RESUMO
Activated Cdc42-associated kinase (ACK), a non-receptor tyrosine kinase, is an effector for the small GTPase Cdc42. ACK is emerging as an important component of the cancer landscape and thus, a promising target for the treatment of many malignancies. ACK is also being increasingly recognized as a potentially influential player in the regulation of protein homoeostasis. The delicate equilibrium between protein synthesis and protein degradation is crucial for healthy cell function and dysregulation of protein homoeostasis is a common occurrence in human disease. Here, we review the molecular mechanisms by which ACK regulates the stability of diverse cellular proteins (e.g. EGFR, p27, p53, p85 isoforms and RhoGDI-3), some of which rely on the kinase activity of ACK while others, interestingly, do not. Ultimately, further research will be required to bridge our knowledge gaps and determine if ACK regulates the stability of further cellular proteins but collectively, such mechanistic interrogation would contribute to determining whether ACK is a promising target for anti-cancer therapy. In therapeutics, proteasome inhibitors are an efficacious but problematic class of drugs. Targeting other modulators of proteostasis, like ACK, could open novel avenues for intervention.
Assuntos
Neoplasias , Proteínas Tirosina Quinases , Humanos , Proteína cdc42 de Ligação ao GTP/metabolismo , Fosforilação , Estabilidade Proteica , Proteínas Tirosina Quinases/metabolismoRESUMO
Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85ß, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85ß promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.
Assuntos
Fosfatidilinositol 3-Quinases , Proteínas Tirosina Quinases , Núcleo Celular/enzimologia , Células HEK293 , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Multimerização Proteica , Proteínas Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
RalA is a small GTPase and a member of the Ras family. This molecular switch is activated downstream of Ras and is widely implicated in tumor formation and growth. Previous work has shown that the ubiquitous Ca2+-sensor calmodulin (CaM) binds to small GTPases such as RalA and K-Ras4B, but a lack of structural information has obscured the functional consequences of these interactions. Here, we have investigated the binding of CaM to RalA and found that CaM interacts exclusively with the C terminus of RalA, which is lipidated with a prenyl group in vivo to aid membrane attachment. Biophysical and structural analyses show that the two RalA membrane-targeting motifs (the prenyl anchor and the polybasic motif) are engaged by distinct lobes of CaM and that CaM binding leads to removal of RalA from its membrane environment. The structure of this complex, along with a biophysical investigation into membrane removal, provides a framework with which to understand how CaM regulates the function of RalA and sheds light on the interaction of CaM with other small GTPases, including K-Ras4B.
Assuntos
Calmodulina/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Calmodulina/química , Membrana Celular/metabolismo , Humanos , Bicamadas Lipídicas/química , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Ligação Proteica , Prenilação de Proteína , Serina/metabolismo , Proteínas ral de Ligação ao GTP/químicaRESUMO
Cdc42 is a member of the Rho family of small GTPases and a master regulator of the actin cytoskeleton, controlling cell motility, polarity and cell cycle progression. This small G protein and its regulators have been the subject of many years of fruitful investigation and the advent of functional genomics and proteomics has opened up new avenues of exploration including how it functions at specific locations in the cell. This has coincided with the introduction of new structural techniques with the ability to study small GTPases in the context of the membrane. The role of Cdc42 in cancer is well established but the molecular details of its action are still being uncovered. Here we review alterations found to Cdc42 itself and to key components of the signal transduction pathways it controls in cancer. Given the challenges encountered with targeting small G proteins directly therapeutically, it is arguably the regulators of Cdc42 and the effector signalling pathways downstream of the small G protein which will be the most tractable targets for therapeutic intervention. These will require interrogation in order to fully understand the global signalling contribution of Cdc42, unlock the potential for mapping new signalling axes and ultimately produce inhibitors of Cdc42 driven signalling.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mutação , Neoplasias/genética , Transdução de Sinais/genética , Proteína cdc42 de Ligação ao GTP/genética , Citoesqueleto de Actina/metabolismo , Animais , Humanos , Microdomínios da Membrana/metabolismo , Microtúbulos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/uso terapêutico , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
