RESUMEN
The development of cancer control plans as a clearly defined concept began in the U.S. in the early 1990s. On an international level, the same concept has been described as "national cancer control planning" or national cancer control plan (NCCP) development and implementation. Recent efforts by the National Cancer Institute's Center for Global Health and its partners have increased international and country-level interest in NCCPs. Central to the development of these plans has been a need for countries to understand the crucial factors and foundational elements necessary to develop and successfully implement a national cancer plan. This article describes the process by which a tool developed by the International Cancer Control Partnership (ICCP) helps countries and international partners assess their efforts to develop and implement a NCCP.
Asunto(s)
Salud Global , Neoplasias/prevención & control , Humanos , Autoevaluación (Psicología) , Planificación SocialRESUMEN
The aPKC [atypical PKC (protein kinase C)] isoforms ι and ζ play crucial roles in the formation and maintenance of cell polarity and represent attractive anti-oncogenic drug targets in Ras-dependent tumours. To date, few isoform-specific chemical biology tools are available to inhibit aPKC catalytic activity. In the present paper, we describe the identification and functional characterization of potent and selective thieno[2,3-d]pyrimidine-based chemical inhibitors of aPKCs. A crystal structure of human PKCι kinase domain bound to a representative compound, CRT0066854, reveals the basis for potent and selective chemical inhibition. Furthermore, CRT0066854 displaces a crucial Asn-Phe-Asp motif that is part of the adenosine-binding pocket and engages an acidic patch used by arginine-rich PKC substrates. We show that CRT0066854 inhibits the LLGL2 (lethal giant larvae 2) phosphorylation in cell lines and exhibits phenotypic effects in a range of cell-based assays. We conclude that this compound can be used as a chemical tool to modulate aPKC activity in vitro and in vivo and may guide the search for further aPKC-selective inhibitors.
Asunto(s)
Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/química , Tiofenos/farmacología , Adenosina/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Proteínas del Citoesqueleto/metabolismo , Perros , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Isoenzimas/antagonistas & inhibidores , Imitación Molecular , Datos de Secuencia Molecular , Fosforilación , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Pirimidinas/farmacología , Tiofenos/químicaRESUMEN
Mitochondria are highly dynamic organelles that mediate essential cell functions such as apoptosis and cell-cycle control in addition to their role as efficient ATP generators. Mitochondrial morphology changes are tightly regulated, and their shape can shift between small, fragmented units and larger networks of elongated mitochondria. We demonstrate that mitochondrial elements become significantly elongated and interconnected shortly after nutrient depletion. This mitochondrial morphological shift depends on the type of starvation, with an additive effect observed when multiple nutrients are depleted simultaneously. We further show that starvation-induced mitochondrial elongation is mediated by down-regulation of dynamin-related protein 1 (Drp1) through modulation of two Drp1 phosphorylation sites, leading to unopposed mitochondrial fusion. Finally, we establish that mitochondrial tubulation upon nutrient deprivation protects mitochondria from autophagosomal degradation, which could permit mitochondria to maximize energy production and supply autophagosomal membranes during starvation.
Asunto(s)
Autofagia/fisiología , Mitocondrias/metabolismo , Fagosomas/metabolismo , Inanición/metabolismo , Animales , Células Cultivadas , Dinaminas/genética , Dinaminas/metabolismo , Fibroblastos/citología , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/ultraestructura , Proteínas Mitocondriales/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
The phosphoserine/threonine binding protein 14-3-3 stimulates the catalytic activity of protein kinase C-epsilon (PKCepsilon) by engaging two tandem phosphoserine-containing motifs located between the PKCepsilon regulatory and catalytic domains (V3 region). Interaction between 14-3-3 and this region of PKCepsilon is essential for the completion of cytokinesis. Here, we report the crystal structure of 14-3-3zeta bound to a synthetic diphosphorylated PKCepsilon V3 region revealing how a consensus 14-3-3 site and a divergent 14-3-3 site cooperate to bind to 14-3-3 and so activate PKCepsilon. Thermodynamic data show a markedly enhanced binding affinity for two-site phosphopeptides over single-site 14-3-3 binding motifs and identifies Ser 368 as a gatekeeper phosphorylation site in this physiologically relevant 14-3-3 ligand. This dual-site intra-chain recognition has implications for other 14-3-3 targets, which seem to have only a single 14-3-3 motif, as other lower affinity and cryptic 14-3-3 gatekeeper sites might exist.
