56 research outputs found
Hydroxycitric Acid Inhibits Chronic Myelogenous Leukemia Growth through Activation of AMPK and mTOR Pathway
No full textMetabolic regulation of cancer cell growth via AMP-activated protein kinase (AMPK) activation is a widely studied strategy for cancer treatment, including leukemias. Recent notions that naturally occurring compounds might have AMPK activity led to the search for nutraceuticals with potential AMPK-stimulating activity. We found that hydroxycitric acid (HCA), a natural, safe bioactive from the plant Garcinia gummi-gutta (cambogia), has potent AMPK activity in chronic myelogenous leukemia (CML) cell line K562. HCA is a known competitive inhibitor of ATP citrate lyase (ACLY) and is widely used as a weight loss inducer. We found that HCA was able to inhibit the growth of K562 cells in in vitro and in vivo xenograft models. At the mechanistic level, we identified a direct interaction between AMPK and ACLY that seems to be sensitive to HCA treatment. Additionally, HCA treatment resulted in the co-activation of AMPK and the mammalian target of rapamycin (mTOR) pathways. Moreover, we found an enhanced unfolded protein response as observed by activation of the eIF2α/ATF4 pathway that could explain the induction of cell cycle arrest at the G2/M phase and DNA fragmentation upon HCA treatment in K562 cells. Overall, these findings suggest HCA as a nutraceutical approach for the treatment of CMLs
Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation
No full textSeveral mechanisms have been proposed for the synthesis of substrate-linked ubiquitin chains. HECT ligases directly catalyse protein ubiquitination and have been found to non-covalently interact with ubiquitin. We report crystal structures of the Nedd4 HECT domain, alone and in complex with ubiquitin, which show a new binding mode involving two surfaces on ubiquitin and both subdomains of the HECT N-lobe. The structures suggest a model for HECT-to-substrate ubiquitin transfer, in which the growing chain on the substrate is kept close to the catalytic cysteine to promote processivity. Mutational analysis highlights differences between the processes of substrate polyubiquitination and self-ubiquitination
Structure of a ubiquitin-loaded HECT ligase reveals the molecular basis for catalytic priming
No full textHomologous to E6-AP C terminus (HECT) E3 ligases recognize and directly catalyze ligation of ubiquitin (Ub) to their substrates. Molecular details of this process remain unknown. We report the first structure, to our knowledge, of a Ub-loaded E3, the human neural precursor cell-expressed developmentally downregulated protein 4 (Nedd4). The HECT(Nedd4)~Ub transitory intermediate provides a structural basis for the proposed sequential addition mechanism. The donor Ub, transferred from the E2, is bound to the Nedd4 C lobe with its C-terminal tail locked in an extended conformation, primed for catalysis. We provide evidence that the Nedd4-family members are Lys63-specific enzymes whose catalysis is mediated by an essential C-terminal acidic residue
Crystallographic Evidence for Substrate-Assisted GTP Hydrolysis by a Small GTP Binding Protein
No full textSummaryGTP hydrolysis by small GTP binding proteins of the Ras superfamily is a universal reaction that controls multiple cellular regulations. Its enzymic mechanism has been the subject of long-standing debates as to the existence/identity of the general base and the electronic nature of its transition state. Here we report the high-resolution crystal structure of a small GTP binding protein, Rab11, solved in complex with GDP and Pi. Unexpectedly, a Pi oxygen and the GDP-cleaved oxygen are located less than 2.5 Å apart, suggesting that they share a proton, likely in the form of a low-barrier hydrogen bond. This implies that the γ-phosphate of GTP was protonated; hence, that GTP acts as a general base. Furthermore, this interaction should establish at, and stabilize, the transition state. Altogether, we propose a revised model for the GTPase reaction that should reconcile earlier models into a unique substrate-assisted mechanism
Etudes structurales de Arf6 et Rab11a (deux petites protéines G impliquées dans la régulation du trafic intracellulaire)
No full textLe transport de protéines et membranes entre les différents compartiments de la cellule est régulé par les petites protéines G des familles Arf et Rab. Comme les autres protéines G, elles basculent entre une forme inactive liée au GDP et une active liée au GTP, et fonctionnent ainsi comme des interrupteurs moléculaires. Les diverses protéines Arf et Rab sont spécifiquement associées à des voies cellulaires distinctes et se localisent sur des compartiments cellulaires spécifiques. Nous avons étudié les bases de ces spécificités d'action grâce à la résolution des cycles structuraux GDP/GTP de deux de ces protéines, Arf6 et Rab11a. Les fonctions cellulaires de Arf6 sont différentes de celles de Arf1, malgré le fait que ces deux protéines partagent 67% d'identité de séquence. Nous avons résolu la structure cristalline de la forme active de Arf6. De façon surprenante, alors que les formes inactives de Arf1 et Arf6 peuvent être distinguées en structure, leurs formes actives apparaissent remarquablement similaires. En particulier, leurs régions switch sont non seulement presque identiques en séquence, mais présentent aussi la même structure. Nous proposons que la spécificité d'action des protéines Arf soit basée sur des interactions (i) de leurs formes liées au GDP avec leurs partenaires spécifiques et/ou (ii) qui impliquent outre aux régions switch classiques (qui portent l'information sur le nucléotide lié) des régions spécifiques aux différentes protéines Arf. Les structures de Rab11a dans sa forme active et inactive montrent des particularités distinctives de cette protéine G. Rab11aGDP cristallise comme un dimère. Nous suggérons que ce dimère puisse exister in vivo, et représenter ainsi un pool associé à la membrane sous forme GDP. D'autre part, la structure de Rab11aGTP permet l'identification d'une surface formée de résidus hautement variables entre protéines Rab. Nous proposons que cette surface puisse être une région de reconnaissance