RESUMEN
RAS proteins are binary switches, cycling between ON and OFF states during signal transduction. These switches are normally tightly controlled, but in RAS-related diseases, such as cancer, RASopathies, and many psychiatric disorders, mutations in the RAS genes or their regulators render RAS proteins persistently active. The structural basis of the switch and many of the pathways that RAS controls are well known, but the precise mechanisms by which RAS proteins function are less clear. All RAS biology occurs in membranes: a precise understanding of RAS' interaction with membranes is essential to understand RAS action and to intervene in RAS-driven diseases.
Asunto(s)
Proteínas ras/metabolismo , Animales , Membrana Celular/metabolismo , Anomalías Congénitas/metabolismo , Humanos , Trastornos Mentales/metabolismo , Mutación , Neoplasias/metabolismo , Filogenia , Transducción de Señal , Levaduras , Proteínas ras/química , Proteínas ras/genéticaRESUMEN
RAS proteins are conserved guanosine triphosphate (GTP) hydrolases (GTPases) that act as molecular binary switches and play vital roles in numerous cellular processes. Upon GTP binding, RAS GTPases adopt an active conformation and interact with specific proteins termed RAS effectors that contain a conserved ubiquitin-like domain, thereby facilitating downstream signaling. Over 50 effector proteins have been identified in the human proteome, and many have been studied as potential mediators of RAS-dependent signaling pathways. Biochemical and structural analyses have provided mechanistic insights into these effectors, and studies using model organisms have complemented our understanding of their role in physiology and disease. Yet, many critical aspects regarding the dynamics and biological function of RAS-effector complexes remain to be elucidated. In this review, we discuss the mechanisms and functions of known RAS effector proteins, provide structural perspectives on RAS-effector interactions, evaluate their significance in RAS-mediated signaling, and explore their potential as therapeutic targets.
Asunto(s)
Transducción de Señal , Proteínas ras , Humanos , Proteínas ras/metabolismo , Proteínas ras/química , Animales , Unión Proteica , Modelos Moleculares , Relación Estructura-Actividad , Conformación Proteica , Guanosina Trifosfato/metabolismoRESUMEN
One of the open questions in RAS biology is the existence of RAS dimers and their role in RAF dimerization and activation. The idea of RAS dimers arose from the discovery that RAF kinases function as obligate dimers, which generated the hypothesis that RAF dimer formation might be nucleated by G-domain-mediated RAS dimerization. Here, we review the evidence for RAS dimerization and describe a recent discussion among RAS researchers that led to a consensus that the clustering of two or more RAS proteins is not due to the stable association of G-domains but, instead, is a consequence of RAS C-terminal membrane anchors and the membrane phospholipids with which they interact.
Asunto(s)
Quinasas raf , Proteínas ras , Dimerización , Consenso , Proteínas ras/genética , Proteínas ras/metabolismo , Quinasas raf/genética , Quinasas raf/metabolismo , Lípidos , Proteínas Proto-Oncogénicas c-raf/metabolismoRESUMEN
Uridylation occurs pervasively on mRNAs, yet its mechanism and significance remain unknown. By applying TAIL-seq, we identify TUT4 and TUT7 (TUT4/7), also known as ZCCHC11 and ZCCHC6, respectively, as mRNA uridylation enzymes. Uridylation readily occurs on deadenylated mRNAs in cells. Consistently, purified TUT4/7 selectively recognize and uridylate RNAs with short A-tails (less than â¼ 25 nt) in vitro. PABPC1 antagonizes uridylation of polyadenylated mRNAs, contributing to the specificity for short A-tails. In cells depleted of TUT4/7, the vast majority of mRNAs lose the oligo-U-tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of oligo-uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 are required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs and demonstrates a fundamental role of oligo-U-tail as a molecular mark for global mRNA decay.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ARN Nucleotidiltransferasas/metabolismo , Estabilidad del ARN , Células HeLa , Humanos , MicroARNs/metabolismo , Poli A/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , ARN Mensajero/metabolismo , Uridina Monofosfato/metabolismoRESUMEN
RAS is a signaling protein associated with the cell membrane that is mutated in up to 30% of human cancers. RAS signaling has been proposed to be regulated by dynamic heterogeneity of the cell membrane. Investigating such a mechanism requires near-atomistic detail at macroscopic temporal and spatial scales, which is not possible with conventional computational or experimental techniques. We demonstrate here a multiscale simulation infrastructure that uses machine learning to create a scale-bridging ensemble of over 100,000 simulations of active wild-type KRAS on a complex, asymmetric membrane. Initialized and validated with experimental data (including a new structure of active wild-type KRAS), these simulations represent a substantial advance in the ability to characterize RAS-membrane biology. We report distinctive patterns of local lipid composition that correlate with interfacially promiscuous RAS multimerization. These lipid fingerprints are coupled to RAS dynamics, predicted to influence effector binding, and therefore may be a mechanism for regulating cell signaling cascades.
