Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.     

Chromosome cohesion is regulated by a clock gene paralogue TIM-1

Faithful transmission of the genome requires that a protein complex called cohesin establishes and maintains the regulated linkage between replicated chromosomes before their segregation. Here we report the unforeseen participation of Caenorhabditis elegans TIM-1, a paralogue of the Drosophila clock protein TIMELESS, in the regulation of chromosome cohesion. Our biochemical experiments defined the C. elegans cohesin complex and revealed its physical association with TIM-1. Functional relevance of the interaction was demonstrated by aberrant mitotic chromosome behaviour, embryonic lethality and defective meiotic chromosome cohesion caused by the disruption of either TIM-1 or cohesin. TIM-1 depletion prevented the assembly of non-SMC (structural maintenance of chromosome) cohesin subunits onto meiotic chromosomes; however, unexpectedly, a partial cohesin complex composed of SMC components still loaded. Further disruption of cohesin activity in meiosis by the simultaneous depletion of TIM-1 and an SMC subunit decreased homologous chromosome pairing before synapsis, revealing a new role for cohesin in metazoans. On the basis of comparisons between TIMELESS homologues in worms, flies and mice, we propose that chromosome cohesion, rather than circadian clock regulation, is the ancient and conserved function for TIMELESS-like proteins.     

Stem cell division is regulated by the microRNA pathway

One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. Here, we report the necessity of the microRNA (miRNA) pathway for proper control of germline stem cell (GSC) division in Drosophila melanogaster. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, we conclude that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle at the G1/S transition.     

Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities

The spindle checkpoint prevents chromosome mis-segregation by delaying sister chromatid separation until all chromosomes have achieved bipolar attachment to the mitotic spindle. Its operation is essential for accurate chromosome segregation, whereas its dysregulation can contribute to birth defects and tumorigenesis. The target of the spindle checkpoint is the anaphase-promoting complex (APC), a ubiquitin ligase that promotes sister chromatid separation and progression to anaphase. Using a short hairpin RNA screen targeting components of the ubiquitin-proteasome pathway in human cells, we identified the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) as a critical regulator of the spindle checkpoint. USP44 is not required for the initial recognition of unattached kinetochores and the subsequent recruitment of checkpoint components. Instead, it prevents the premature activation of the APC by stabilizing the APC-inhibitory Mad2-Cdc20 complex. USP44 deubiquitinates the APC coactivator Cdc20 both in vitro and in vivo, and thereby directly counteracts the APC-driven disassembly of Mad2-Cdc20 complexes (discussed in an accompanying paper). Our findings suggest that a dynamic balance of ubiquitination by the APC and deubiquitination by USP44 contributes to the generation of the switch-like transition controlling anaphase entry, analogous to the way that phosphorylation and dephosphorylation of Cdk1 by Wee1 and Cdc25 controls entry into mitosis.     

Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in obesity

A subset of neurons in the brain, known as 'glucose-excited' neurons, depolarize and increase their firing rate in response to increases in extracellular glucose. Similar to insulin secretion by pancreatic beta-cells, glucose excitation of neurons is driven by ATP-mediated closure of ATP-sensitive potassium (K(ATP)) channels. Although beta-cell-like glucose sensing in neurons is well established, its physiological relevance and contribution to disease states such as type 2 diabetes remain unknown. To address these issues, we disrupted glucose sensing in glucose-excited pro-opiomelanocortin (POMC) neurons via transgenic expression of a mutant Kir6.2 subunit (encoded by the Kcnj11 gene) that prevents ATP-mediated closure of K(ATP) channels. Here we show that this genetic manipulation impaired the whole-body response to a systemic glucose load, demonstrating a role for glucose sensing by POMC neurons in the overall physiological control of blood glucose. We also found that glucose sensing by POMC neurons became defective in obese mice on a high-fat diet, suggesting that loss of glucose sensing by neurons has a role in the development of type 2 diabetes. The mechanism for obesity-induced loss of glucose sensing in POMC neurons involves uncoupling protein 2 (UCP2), a mitochondrial protein that impairs glucose-stimulated ATP production. UCP2 negatively regulates glucose sensing in POMC neurons. We found that genetic deletion of Ucp2 prevents obesity-induced loss of glucose sensing, and that acute pharmacological inhibition of UCP2 reverses loss of glucose sensing. We conclude that obesity-induced, UCP2-mediated loss of glucose sensing in glucose-excited neurons might have a pathogenic role in the development of type 2 diabetes.     