RLIP76/RalBP1 is an ATP-dependent transporter of glutathione conjugates, which is overexpressed in various human cancers, but its diverse functions in normal cells, which include endocytosis, stress response and mitochondrial dynamics, are still not fully understood. The protein can be divided into three distinct regions, each with its own structural properties. At the centre of the protein are two well-defined domains, a GTPase activating protein domain targeting Rho family small G proteins and a small coiled-coil that binds to the Ras family small GTPases RalA and RalB. In engaging with Rho and Ral proteins, RLIP76 bridges these two distinct G protein families. The N-terminal region is predicted to be disordered and is rich in basic amino acids, which may mediate membrane association, consistent with its role in transport. RLIP76 is an ATP-dependent transporter with ATP-binding sites within the N-terminus and the Ral binding domain. Furthermore, RLIP76 is subject to extensive phosphorylation, particularly in the N-terminal region. In contrast, the C-terminal region is thought to form an extensive coiled-coil that could mediate dimerization. Here, we review the structural features of RLIP76, including experimental data and computational predictions, and discuss the implications of its various post-translational modifications.
RESUMO
Cdc42 is a member of the Rho family of small GTPases and a key regulator of the actin cytoskeleton, controlling cell motility, polarity and cell cycle progression. It signals downstream of the master regulator Ras and is essential for cell transformation by this potent oncogene. Overexpression of Cdc42 is observed in several cancers, where it is linked to poor prognosis. As a regulator of both cell architecture and motility, deregulation of Cdc42 is also linked to tumour metastasis. Like Ras, Cdc42 and other components of the signalling pathways it controls represent important potential targets for cancer therapeutics. In this review, we consider the progress that has been made targeting Cdc42, its regulators and effectors, including new modalities and new approaches to inhibition. Strategies under consideration include inhibition of lipid modification, modulation of Cdc42-GEF, Cdc42-GDI and Cdc42-effector interactions, and direct inhibition of downstream effectors.
Assuntos
Citoesqueleto de Actina/metabolismo , Neoplasias/metabolismo , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Aminoquinolinas/uso terapêutico , Animais , Benzamidas/uso terapêutico , Benzazepinas/uso terapêutico , Humanos , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Oximas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sulfonamidas/uso terapêutico , Tioureia/análogos & derivados , Tioureia/uso terapêutico , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/genéticaRESUMO
There are three RhoGDIs in mammalian cells, which were initially defined as negative regulators of Rho family small GTPases. However, it is now accepted that RhoGDIs not only maintain small GTPases in their inactive GDP-bound form but also act as chaperones for small GTPases, targeting them to specific intracellular membranes and protecting them from degradation. Studies to date with RhoGDIs have usually focused on the interactions between the "typical" or "classical" small GTPases, such as the Rho, Rac, and Cdc42 subfamily members, and either the widely expressed RhoGDI-1 or the hematopoietic-specific RhoGDI-2. Less is known about the third member of the family, RhoGDI-3 and its interacting partners. RhoGDI-3 has a unique N-terminal extension and is found to localize in both the cytoplasm and the Golgi. RhoGDI-3 has been shown to target RhoB and RhoG to endomembranes. In order to facilitate a more thorough understanding of RhoGDI function, we undertook a systematic study to determine all possible Rho family small GTPases that interact with the RhoGDIs. RhoGDI-1 and RhoGDI-2 were found to have relatively restricted activity, mainly binding members of the Rho and Rac subfamilies. RhoGDI-3 displayed wider specificity, interacting with the members of Rho, Rac, and Cdc42 subfamilies but also forming complexes with "atypical" small Rho GTPases such as Wrch2/RhoV, Rnd2, Miro2, and RhoH. Levels of RhoA, RhoB, RhoC, Rac1, RhoH, and Wrch2/RhoV bound to GTP were found to decrease following coexpression with RhoGDI-3, confirming its role as a negative regulator of these small Rho GTPases.