spécifique des protéines Rab.Proteins and membranes are transported between the different compartments of the cell through processes that are regulated by small GTP-binding proteins of the Arf and Rab families. Like other small G proteins, they function as molecular switches, cycling between an inactive GDP-bound form and an active GTP-bound form, which differ in structure in their 'switch regions'. The diverse Arf and Rab proteins are specifically associated with distinct cellular pathways and localize to precise cellular compartments. We have investigated the bases of these specificities of action by elucidating the GDP/GTP structural cycle of two of these proteins, Arf6 and Rab11a. We have solved the crystal structure of the active form of full length human Arf6, which displays cellular functions unrelated to that of well-characterized Arfl, despite sharing with it ~670% sequence identity. We round that, while Arfl and Arf6 GDP-bound forms are structurally recognizable, their active forms are remarkably similar in structure. Moreover, their switch regions are not only almost identical in sequence, but also fold in the same structure. Therefore, we propose that specificity of action of Arf proteins is based on (i) interactions of their GDP-bound forms with specific partners and/or (ii) interactions that involve both the classical switch regions (that provide information on the bound nucleotide) and Arf-specific regions. Rab11a is a member of a previously structurally uncharacterized Rab protein subfamily. The structures of human Rab11a in its inactive and active forms reveal features peculiar to this Rab subfamily. Rab11a-GDP crystallizes as a dimer. We suggest that this dimer could exist in vivo, and represent a membrane-associated GDP-bound pool. On the other hand Rab11a-GTP structure allows the identification of a surface shaped by residues highly variable between Rab proteins. We propose that this area could be a putative region for Rab protein specific recognition.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF
The MIS12 complex is a protein interaction hub for outer kinetochore assembly.
No full textKinetochores are nucleoprotein assemblies responsible for the attachment of chromosomes to spindle microtubules during mitosis. The KMN network, a crucial constituent of the outer kinetochore, creates an interface that connects microtubules to centromeric chromatin. The NDC80, MIS12, and KNL1 complexes form the core of the KMN network. We recently reported the structural organization of the human NDC80 complex. In this study, we extend our analysis to the human MIS12 complex and show that it has an elongated structure with a long axis of similar to 22 nm. Through biochemical analysis, cross-linking-based methods, and negative-stain electron microscopy, we investigated the reciprocal organization of the subunits of the MIS12 complex and their contacts with the rest of the KMN network. A highlight of our findings is the identification of the NSL1 subunit as a scaffold supporting interactions of the MIS12 complex with the NDC80 and KNL1 complexes. Our analysis has important implications for understanding kinetochore organization in different organisms
Structural and Functional Framework for the Autoinhibition of Nedd4-Family Ubiquitin Ligases
No full textSummaryNedd4-family ubiquitin ligases are key regulators of cell surface receptor signaling. Their dysregulation is associated with several human diseases, including cancer. Under normal conditions, the activity of various Nedd4 E3s is controlled through an autoinhibitory interaction of the N-terminal C2 domain with the C-terminal catalytic HECT domain. Here, we report the structural and functional framework for this intramolecular interaction. Our nuclear magnetic resonance (NMR) data and biochemical analyses on Smurf2 and Nedd4 show that the C2 domain has the potential to regulate E3 activity by maintaining the HECT domain in a low-activity state where its ability for transthiolation and noncovalent Ub binding are impaired
GTP-binding proteins with a structural device for ‘front–back ’ communication
No full textArf proteins are important regulators of cellular traffic and the founding members of an expanding family of homologous proteins and genomic sequences. They depart from other small GTP-binding proteins by a unique structural device, which we call the ‘interswitch toggle’, that implements front– back communication from the N-terminus to the nucleotide binding site. Here we define the sequence and structural determinants that propagate information across the protein and identify them in all of the Arf family proteins other than Arl6 and Arl4/Arl7. The positions of these determinants lead us to propose that Arf family members with the interswitch toggle device are activated by a bipartite mechanism acting on oppo-site sides of the protein. The presence of this communication device might provide a more useful basis for unifying Arf homologs as a family than do the cellular functions of these proteins, which are mostly unrelated. We review available genomic sequences and functional data from this perspective, and identify a novel subfamily that we call Arl8
Purification and characterization of a DNA-binding recombinant PREP1:PBX1 complex.
No full textHuman PREP1 and PBX1 are homeodomain transcriptional factors, whose biochemical and structural characterization has not yet been fully described. Expression of full-length recombinant PREP1 (47.6 kDa) and PBX1 (46.6 kDa) in E. coli is difficult because of poor yield, high instability and insufficient purity, in particular for structural studies. We cloned the cDNA of both proteins into a dicistronic vector containing an N-terminal glutathione S-transferase (GST) tag and co-expressed and co-purified a stable PBX1:PREP1 complex. For structural studies, we produced two C-terminally truncated complexes that retain their ability to bind DNA and are more stable than the full-length proteins through various purification steps. Here we report the production of large amounts of soluble and pure recombinant human PBX1:PREP1 complex in an active form capable of binding DNA
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