Asunto(s)
Membrana Celular/enzimología , Lípidos/química , Aprendizaje Automático , Simulación de Dinámica Molecular , Multimerización de Proteína , Proteínas Proto-Oncogénicas p21(ras)/química , Transducción de Señal , HumanosRESUMEN
Sprouty-related EVH-1 domain-containing (SPRED) proteins are a family of proteins that negatively regulate the RAS-Mitogen-Activated Protein Kinase (MAPK) pathway, which is involved in the regulation of the mitogenic response and cell proliferation. However, the mechanism by which these proteins affect RAS-MAPK signaling has not been elucidated. Patients with mutations in SPRED give rise to unique disease phenotypes; thus, we hypothesized that distinct interactions across SPRED proteins may account for alternative nodes of regulation. To characterize the SPRED interactome and evaluate how members of the SPRED family function through unique binding partners, we performed affinity purification mass spectrometry. We identified 90-kDa ribosomal S6 kinase 2 (RSK2) as a specific interactor of SPRED2 but not SPRED1 or SPRED3. We identified that the N-terminal kinase domain of RSK2 mediates the interaction between amino acids 123 to 201 of SPRED2. Using X-ray crystallography, we determined the structure of the SPRED2-RSK2 complex and identified the SPRED2 motif, F145A, as critical for interaction. We found that the formation of this interaction is regulated by MAPK signaling events. We also find that this interaction between SPRED2 and RSK2 has functional consequences, whereby the knockdown of SPRED2 resulted in increased phosphorylation of RSK substrates, YB1 and CREB. Furthermore, SPRED2 knockdown hindered phospho-RSK membrane and nuclear subcellular localization. We report that disruption of the SPRED2-RSK complex has effects on RAS-MAPK signaling dynamics. Our analysis reveals that members of the SPRED family have unique protein binding partners and describes the molecular and functional determinants of SPRED2-RSK2 complex dynamics.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos , Proteínas Represoras , Proteínas Quinasas S6 Ribosómicas 90-kDa , Transducción de Señal , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Quinasas S6 Ribosómicas 90-kDa/química , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de Señal/genética , Humanos , Línea Celular , Dominios Proteicos , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Técnicas de Silenciamiento del Gen , Transporte de Proteínas/genética , Unión Proteica , Estructura Terciaria de Proteína , Modelos Moleculares , Neurofibromina 1/metabolismoRESUMEN
RAS proteins are molecular switches that interact with effector proteins when bound to guanosine triphosphate, stimulating downstream signaling in response to multiple stimuli. Although several canonical downstream effectors have been extensively studied and tested as potential targets for RAS-driven cancers, many of these remain poorly characterized. In this study, we undertook a biochemical and structural approach to further study the role of Sin1 as a RAS effector. Sin1 interacted predominantly with KRAS isoform 4A in cells through an atypical RAS-binding domain that we have characterized by X-ray crystallography. Despite the essential role of Sin1 in the assembly and activity of mTORC2, we find that the interaction with RAS is not required for these functions. Cells and mice expressing a mutant of Sin1 that is unable to bind RAS are proficient for activation and assembly of mTORC2. Our results suggest that Sin1 is a bona fide RAS effector that regulates downstream signaling in an mTORC2-independent manner.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Espectrometría de Masas , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Modelos Moleculares , Conformación Proteica , Isoformas de Proteínas , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de SeñalRESUMEN
The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as "membrane-distal" and estimate to comprise â¼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Quinasas raf/metabolismo , Membrana Celular/metabolismo , Simulación de Dinámica MolecularRESUMEN