Assuntos
Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Inibidor gama de Dissociação do Nucleotídeo Guanina rho/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/química , Células HEK293 , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ligação Proteica , Proteínas rho de Ligação ao GTP/química , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/fisiologia , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/fisiologia , Inibidor gama de Dissociação do Nucleotídeo Guanina rho/fisiologia , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-Específico/fisiologiaRESUMO
The protein kinase C-related kinase (PRK) family of serine/threonine kinases, PRK1, PRK2 and PRK3, are effectors for the Rho family small G proteins. An array of studies have linked these kinases to multiple signalling pathways and physiological roles, but while PRK1 is relatively well-characterized, the entire PRK family remains understudied. Here, we provide a holistic overview of the structure and function of PRKs and describe the molecular events that govern activation and autoregulation of catalytic activity, including phosphorylation, protein interactions and lipid binding. We begin with a structural description of the regulatory and catalytic domains, which facilitates the understanding of their regulation in molecular detail. We then examine their diverse physiological roles in cytoskeletal reorganization, cell adhesion, chromatin remodelling, androgen receptor signalling, cell cycle regulation, the immune response, glucose metabolism and development, highlighting isoform redundancy but also isoform specificity. Finally, we consider the involvement of PRKs in pathologies, including cancer, heart disease and bacterial infections. The abundance of PRK-driven pathologies suggests that these enzymes will be good therapeutic targets and we briefly report some of the progress to date.
Assuntos
Proteína Quinase C/química , Proteínas Quinases/química , Proteínas Quinases/fisiologia , Animais , Catálise , Humanos , Conformação Proteica , Transdução de Sinais/fisiologia , Relação Estrutura-AtividadeRESUMO
Ral GTPases have been implicated as critical drivers of cell growth and metastasis in numerous Ras-driven cancers. We have previously reported stapled peptides, based on the Ral effector RLIP76, that can disrupt Ral signaling. Stapled peptides are short peptides that are locked into their bioactive form using a synthetic brace. Here, using an affinity maturation of the RLIP76 Ral-binding domain, we identified several sequence substitutions that together improve binding to Ral proteins by more than 20-fold. Hits from the selection were rigorously analyzed to determine the contributions of individual residues and two 1.5 Å cocrystal structures of the tightest-binding mutants in complex with RalB revealed key interactions. Insights gained from this maturation were used to design second-generation stapled peptides based on RLIP76 that exhibited vastly improved selectivity for Ral GTPases when compared with the first-generation lead peptide. The binding of second-generation peptides to Ral proteins was quantified and the binding site of the lead peptide on RalB was determined by NMR. Stapled peptides successfully competed with multiple Ral-effector interactions in cellular lysates. Our findings demonstrate how manipulation of a native binding partner can assist in the rational design of stapled peptide inhibitors targeting a protein-protein interaction.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas ral de Ligação ao GTP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Calorimetria , Dicroísmo Circular , Fluorescência , Proteínas Ativadoras de GTPase/química , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Transdução de Sinais , Proteínas ral de Ligação ao GTP/químicaRESUMO
The Rho-family of small GTPases are biological molecular switches that are best known for their regulation of the actin cytoskeleton. Through their activation and stimulation of downstream effectors, the Rho-family control pathways involved in cellular morphology, which are commonly activated in cancer cell invasion and metastasis. While this makes them excellent potential therapeutic targets, a deeper understanding of the downstream signalling pathways they influence will be required for successful drug targeting. Signal transducers and activators of transcription (STATs) are a family of transcription factors that are hyper-activated in most cancer types and while STATs are widely understood to be activated by the JAK family of kinases, many additional activators have been discovered. A growing number of examples of Rho-family driven STAT activation, largely of the oncogenic family members, STAT3 and STAT5, are being identified. Cdc42, Rac1, RhoA, RhoC and RhoH have all been implicated in STAT activation, contributing to Rho GTPase-driven changes in cellular morphology that lead to cell proliferation, invasion and metastasis. This highlights the importance and therapeutic potential of the Rho-family as regulators of non-canonical activation of STAT signalling.