Asunto(s)
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Proteína Quinasa C-epsilon/química , Proteína Quinasa C-epsilon/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosfoserina/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , TermodinámicaRESUMEN
According to the Pan American Health Organization, noncommunicable diseases, including cancer, are the leading causes of preventable and premature death in the Americas. Governments and health care systems in Latin America face numerous challenges as a result of increasing morbidity and mortality from cancer. Multiple international organizations have recognized the need for collaborative action on and technical support for cancer research and control in Latin America. The Center for Global Health at the US National Cancer Institute (NCI-CGH) is one entity among many that are working in the region and has sought to develop a strategy for working in Latin America that draws on and expands the collaborative potential of engaged, skilled, and diverse partners. NCI-CGH has worked toward developing and implementing initiatives in collaboration with global partners that share the common objectives of building a global cancer research community and translating research results into evidence-informed policy and practice. Both objectives are complementary and synergistic and are additionally supported by an overarching strategic framework that is focused on partnerships and science diplomacy. This work highlights the overall strategy for NCI-CGH engagement in Latin America through partnerships and diplomacy, and highlights selected collaborative efforts that are aimed at improving cancer outcomes in the region.
Asunto(s)
Diplomacia , Cooperación Internacional , Neoplasias/epidemiología , Neoplasias/prevención & control , Investigación , Animales , Financiación del Capital , Salud Global , Planificación en Salud , Humanos , América Latina/epidemiología , Vigilancia en Salud Pública , Investigación/economía , Investigación/legislación & jurisprudencia , Investigación/organización & administraciónRESUMEN
ZiPD (zinc phosphodiesterase; synonyms are ElaC, ecoZ, RNaseZ and 3' tRNase) and the iron-dependent redox enzyme FlRd (flavorubredoxin) from Escherichia coli represent prototypical cases of proteins sharing the metallo-beta-lactamase fold that require strict metal selectivity for catalytic activity, yet their metal selectivity has only been partially understood. In contrast with hydrolytic metallo-beta-lactamase proteins, iron-dependent FlRd-like enzymes have an atypical glutamate ligand, which replaces one otherwise conserved histidine ligand. X-ray absorption spectroscopy revealed that the FlRd metallo-beta-lactamase domain is capable of incorporating two zinc ions into the binuclear metal-binding site. Zinc dissociation constants, determined by isothermal titration calorimetry are similar for zinc binding to E. coli ZiPD (K(d1)=2.2+/-0.2 microM and K(d2)=23.0+/-0.6 microM) and to the E. coli FlRd metallo-beta-lactamase domain (K(d1)=0.7+/-0.1 microM and K(d2)=26.0+/-0.1 microM). In good correspondence, apo-ZiPD requires incubation with 10 microM zinc for full reconstitution of the phosphodiesterase activity. Accordingly, metal selectivity of ZiPD and FlRd only partially relies on first shell metal ligands. Back mutation of the atypical glutamate in FlRd to a histidine unexpectedly resulted in an increased first zinc dissociation constant (K(d1)=30+/-4 microM and K(d2)=23+/-2 microM). In combination with a recent mutational study on ZiPD [Vogel, Schilling and Meyer-Klaucke (2004) Biochemistry 43, 10379-10386], we conclude that the atypical glutamate does not guide metal selectivity of the FlRd metallo-beta-lactamase domain but suppresses possible hydrolytic cross-activity.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glutamatos/metabolismo , Hierro/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Zinc/metabolismo , beta-Lactamasas/química , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sitios de Unión , Citosol/enzimología , Activación Enzimática/efectos de los fármacos , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Hierro/farmacología , Cinética , Mutación/genética , Hidrolasas Diéster Fosfóricas/genética , Unión Proteica , Estructura Terciaria de Proteína , Factores de Transcripción/genética , Zinc/farmacologíaRESUMEN
Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.
Asunto(s)
Uniones Adherentes/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Células Epiteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Polaridad Celular/fisiología , Drosophila , Proteínas de Drosophila/genética , Epitelio/crecimiento & desarrollo , Células HCT116 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Fosforilación , Unión Proteica/genética , Estructura Terciaria de ProteínaRESUMEN
Infectious Agents and Cancer is introducing a new section on Cancer Centers in Low- and Middle-Income Countries intended to provide the oncology community with detailed information about lessons learned in cancer control in resource-limited settings. The growing burden of cancer and the high rates of infection-related cancers in Low- and Middle-Income Countries (LMICs) argue for exploring the successes and challenges of cancer centers in low-resource settings. Detailed analyses are needed on how successful cancer centers have developed and managed such key components as strategic partnerships, trained cancer professionals, sustainable funding, appropriate technology, and research capacity. Many examples exist wherein local cancer centers have made significant progress and as such, the series will provide a platform to showcase detailed features of cancer institutes in LMICs and provide valuable information for those seeking to replicate successful models and to help invigorate efforts to build cancer capacity.
RESUMEN
Starvation induces a protective process of self-cannibalization called autophagy that is thought to mediate nonselective degradation of cytoplasmic material. We recently reported that mitochondria escape autophagosomal degradation through extensive fusion into mitochondrial networks upon certain starvation conditions. The extent of mitochondrial elongation is dependent on the type of nutrient deprivation, with amino acid depletion having a particularly strong effect. Downregulation of the mitochondrial fission protein Drp1 was determined to be important in bringing about starvation-induced mitochondrial fusion. The formation of mitochondrial networks during nutrient depletion selectively blocked their autophagic degradation, presumably allowing cells to sustain efficient ATP production and thereby survive starvation.