The orthodenticle gene is regulated by bicoid and torso and specifies Drosophila head development

In the Drosophila embryo, cell fate along the anterior-posterior axis is determined by maternally expressed genes. The activity of the bicoid (bcd) gene is required for the development of larval head and thoracic structures, and that of maternal torso (tor) for the development of the unsegmented region of the head (acron). In contrast to the case of thoracic and abdominal segmentation, the hierarchy of zygotically expressed genes controlling head development has not been clearly defined. The bcd protein, which is expressed in a gradient, activates zygotic expression of the gap gene hunchback (hb), but hb alone is not sufficient to specify head development. Driever et al. proposed that at least one other bcd-activated gene controls the development of head regions anterior to the hb domain. We report here that the homeobox gene orthodenticle (otd), which is involved in head development, could be such a gene. We also show that otd expression responds to the activity of the maternal tor gene at the anterior pole of the embryo.     

Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1

The evolutionary conservation of T lymphocyte subsets bearing T-cell receptors (TCRs) using invariant alpha-chains is indicative of unique functions. CD1d-restricted natural killer T (NK-T) cells that express an invariant Valpha14 TCRalpha chain have been implicated in microbial and tumour responses, as well as in auto-immunity. Here we show that T cells that express the canonical hValpha7.2-Jalpha33 or mValpha19-Jalpha33 TCR rearrangement are preferentially located in the gut lamina propria of humans and mice, respectively, and are therefore genuine mucosal-associated invariant T (MAIT) cells. Selection and/or expansion of this population requires B lymphocytes, as MAIT cells are absent in B-cell-deficient patients and mice. In addition, we show that MAIT cells are selected and/or restricted by MR1, a monomorphic major histocompatibility complex class I-related molecule that is markedly conserved in diverse mammalian species. MAIT cells are not present in germ-free mice, indicating that commensal flora is required for their expansion in the gut lamina propria. This indicates that MAIT cells are probably involved in the host response at the site of pathogen entry, and may regulate intestinal B-cell activity.     

原始蛋白结构与功能研究进展

近年来在生命起源研究方面,蛋白质起源研究来有很大进展,因为各种“组学（-omics）”的快速发展提供了关于蛋白质结构与功能的大量信息,据此可以初步推断原始蛋白的结构与功能特征．文章就该领域的最新研究进展进行了综述．     

Pattern formation in the developing eye of Drosophila melanogaster is regulated by the homoeo-box gene, rough

Homoeo-box genes play a central role in the regulation of embryogenesis in Drosophila melanogaster. Their widespread phylogenetic distribution, and the tissue and stage specificity of their expression in other organisms, argue that they play a general and significant role in animal development. In D. melanogaster, all homoeo-box genes characterized to date are involved in major aspects of embryogenesis. We report here the molecular characterization of a Drosophila homoeo-box gene that has no apparent involvement in early embryogenesis. The gene appears to be rough, a gene implicated in pattern formation in the developing eye. It is expressed in cells within, and posterior to, the morphogenetic furrow, the site of the primary pattern forming events in the developing retina, and also in a region of the brain of the third instar larva. We have found no genetic or molecular evidence of a role for this gene in other aspects of fly development.     

MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin.

AGONISTS that stimulate protein kinase C (PKC) induce profound changes in cell morphology correlating with the reorganization of submembranous actin, but no direct connection between PKC and actin assembly has been identified. The myristoylated, alanine-rich C kinase substrate (MARCKS) binds calmodulin and is a predominant, specific substrate of PKC which is phosphorylated during macrophage and neutrophil activation , growth factor-dependent mitogenesis and neurosecretion; it is redistributed from plasma membrane to cytoplasm when phosphorylated and is involved in leukocyte motility. Here we report that MARCKS is a filamentous (F) actin crosslinking protein, with activity that is inhibited by PKC-mediated phosphorylation and by binding to calcium-calmodulin. MARCKS may be a regulated crossbridge between actin and the plasma membrane, and modulation of the actin crosslinking activity of the MARCKS protein by calmodulin and phosphorylation represents a potential convergence of the calcium-calmodulin and PKC signal transduction pathways in the regulation of the actin cytoskeleton.     