Assuntos
Fator de Transcrição STAT3/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Progressão da Doença , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/metabolismo , Domínios Proteicos , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The phosphatidylinositol 3-kinase (PI3K) pathway is a critical regulator of many cellular processes including cell survival, growth, proliferation and motility. Not surprisingly therefore, the PI3K pathway is one of the most frequently mutated pathways in human cancers. In addition to their canonical role as part of the PI3K holoenzyme, the class IA PI3K regulatory subunits undertake critical functions independent of PI3K. The PI3K regulatory subunits exist in excess over the p110 catalytic subunits and therefore free in the cell. p110-independent p85 is unstable and exists in a monomer-dimer equilibrium. Two conformations of dimeric p85 have been reported that are mediated by N-terminal and C-terminal protein domain interactions, respectively. The role of p110-independent p85 is under investigation and it has been found to perform critical adaptor functions, sequestering or influencing compartmentalisation of key signalling proteins. Free p85 has roles in glucose homeostasis, cellular stress pathways, receptor trafficking and cell migration. As a regulator of fundamental pathways, the amount of p110-independent p85 in the cell is critical. Factors that influence the monomer-dimer equilibrium of p110-independent p85 offer additional control over this system, disruption to which likely results in disease. Here we review the current knowledge of the structure and functions of p110-independent class IA PI3K regulatory subunits.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Animais , Movimento Celular , Dimerização , Humanos , Insulina/metabolismo , Mutação , Neoplasias/enzimologia , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Conformação Proteica , Transporte Proteico , Transdução de SinaisRESUMO
PRK1 is a member of the protein kinase C-related kinase (PRK) family of serine/threonine kinases and a downstream effector of Rho GTPases. PRK1 has three N-terminal Homology Region 1 (HR1) domains (HR1a, HR1b and HR1c), which form antiparallel coiled coils that interact with Rho family GTPases. PRK1 also has a C2-like domain that targets it to the plasma membrane and a kinase domain, which is a member of the protein kinase C superfamily. PRK1 is involved in cytoskeletal regulation, cell adhesion, cell cycle progression and the immune response, and is implicated in cancer. There is currently no structural information for the HR1c domain. The 1H, 15N and 13C NMR backbone and sidechain resonance assignment of the HR1c domain presented here forms the basis for this domain's structural characterisation. This work will also enable studies of interactions between the three HR1 domains in an effort to obtain structural insight into the regulation of PRK1 activity.
Assuntos
Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Proteína Quinase C/química , Espectroscopia de Prótons por Ressonância Magnética , Sequência de Aminoácidos , Humanos , Isótopos de Nitrogênio , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
The Ral proteins (RalA and RalB) are small G proteins of the Ras family that have been implicated in exocytosis, endocytosis, transcriptional regulation and mitochondrial fission, as well as having a role in tumourigenesis. RalA and RalB are activated downstream of the master regulator, Ras, which causes the nucleotide exchange of GDP for GTP. Here we report the 1H, 15 N and 13C resonance assignments of RalA in its active form bound to the GTP analogue GMPPNP. We also report the backbone assignments of RalA in its inactive, GDP-bound form. The assignments give insight into the switch regions, which change conformation upon nucleotide exchange. These switch regions are invisible in the spectra of the active, GMPPNP bound form but the residues proximal to the switches can be monitored. RalA is also an important drug target due to its over activation in some cancers and these assignments will be extremely useful for NMR-based screening approaches.