Asunto(s)
Autofagia , Fusión de Membrana , Mitocondrias/metabolismo , Fagosomas/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico , Ratones , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Mitochondria continuously change their shape and thereby influence different cellular processes like cell death or development. Recently, we showed that during starvation mitochondria fuse into a highly connected network. The change in mitochondrial shape was dependent on inactivation of the fission protein Drp1, through targeting of two different phosphorylation sites. This rapid inhibition of mitochondrial fission led to unopposed fusion, protecting mitochondria from starvation-induced degradation and enabling the cell to survive nutrient scarce conditions.
RESUMEN
The protein kinase C (PKC) Ser/Thr kinases account for approximately 2% of the human kinome and regulate diverse cellular behaviors. PKC catalytic activity requires priming phosphorylations at three conserved sites within the kinase domain. Here we demonstrate that priming of PKC is dependent on the conformation of the nucleotide binding pocket but not on its intrinsic kinase activity. Inactive ATP binding site mutants are unprimed, but they become phosphorylated upon occupancy of the ATP binding pocket with inhibitors of PKC. We have exploited this property to screen for PKC inhibitors in vivo. Further, we generated a distinct class of kinase-inactive mutants that maintain the integrity of the ATP binding pocket; such mutants are constitutively primed and functionally distinct from ATP binding site mutants. These data demonstrate that autophosphorylation is not required for PKC priming and show how ATP pocket occupation can enable a kinase to mature as well as function.
Asunto(s)
Nucleótidos/metabolismo , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Adenosina Trifosfato , Sitios de Unión/genética , Catálisis , Humanos , Proteínas Mutantes , Fosforilación , Conformación Proteica , Proteína Quinasa C/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
The elaC gene product from Escherichia coli, ZiPD, is a 3' tRNA-processing endonuclease belonging to the tRNase Z family of enzymes that have been identified in a wide variety of organisms. In contrast to the elaC homologue from Bacillus subtilis, E. coli elaC is not essential for viability, and although both enzymes process only precursor tRNA (pre-tRNA) lacking a CCA triplet at the 3' end in vitro, the physiological role of ZiPD remains enigmatic because all pre-tRNA species in E. coli are transcribed with the CCA triplet. We present the first crystal structure of ZiPD determined by multiple anomalous diffraction at a resolution of 2.9 A. This structure shares many features with the tRNase Z enzymes from B. subtilis and Thermotoga maritima, but there are distinct differences in metal binding and overall domain organization. Unlike the previously described homologous structures, ZiPD dimers display crystallographic symmetry and fully loaded metal sites. The ZiPD exosite is similar to that of the B. subtilis enzyme structurally, but its position with respect to the protein core differs substantially, illustrating its ability to act as a clamp in binding tRNA. Furthermore, the ZiPD crystal structure presented here provides insight into the enzyme's cooperativity and assists the ongoing attempt to elucidate the physiological function of this protein.
Asunto(s)
Escherichia coli/enzimología , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/química , Secuencia de Aminoácidos , Cristalografía , Datos de Secuencia Molecular , Hidrolasas Diéster Fosfóricas/genética , Alineación de SecuenciaRESUMEN
Escherichia coli ZiPD is the best characterized protein encoded by the elaC gene family and is a model for the 3'-pre-tRNA processing endoribonucleases (tRNase Z). A metal ligand-based sequence alignment of ZiPD with metallo-beta-lactamase domain proteins of known crystallographic structure identifies a ZiPD-specific sequence insertion of approximately 50 residues, which we will refer to as the ZiPD exosite. Functionally characterized ZiPD homologs from Bacillus subtilis, Methanococcus janaschii, and human share the presence of the ZiPD exosite, which is also present in the amino-terminal, but not in the carboxyl-terminal, domain of ElaC2 proteins. Another class of functionally characterized tRNase Z enzymes from Thermotoga maritima and Arabidopsis thaliana lack characteristic motifs in the exosite but possess a sequence segment with clustered basic amino acid residues. As an experimental attempt to investigate the function of the exosite we constructed a ZiPD variant that lacks this module (ZiPDDelta). ZiPDDelta has almost wild-type-like catalytic properties for hydrolysis of the small, chromogenic substrate bis(p-nitrophenyl) phosphate. Removal of the ZiPD exosite only affects k(cat), which is reduced by less than 40%, whereas both K' andthe Hill coefficient (measures of the substrate affinity and cooperativity, respectively) remain unchanged. Hence, the exosite is not required for the intrinsic phosphodiesterase activity of ZiPD. Removal of the exosite also does not affect the dimerization properties of ZiPD. In contrast to the wild-type enzyme, ZiPDDelta does not process pre-tRNA, and gel shift assays demonstrate that only the wild-type enzyme, but not ZiPDDelta, binds mature tRNA. These findings show that the exosite is essential for pre-tRNA recognition. In conclusion, we identify a ZiPD exosite that guides physiological substrate recognition in the ZiPD/ElaC protein family.