Somite-specific expression of a novel fibronectin variant FN3 is negatively regulated by SHH

Fibronectins (FNs) are major extracellular proteins in blood plasma and many tissues of vertebrates, and play important roles in adhesion, migration and differentiation of cells. We have identified a novel variant (FN3) of fibronectin in zebrafish. FN3 mRNA is abundant, as detected by whole-mount in situ hybridization, in the presomitic mesoderm and the newly formed somites, but less abundant in mature somites. Ectopic expression of Sonic Hedgehog (SHH) results in a decrease of FN3 expression, whereas the expression level of FN3 increases in the flh mutants that lack the notochord. Our results suggest that FN3 may be involved in the formation of somites, but during somite differentiation its expression needs to be downregulated by signals derived from the axial tissues.     

Surface expression of MHC class II in dendritic cells is controlled by regulated ubiquitination

Dendritic cells have a unique function in the immune response owing to their ability to stimulate immunologically naive T lymphocytes. In response to microbial and inflammatory stimuli, dendritic cells enhance their capacity for antigen presentation by a process of terminal differentiation, termed maturation. The conversion of immature to mature dendritic cells is accompanied by a marked cellular reorganization, including the redistribution of major histocompatibility complex class II molecules (MHC II) from late endosomal and lysosomal compartments to the plasma membrane and the downregulation of some forms of endocytosis, which has been thought to slow the clearance of MHC II from the surface. The relative extent to which these or other mechanisms contribute to the regulation of surface MHC II remains unclear, however. Here we find that the MHC II beta-chain cytoplasmic tail is ubiquitinated in mouse immature dendritic cells. Although only partly required for the sequestration of MHC II in multivesicular bodies, this modification is essential for endocytosis. Notably, ubiquitination of MHC II ceased upon maturation, resulting in the accumulation of MHC II at the cell surface. Dendritic cells thus exhibit a unique ability to regulate MHC II surface expression by selectively controlling MHC II ubiquitination.     

Computational design of receptor and sensor proteins with novel functions

The formation of complexes between proteins and ligands is fundamental to biological processes at the molecular level. Manipulation of molecular recognition between ligands and proteins is therefore important for basic biological studies and has many biotechnological applications, including the construction of enzymes, biosensors, genetic circuits, signal transduction pathways and chiral separations. The systematic manipulation of binding sites remains a major challenge. Computational design offers enormous generality for engineering protein structure and function. Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities. This method was used to construct soluble receptors that bind trinitrotoluene, l-lactate or serotonin with high selectivity and affinity. These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or l-lactate. The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally. The biological and biosensing activities of the designed receptors illustrate potential applications of computational design.     

p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage.

The protein p73 is a structural and functional homologue of the p53 tumour-suppressor protein but, unlike p53, it is not induced in response to DNA damage. The tyrosine kinase c-Abl is activated by certain DNA-damaging agents and contributes to the induction of programmed cell death (apoptosis) by p53-dependent and p53-independent mechanisms. Here we show that c-Abl binds to p73 in cells, interacting through its SH3 domain with the carboxy-terminal homo-oligomerization domain of p73. c-Abl phosphorylates p73 on a tyrosine residue at position 99 both in vitro and in cells that have been exposed to ionizing radiation. Our results show that c-Abl stimulates p73-mediated transactivation and apoptosis. This regulation of p73 by c-Abl in response to DNA damage is also demonstrated by a failure of ionizing-radiation-induced apoptosis after disruption of the c-Abl-p73 interaction. These findings show that p73 is regulated by a c-Abl-dependent mechanism and that p73 participates in the apoptotic response to DNA damage.     

由算子I_(n+p-1)定义的解析函数的新子类

利用算子In p-1刻划了强星形函数和强凸函数的新子类Sn*,p(β,γ)和Cn,p(β,γ),建立了包含关系和函数在积分算子Lc(f)作用下的保持关系.     

The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation) and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.