Assuntos
Proteínas Monoméricas de Ligação ao GTP/química , Ressonância Magnética Nuclear Biomolecular , Proteínas ral de Ligação ao GTP/química , Guanosina Difosfato/química , Conformação ProteicaRESUMO
Aberrant Ras signaling drives 30% of cancers, and inhibition of the Rho family small GTPase signaling has been shown to combat Ras-driven cancers. Here, we present the discovery of a 16-mer cyclic peptide that binds to Cdc42 with nanomolar affinity. Affinity maturation of this sequence has produced a panel of derived candidates with increased affinity and modulated specificity for other closely-related small GTPases. The structure of the tightest binding peptide was solved by NMR, and its binding site on Cdc42 was determined. Addition of a cell-penetrating sequence allowed the peptides to access the cell interior and engage with their target(s), modulating signaling pathways. In Ras-driven cancer cell models, the peptides have an inhibitory effect on proliferation and show suppression of both invasion and motility. As such, they represent promising candidates for Rho-family small GTPase inhibitors and therapeutics targeting Ras-driven cancers. Our data add to the growing literature demonstrating that peptides are establishing their place in the biologics arm of drug discovery.
Assuntos
Descoberta de Drogas , Peptídeos Cíclicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteínas ras/metabolismo , Sítios de Ligação , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células , GTP Fosfo-Hidrolases/antagonistas & inibidores , Humanos , Estrutura Molecular , Invasividade Neoplásica/prevenção & controle , Neoplasias/tratamento farmacológico , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Inhibition of Ras signalling has been a goal almost since its central role in cell signalling and its deregulation in disease were discovered. Early attempts at inhibiting its post-translational modification using peptidomimetics were successful in cell culture but failed spectacularly in clinical trials, making industry wary of targeting this critical oncoprotein. Small molecule inhibition of the protein-protein interactions involving Ras has also been difficult due to the nature of the interaction interface. Recent improvements in design, synthesis and selection of stabilised peptides, peptidomimetics and macrocycles have suggested that these biologics may represent a new hope in Ras inhibition. Here we review the various ways in which Ras has been targeted with these molecules. We also describe work on related small G proteins of the Ras superfamily, since many of the principles may be applicable to Ras, and these also provide inhibition of pathways downstream of Ras.
Assuntos
Descoberta de Drogas , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Humanos , Proteínas Monoméricas de Ligação ao GTP/química , Família Multigênica , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Son Of Sevenless/química , Proteínas Son Of Sevenless/metabolismo , Relação Estrutura-Atividade , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Wiskott-Aldrich syndrome protein (WASP) activates the actin-related protein 2/3 homolog (Arp2/3) complex and regulates actin polymerization in a physiological setting. Cell division cycle 42 (Cdc42) is a key activator of WASP, which binds Cdc42 through a Cdc42/Rac-interactive binding (CRIB)-containing region that defines a subset of Cdc42 effectors. Here, using site-directed mutagenesis and binding affinity determination and kinetic assays, we report the results of an investigation into the energetic contributions of individual WASP residues to both the Cdc42-WASP binding interface and the kinetics of complex formation. Our results support the previously proposed dock-and-coalesce binding mechanism, initiated by electrostatic steering driven by WASP's basic region and followed by a coalescence phase likely driven by the conserved CRIB motif. The WASP basic region, however, appears also to play a role in the final complex, as its mutation affected both on- and off-rates, suggesting a more comprehensive physiological role for this region centered on the C-terminal triad of positive residues. These results highlight the expanding roles of the basic region in WASP and other CRIB-containing effector proteins in regulating complex cellular processes and coordinating multiple input signals. The data presented improve our understanding of the Cdc42-WASP interface and also add to the body of information available for Cdc42-effector complex formation, therapeutic targeting of which has promise for Ras-driven cancers. Our findings suggest that combining high-affinity peptide-binding sequences with short electrostatic steering sequences could increase the efficacy of peptidomimetic candidates designed to interfere with Cdc42 signaling in cancer.
Assuntos
Neoplasias/genética , Proteína da Síndrome de Wiskott-Aldrich/química , Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/química , Actinas/química , Actinas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Cinética , Neoplasias/química , Neoplasias/patologia , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Transdução de Sinais , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas ras/química , Proteínas ras/genéticaRESUMO
The CRIB (Cdc42/Rac interactive binding) family of small G-protein effectors contain significant regions with intrinsic disorder. The G-protein-binding regions are contained within these intrinsically disordered regions. Most CRIB proteins also contain stretches of basic residues associated with their G-protein-binding regions. The basic region (BR) and G-protein-binding region together allow the CRIB effectors to bind to their cognate G-protein via a dock- and coalesce-binding mechanism. The BRs of these proteins take on multiple roles: steering G-protein binding, interacting with elements of the membrane and regulating intramolecular regulatory interactions. The ability of these regions of the CRIBs to undergo multivalent interactions and mediate charge neutralizations equips them with all the properties required to drive liquid-liquid phase separation and therefore to initiate and drive signalosome formation. It is only recently that the structural plasticity in these proteins is being appreciated as the driving force for these vital cellular processes.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , GTP Fosfo-Hidrolases/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dinâmica não Linear , Polilisina/química , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Transdução de Sinais , Eletricidade Estática , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Bladder cancer incurs a higher lifetime treatment cost than other cancers due to frequent recurrence of non-invasive disease. Improved prognostic biomarkers and localized therapy are needed for this large patient group. We defined two major genomic subtypes of primary stage Ta tumors. One of these was characterized by loss of 9q including TSC1, increased KI67 labeling index, upregulated glycolysis, DNA repair, mTORC1 signaling, features of the unfolded protein response, and altered cholesterol homeostasis. Comparison with muscle-invasive bladder cancer mutation profiles revealed lower overall mutation rates and more frequent mutations in RHOB and chromatin modifier genes. More mutations in the histone lysine demethylase KDM6A were present in non-invasive tumors from females than males.
Assuntos
Carcinoma de Células de Transição/metabolismo , Histona Desmetilases/genética , Metabolômica/métodos , Mutação , Proteínas Nucleares/genética , Neoplasias da Bexiga Urinária/metabolismo , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/patologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Frequência do Gene , Genômica/métodos , Células HEK293 , Histona Desmetilases/metabolismo , Humanos , Masculino , Metaboloma/genética , Proteínas Nucleares/metabolismo , Fatores Sexuais , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
Cdc42 is a Rho-family small G protein that has been widely studied for its role in controlling the actin cytoskeleton and plays a part in several potentially oncogenic signaling networks. Similar to most other small G proteins, Cdc42 binds to many downstream effector proteins to elicit its cellular effects. These effector proteins all engage the same face of Cdc42, the conformation of which is governed by the activation state of the G protein. Previously, the importance of individual residues in conferring binding affinity has been explored for residues within Cdc42 for three of its Cdc42/Rac interactive binding (CRIB) effectors, activated Cdc42 kinase (ACK), p21-activated kinase (PAK), and Wiskott-Aldrich syndrome protein (WASP). Here, in a complementary study, we have used our structure of Cdc42 bound to ACK via an intrinsically disordered ACK region to guide an analysis of the Cdc42 interface on ACK, creating a panel of mutant proteins with which we can now describe the complete energetic landscape of the Cdc42-binding site on ACK. Our data suggest that the binding affinity of ACK relies on several conserved residues that are critical for stabilizing the quaternary structure. These residues are centered on the CRIB region, with the complete binding region anchored at each end by hydrophobic interactions. These findings suggest that ACK adopts a dock and coalesce binding mechanism with Cdc42. In contrast to other CRIB-family effectors and indeed other intrinsically disordered proteins, hydrophobic residues likely drive Cdc42-ACK binding.
Assuntos
Proteínas Tirosina Quinases/química , Proteína cdc42 de Ligação ao GTP/química , Sítios de Ligação , Humanos , Mutação , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Aberrant Ras signaling drives numerous cancers, and drugs to inhibit this are urgently required. This compelling clinical need combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly, and the focus has moved to the main downstream Ras-signaling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets that were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development, and there have, therefore, been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of α-helical-stapled peptides that bind selectively to active, GTP-bound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This, therefore, provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.