During the activation of mitogen-activated protein kinase (MAPK) signaling, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) of RAF bind to active RAS at the plasma membrane. The orientation of RAS at the membrane may be critical for formation of the RAS-RBDCRD complex and subsequent signaling. To explore how RAS membrane orientation relates to the protein dynamics within the RAS-RBDCRD complex, we perform multiscale coarse-grained and all-atom molecular dynamics (MD) simulations of KRAS4b bound to the RBD and CRD domains of RAF-1, both in solution and anchored to a model plasma membrane. Solution MD simulations describe dynamic KRAS4b-CRD conformations, suggesting that the CRD has sufficient flexibility in this environment to substantially change its binding interface with KRAS4b. In contrast, when the ternary complex is anchored to the membrane, the mobility of the CRD relative to KRAS4b is restricted, resulting in fewer distinct KRAS4b-CRD conformations. These simulations implicate membrane orientations of the ternary complex that are consistent with NMR measurements. While a crystal structure-like conformation is observed in both solution and membrane simulations, a particular intermolecular rearrangement of the ternary complex is observed only when it is anchored to the membrane. This configuration emerges when the CRD hydrophobic loops are inserted into the membrane and helices α3-5 of KRAS4b are solvent exposed. This membrane-specific configuration is stabilized by KRAS4b-CRD contacts that are not observed in the crystal structure. These results suggest modulatory interplay between the CRD and plasma membrane that correlate with RAS/RAF complex structure and dynamics, and potentially influence subsequent steps in the activation of MAPK signaling.
Asunto(s)
Cisteína , Proteínas Proto-Oncogénicas c-raf , Sitios de Unión , Membrana Celular/metabolismo , Cisteína/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Solventes/metabolismoRESUMEN
KRAS is the most frequently mutated RAS protein in cancer patients, and it is estimated that about 20% of the cancer patients in the United States carried mutant RAS proteins. To accelerate therapeutic development, structures and dynamics of RAS proteins had been extensively studied by various biophysical techniques for decades. Although 31P NMR studies revealed population equilibrium of the two major states in the active GMPPNP-bound form, more complex conformational dynamics in RAS proteins and oncogenic mutants subtly modulate the interactions with their downstream effectors. We established a set of customized NMR relaxation dispersion techniques to efficiently and systematically examine the ms-µs conformational dynamics of RAS proteins. This method allowed us to observe varying synchronized motions that connect the effector and allosteric lobes in KRAS. We demonstrated the role of conformational dynamics of KRAS in controlling its interaction with the Ras-binding domain of the downstream effector RAF1, the first kinase in the MAPK pathway. This allows one to explain, as well as to predict, the altered binding affinities of various KRAS mutants, which was neither previously reported nor apparent from the structural perspective.
Asunto(s)
Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Fenómenos Fisiológicos Celulares , Humanos , Conformación Molecular , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas ras/químicaRESUMEN
We have solved two families of crystal structures of the human Dicer "platform-PAZ-connector helix" cassette in complex with small interfering RNAs (siRNAs). The structures possess two adjacently positioned pockets: a 2 nt 3'-overhang-binding pocket within the PAZ domain (3' pocket) and a phosphate-binding pocket within the platform domain (phosphate pocket). One family of complexes contains a knob-like α-helical protrusion, designated "hDicer-specific helix," that separates the two pockets and orients the bound siRNA away from the surface of Dicer, which could be indicative of a product release/transfer state. In the second complex, the helical protrusion is melted/disordered and the bound siRNA is aligned toward the surface of Dicer, suggestive of a cleavage-competent state. These structures allow us to propose that the transition from the cleavage-competent to the postulated product release/transfer state may involve release of the 5'-phosphate from the phosphate pocket while retaining the 3' overhang in the 3' pocket.
