The Deubiquitylase MATH-33 Controls DAF-16 Stability and Function in Metabolism and Longevity

SummaryFOXO family transcription factors are downstream effectors of Insulin/IGF-1 signaling (IIS) and major determinants of aging in organisms ranging from worms to man. The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response, and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity

Clinical and Functional Characterization of a Patient Carrying a Compound Heterozygous Pericentrin Mutation and a Heterozygous IGF1 Receptor Mutation

Intrauterine and postnatal longitudinal growth is controlled by a strong genetic component that regulates a complex network of endocrine factors integrating them with cellular proliferation, differentiation and apoptotic processes in target tissues, particularly the growth centers of the long bones. Here we report on a patient born small for gestational age (SGA) with severe, proportionate postnatal growth retardation, discreet signs of skeletal dysplasia, microcephaly and moyamoya disease. Initial genetic evaluation revealed a novel heterozygous IGF1R p.Leu1361Arg mutation affecting a highly conserved residue with the insulin-like growth factor type 1 receptor suggestive for a disturbance within the somatotropic axis. However, because the mutation did not co-segregate with the phenotype and functional characterization did not reveal an obvious impairment of the ligand depending major IGF1R signaling capabilities a second-site mutation was assumed. Mutational screening of components of the somatotropic axis, constituents of the IGF signaling system and factors involved in cellular proliferation, which are described or suggested to provoke syndromic dwarfism phenotypes, was performed. Two compound heterozygous PCNT mutations (p.[Arg585X];[Glu1774X]) were identified leading to the specification of the diagnosis to MOPD II. These investigations underline the need for careful assessment of all available information to derive a firm diagnosis from a sequence aberration

Designer Lipid-Like Peptides: A Class of Detergents for Studying Functional Olfactory Receptors Using Commercial Cell-Free Systems

A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.United States. Defense Advanced Research Projects Agency (DARPA-HR0011-09-C-0012)Massachusetts Institute of Technology. Undergraduate Research Opportunities Progra

Liposome-Mediated Cellular Delivery of Active gp91phox

International audienceBACKGROUND: Gp91(phox) is a transmembrane protein and the catalytic core of the NADPH oxidase complex of neutrophils. Lack of this protein causes chronic granulomatous disease (CGD), a rare genetic disorder characterized by severe and recurrent infections due to the incapacity of phagocytes to kill microorganisms. METHODOLOGY: Here we optimize a prokaryotic cell-free expression system to produce integral mammalian membrane proteins. CONCLUSIONS: Using this system, we over-express truncated forms of the gp91(phox) protein under soluble form in the presence of detergents or lipids resulting in active proteins with a "native-like" conformation. All the proteins exhibit diaphorase activity in the presence of cytosolic factors (p67(phox), p47(phox), p40(phox) and Rac) and arachidonic acid. We also produce proteoliposomes containing gp91(phox) protein and demonstrate that these proteins exhibit activities similar to their cellular counterpart. The proteoliposomes induce rapid cellular delivery and relocation of recombinant gp91(phox) proteins to the plasma membrane. Our data support the concept of cell-free expression technology for producing recombinant proteoliposomes and their use for functional and structural studies or protein therapy by complementing deficient cells in gp91(phox) protein

Novel Regulatory Mechanisms for Generation of the Soluble Leptin Receptor: Implications for Leptin Action

The adipokine leptin realizes signal transduction via four different membrane-anchored leptin receptor (Ob-R) isoforms in humans. However, the amount of functionally active Ob-R is affected by constitutive shedding of the extracellular domain via a so far unknown mechanism. The product of the cleavage process the so-called soluble leptin receptor (sOb-R) is the main binding protein for leptin in human blood and modulates its bioavailability. sOb-R levels are differentially regulated in metabolic disorders like type 1 diabetes mellitus or obesity and can, therefore, enhance or reduce leptin sensitivity.To describe mechanisms of Ob-R cleavage and to investigate the functional significance of differential sOb-R levels we established a model of HEK293 cells transiently transfected with different human Ob-R isoforms. Using siRNA knockdown experiments we identified ADAM10 (A Disintegrin And Metalloproteinase 10) as a major protease for constitutive and activated Ob-R cleavage. Additionally, the induction of lipotoxicity and apoptosis led to enhanced shedding shown by increased levels of the soluble leptin receptor (sOb-R) in cell supernatants. Conversely, high leptin concentrations and ER stress reduced sOb-R levels. Decreased amounts of sOb-R due to ER stress were accompanied by impaired leptin signaling and reduced leptin binding.Lipotoxicity and apoptosis increased Ob-R cleavage via ADAM10-dependent mechanisms. In contrast high leptin levels and ER stress led to reduced sOb-R levels. While increased sOb-R concentrations seem to directly block leptin action, reduced amounts of sOb-R may reflect decreased membrane expression of Ob-R. These findings could explain changes of leptin sensitivity which are associated with variations of serum sOb-R levels in metabolic diseases

The eukaryotic CO2-concentrating organelle is liquid-like and exhibits dynamic reorganization

Approximately 30%–40% of global CO 2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO 2-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a “magic number” effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles

Evaluation of detergents for the soluble expression of α-helical and β-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system

Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation steps. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial alpha-helical multidrug transporter, EmrE, the beta-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a beta-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent