requer mecanismos com maior especificidade para a localização de proteínas. Com o estabelecimento das mitocôndrias e plastídeos como organelas da célula eucariota, grande parte dos genes específicos para sua atividade e manutenção foram transferidos ao núcleo. Após a transferência gênica, a maioria das proteínas passaram a ser codificadas pelo núcleo, sintetizadas no citosol e direcionadas às organelas por uma maquinaria complexa que envolve receptores nas membranas das organelas, sequências de direcionamento nas proteínas e proteínas citossólicas que auxiliam o transporte. A importação depende em grande parte de uma sequência na região N-terminal das proteínas que contém sinais reconhecidos pelas membranas organelares. No entanto, muito ainda não é compreendido sobre o transporte de proteínas organelares e fatores ainda desconhecidos podem influenciar o direcionamento sub-celular. O objetivo deste trabalho foi a caracterização da General Regulatory Factor 9 (GRF9), uma proteína da família 14-3-3 de Arabidopsis thaliana potencialmente envolvida no direcionamento de proteínas organelares, e a geração de um genótipo para ser utilizado na obtenção de uma população mutante para genes que afetam o direcionamento da proteína Tiamina Monofosfato Sintetase (TH-1). Após experimentos in vivo e in planta, foi observado que GRF9 interage com as proteínas duplo-direcionadas Mercaptopyruvate Sulfurtransferase1 (MST1) e a Thiazole Biosynthetic Enzyme (THI1), e com a proteína direcionada aos cloroplastos TH-1. Experimentos de deleção e interação in vivo mostraram que a região Box1 de GRF9 é essencial para a interação com THI1 e MST1. Com a finalidade de dar continuidade a caracterização da GRF9 e para realização de testes com relação a sua função no direcionamento de proteínas organelares foi gerada uma linhagem homozigota que superexpressa GRF9. Plantas expressando o transgene TH-1 fusionado a Green Fluorescent Protein (GFP) em genótipo deficiente na TH-1 (CS3469/TH-1-GFP) foram obtidas para a geração de população mutante que possibilitará a descoberta de componentes genéticos ainda desconhecidos e responsáveis pelo direcionamento de proteínas aos cloroplastos. / In Eukaryotes, the evolution of molecular transport in the cell was essential due to their increase in compartmentalization, which requires more specific mechanisms for the correct localization of proteins. With the establishment of mitochondria and plastids as organelles, a great number of their genes, either specific for their metabolic functions or maintenance of their own transcription/translation processes, were transferred to the nucleus of the cell. These transfers caused most of the organellar proteins to be coded by the nucleus, then synthesized in the cytosol and targeted to the organelles by a complex machinery which involves membrane receptors in the organelles, targeting sequences in the proteins, and cytosolic proteins which assist them with the transport. Protein import depends greatly on an N-terminal sequence in proteins which has recognizable signals for the organellar membrane receptors. However, much is still not understood about the transport of organellar proteins, and unknown factors may still influence subcellular targeting. The goal of this work was the characterization of General Regulatory Factor 9 (GRF9), a protein of the 14-3-3 family in Arabidopsis thaliana potentially involved in the targeting of organellar proteins, and generating a genotype to be used in obtaining a mutant population for genes affecting the targeting of the protein Thiamine Requiring 1 (TH-1). After in vivo and in planta experiments it was observed that GRF9 interacts with the dual-targeted proteins Mercaptopyruvate Sulfurtransferase1 (MST1) and Thiazole Biosynthetic Enzyme (THI1), and with the chloroplast targeted protein TH-1. Deletion experiments followed by in vivo interaction assays showed that Box 1 region of GRF9 is essential for the interaction with THI1 and MST1. For the continuing characterization of GRF9 and for following tests of its function in the targeting of organellar proteins, a homozygous line was generated overexpressing GRF9. Plants expressing the transgene TH-1 fused to the Green Fluorescent Protein (GFP) in a TH-1 deficient genotype (CS3469/TH-1-GFP) were obtained for the generation of a mutant population which will allow the discovery of genetic components still unknown responsible for targeting proteins to the chloroplasts.