Asunto(s)
ARN Helicasas DEAD-box/química , Ribonucleasa III/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Cristalografía por Rayos X , ARN Helicasas DEAD-box/metabolismo , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Fosfatos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/metabolismo , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
KRAS mutations occur in â¼35% of colorectal cancers and promote tumor growth by constitutively activating the mitogen-activated protein kinase (MAPK) pathway. KRAS mutations at codons 12, 13, or 61 are thought to prevent GAP protein-stimulated GTP hydrolysis and render KRAS-mutated colorectal cancers unresponsive to epidermal growth factor receptor (EGFR) inhibitors. We report here that KRAS G13-mutated cancer cells are frequently comutated with NF1 GAP but NF1 is rarely mutated in cancers with KRAS codon 12 or 61 mutations. Neurofibromin protein (encoded by the NF1 gene) hydrolyzes GTP directly in complex with KRAS G13D, and KRAS G13D-mutated cells can respond to EGFR inhibitors in a neurofibromin-dependent manner. Structures of the wild type and G13D mutant of KRAS in complex with neurofibromin (RasGAP domain) provide the structural basis for neurofibromin-mediated GTP hydrolysis. These results reveal that KRAS G13D is responsive to neurofibromin-stimulated hydrolysis and suggest that a subset of KRAS G13-mutated colorectal cancers that are neurofibromin-competent may respond to EGFR therapies.
Asunto(s)
Neoplasias Colorrectales/genética , Receptores ErbB/antagonistas & inhibidores , Guanosina Trifosfato/metabolismo , Neurofibromina 1/química , Proteínas Proto-Oncogénicas p21(ras)/química , Sustitución de Aminoácidos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Dominio Catalítico , Línea Celular , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas Activadoras de GTPasa/metabolismo , Guanosina Trifosfato/química , Humanos , Hidrólisis , Modelos Moleculares , Neurofibromina 1/metabolismo , Neurofibromina 1/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutant active RAS is a driver in many types of solid tumors and hematological malignancies. Yet the biological effects of different RAS mutations and the tissue-specific clinical implications are complex and nuanced. Here, we identified an internal tandem duplication (ITD) in the switch II domain of NRAS from a patient with extremely aggressive colorectal carcinoma. Results of whole-exome DNA sequencing of primary and metastatic tumors indicated that this mutation was present in all analyzed metastases and excluded the presence of any other clear oncogenic driver mutations. Biochemical analysis revealed increased interaction of the RAS ITD with Raf proto-oncogene Ser/Thr kinase (RAF), leading to increased phosphorylation of downstream MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK). The ITD prevented interaction with neurofibromin 1 (NF1)-GTPase-activating protein (GAP), providing a mechanism for sustained activity of the RAS ITD protein. We present the first crystal structures of NRAS and KRAS ITD at 1.65-1.75 Å resolution, respectively, providing insight into the physical interactions of this class of RAS variants with its regulatory and effector proteins. Our in-depth bedside-to-bench analysis uncovers the molecular mechanism underlying a case of highly aggressive colorectal cancer and illustrates the importance of robust biochemical and biophysical approaches in the implementation of individualized medicine.
Asunto(s)
Neoplasias Colorrectales , GTP Fosfohidrolasas , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana , Mutación , Proteínas Proto-Oncogénicas p21(ras) , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Cristalografía por Rayos X , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Dominios Proteicos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Secuenciación del Exoma , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
BACKGROUND: DLC1, a tumor suppressor gene that is downregulated in many cancer types by genetic and nongenetic mechanisms, encodes a protein whose RhoGAP and scaffolding activities contribute to its tumor suppressor functions. The role of the DLC1 START (StAR-related lipid transfer; DLC1-START) domain, other than its binding to Caveolin-1, is poorly understood. In other START domains, a key function is that they bind lipids, but the putative lipid ligand for DLC1-START is unknown. METHODS: Lipid overlay assays and Phosphatidylserine (PS)-pull down assays confirmed the binding of DLC1-START to PS. Co-immunoprecipitation studies demonstrated the interaction between DLC1-START and Phospholipase C delta 1 (PLCD1) or Caveolin-1, and the contribution of PS to those interactions. Rho-GTP, cell proliferation, cell migration, and/or anchorage-independent growth assays were used to investigate the contribution of PS and PLCD1, or the implications of TCGA cancer-associated DLC1-START mutants, to DLC1 functions. Co-immunoprecipitations and PS-pull down assays were used to investigate the molecular mechanisms underlying the impaired functions of DLC1-START mutants. A structural model of DLC1-START was also built to better understand the structural implications of the cancer-associated mutations in DLC1-START. RESULTS: We identified PS as the lipid ligand for DLC1-START and determined that DLC1-START also binds PLCD1 protein in addition to Caveolin-1. PS binding contributes to the interaction of DLC1 with Caveolin-1 and with PLCD1. The importance of these activities for tumorigenesis is supported by our analysis of 7 cancer-associated DLC1-START mutants, each of which has reduced tumor suppressor function but retains wildtype RhoGAP activity. Our structural model of DLC1-START indicates the mutants perturb different elements within the structure, which is correlated with our experimental findings that the mutants are heterogenous with regard to the deficiency of their binding properties. Some have reduced PS binding, others reduced PLCD1 and Caveolin-1 binding, and others are deficient for all of these properties. CONCLUSION: These observations highlight the importance of DLC1-START for the tumor suppressor function of DLC1 that is RhoGAP-independent. They also expand the versatility of START domains, as DLC1-START is the first found to bind PS, which promotes the binding to other proteins.
Asunto(s)
Caveolina 1/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipasa C delta/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Supresoras de Tumor/metabolismo , Sitios de Unión , Proteínas Portadoras , Caveolina 1/química , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Proteínas Activadoras de GTPasa/genética , Humanos , Modelos Moleculares , Mutación , Fosfolipasa C delta/química , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Proteínas Supresoras de Tumor/genéticaRESUMEN
MazF is an mRNA interferase, which, upon activation during stress conditions, cleaves mRNAs in a sequence-specific manner, resulting in cellular growth arrest. During normal growth conditions, the MazF toxin is inactivated through binding to its cognate antitoxin, MazE. How MazF specifically recognizes its mRNA target and carries out cleavage and how the formation of the MazE-MazF complex inactivates MazF remain unclear. We present crystal structures of MazF in complex with mRNA substrate and antitoxin MazE in Bacillus subtilis. The structure of MazF in complex with uncleavable UUdUACAUAA RNA substrate defines the molecular basis underlying the sequence-specific recognition of UACAU and the role of residues involved in the cleavage through site-specific mutational studies. The structure of the heterohexameric (MazF)2-(MazE)2-(MazF)2 complex in Bacillus subtilis, supplemented by mutational data, demonstrates that the positioning of the C-terminal helical segment of MazE within the RNA-binding channel of the MazF dimer prevents mRNA binding and cleavage by MazF.
Asunto(s)
Bacillus subtilis/química , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Endorribonucleasas/química , Proteínas de Escherichia coli/química , ARN Mensajero/química , Antitoxinas/química , Escherichia coli/química , Mutación , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The glycolipid transfer protein (GLTP) fold defines a superfamily of eukaryotic proteins that selectively transport sphingolipids (SLs) between membranes. However, the mechanisms determining the protein selectivity for specific glycosphingolipids (GSLs) are unclear. Here, we report the crystal structure of the GLTP homology (GLTPH) domain of human 4-phosphate adaptor protein 2 (FAPP2) bound with N-oleoyl-galactosylceramide. Using this domain, FAPP2 transports glucosylceramide from its cis-Golgi synthesis site to the trans-Golgi for conversion into complex GSLs. The FAPP2-GLTPH structure revealed an element, termed the ID loop, that controls specificity in the GLTP family. We found that, in accordance with FAPP2 preference for simple GSLs, the ID loop protrudes from behind the SL headgroup-recognition center to mitigate binding by complex GSLs. Mutational analyses including GLTP and FAPP2 chimeras with swapped ID loops supported the proposed restrictive role of the FAPP2 ID loop in GSL selectivity. Comparative analysis revealed distinctly designed ID loops in each GLTP family member. This analysis also disclosed a conserved H-bond triplet that "clasps" both ID-loop ends together to promote structural autonomy and rigidity. The findings indicated that various ID loops work in concert with conserved recognition centers to create different specificities among family members. We also observed four bulky, conserved hydrophobic residues involved in "sensor-like" interactions with lipid chains in protein hydrophobic pockets and FF motifs in GLTP and FAPP2, well-positioned to provide acyl chain-dependent SL selectivity for the hydrophobic pockets. In summary, our study provides mechanistic insights into sphingolipid recognition by the GLTP fold and uncovers the elements involved in this recognition.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Portadoras/química , Esfingolípidos/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Conformación Proteica , Alineación de Secuencia , Esfingolípidos/metabolismoRESUMEN
Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single-molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre-miRNAs) in the absence of Lin28. We find that the overhang of a pre-miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre-miRNAs, which have a 1-nt 3' overhang, TUT7 restores the canonical end structure (2-nt 3' overhang) through mono-uridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 3' end is further recessed into the stem (as in 3' trimmed pre-miRNAs), TUT7 generates an oligo-U tail that leads to degradation. In contrast to Lin28-stimulated oligo-uridylation, which is processive, a distributive mode is employed by TUT7 for both mono- and oligo-uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7-RNA interaction, thus explaining how TUT7 differentiates pre-miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre-miRNAs.
Asunto(s)
MicroARNs/metabolismo , ARN Nucleotidiltransferasas/fisiología , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , Uridina Monofosfato/metabolismo , Nucleótidos de Adenina/metabolismo , Secuencia de Bases , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligorribonucleótidos/metabolismo , Procesamiento Postranscripcional del ARN/genética , Estabilidad del ARN/genética , Nucleótidos de Uracilo/metabolismoRESUMEN
Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.
Asunto(s)
Proteínas Portadoras/metabolismo , Ceramidas/metabolismo , Eicosanoides/metabolismo , Animales , Apoproteínas/química , Ácido Araquidónico/metabolismo , Transporte Biológico , Proteínas Portadoras/química , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Ceramidas/química , Cristalografía por Rayos X , Citosol/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Modelos Moleculares , Ácidos Fosfatidicos/química , Ácidos Fosfatidicos/metabolismo , Proteínas de Transferencia de Fosfolípidos , Conformación Proteica , Pliegue de Proteína , Especificidad por Sustrato , Red trans-Golgi/metabolismoRESUMEN
Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated-methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated-methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated-methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated-methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein-protein interaction interface in KRAS4b-PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/química , 3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Prenilación de Proteína/fisiología , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , 3',5'-GMP Cíclico Fosfodiesterasas/genética , Secuencia de Aminoácidos , Sitios de Unión , Membrana Celular/metabolismo , Cristalografía por Rayos X , Genes ras , Humanos , Metilación , Modelos Moleculares , Conformación Molecular , Mutación , Unión Proteica/fisiología , Proteínas Proto-Oncogénicas p21(ras)/genética , Análisis de Secuencia , Proteína de Unión al GTP rac1/metabolismo , Proteínas de Unión al GTP ral/metabolismoRESUMEN
Cytosine DNA methylation is evolutionarily ancient, and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA (SET- or RING-associated) methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals. The 5-methyl-cytosine (5mC)-binding specificity of several SRA domains have been characterized, and each one has a preference for DNA methylation in different sequence contexts. Here we demonstrate through mobility shift assays and calorimetric measurements that the SU(VAR)3-9 HOMOLOG 5 (SUVH5) SRA domain differs from other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of the SUVH5 SRA domain bound to 5mC-containing DNA in either the fully or hemimethylated CG context or the methylated CHH context revealed a dual flip-out mechanism where both the 5mC and a base (5mC, C, or G, respectively) from the partner strand are simultaneously extruded from the DNA duplex and positioned within binding pockets of individual SRA domains. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9 dimethyl modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci.