Positional Cloning of a Type 2 Diabetes Quantitative Trait Locus; Tomosyn-2, a Negative Regulator of Insulin Secretion

We previously mapped a type 2 diabetes (T2D) locus on chromosome 16 (Chr 16) in an F2 intercross from the BTBR T (+) tf (BTBR) Lepob/ob and C57BL/6 (B6) Lepob/ob mouse strains. Introgression of BTBR Chr 16 into B6 mice resulted in a consomic mouse with reduced fasting plasma insulin and elevated glucose levels. We derived a panel of sub-congenic mice and narrowed the diabetes susceptibility locus to a 1.6 Mb region. Introgression of this 1.6 Mb fragment of the BTBR Chr 16 into lean B6 mice (B6.16BT36–38) replicated the phenotypes of the consomic mice. Pancreatic islets from the B6.16BT36–38 mice were defective in the second phase of the insulin secretion, suggesting that the 1.6 Mb region encodes a regulator of insulin secretion. Within this region, syntaxin-binding protein 5-like (Stxbp5l) or tomosyn-2 was the only gene with an expression difference and a non-synonymous coding single nucleotide polymorphism (SNP) between the B6 and BTBR alleles. Overexpression of the b-tomosyn-2 isoform in the pancreatic β-cell line, INS1 (832/13), resulted in an inhibition of insulin secretion in response to 3 mM 8-bromo cAMP at 7 mM glucose. In vitro binding experiments showed that tomosyn-2 binds recombinant syntaxin-1A and syntaxin-4, key proteins that are involved in insulin secretion via formation of the SNARE complex. The B6 form of tomosyn-2 is more susceptible to proteasomal degradation than the BTBR form, establishing a functional role for the coding SNP in tomosyn-2. We conclude that tomosyn-2 is the major gene responsible for the T2D Chr 16 quantitative trait locus (QTL) we mapped in our mouse cross. Our findings suggest that tomosyn-2 is a key negative regulator of insulin secretion

Anaplasma phagocytophilum APH0032 is exposed on the cytosolic face of the pathogen-occupied vacuole and co-opts host cell SUMOylation

Anaplasma phagocytophilum, a member of the family Anaplasmataceae and the obligate intracellular bacterium that causes granulocytic anaplasmosis, resides in a host cell-derived vacuole. Bacterial proteins that localize to the A. phagocytophilum-occupied vacuole membrane (AVM) are critical host-pathogen interfaces. Of the few bacterial AVM proteins that have been identified, the domains responsible for AVM localization and the host cell pathways that they co-opt are poorly defined. APH0032 is an effector that is expressed and localizes to the AVM late during the infection cycle. Herein, the APH0032 domain that is essential for associating with host cell membranes was mapped. Immunofluorescent labeling of infected cells that had been differentially permeabilized confirmed that APH0032 is exposed on the AVM’s cytosolic face, signifying its potential to interface with host cell processes. SUMOylation is the covalent attachment of a member of the small ubiquitin-like modifier (SUMO) family of proteins to lysines in target substrates. Previous work from our laboratory determined that SUMOylation is important for A. phagocytophilum survival and that SUMOylated proteins decorate the AVM. Algorithmic prediction analyses identified APH0032 as a candidate for SUMOylation. Endogenous APH0032 was precipitated from infected cells using a SUMO affinity matrix, confirming that the effector co-opts SUMOylation during infection. APH0032 pronouncedly colocalized with SUMO1, but not SUMO2/3 moieties on the AVM. Ectopic expression of APH0032 in A. phagocytophilum infected host cells significantly boosted the bacterial load. This study delineates the first domain of any Anaplasmataceae protein that is essential for associating with the pathogen-occupied vacuole membrane, demonstrates the importance of APH0032 to infection, and identifies it as the second A. phagocytophilum effector that co-opts SUMOylation, thus underscoring the relevance of this post-translational modification to infection

Anaplasma phagocytophilum APH0032 Is Exposed on the Cytosolic Face of the Pathogen-Occupied Vacuole and Co-opts Host Cell SUMOylation.

Anaplasma phagocytophilum, a member of the family Anaplasmataceae and the obligate intracellular bacterium that causes granulocytic anaplasmosis, resides in a host cell-derived vacuole. Bacterial proteins that localize to the A. phagocytophilum-occupied vacuole membrane (AVM) are critical host-pathogen interfaces. Of the few bacterial AVM proteins that have been identified, the domains responsible for AVM localization and the host cell pathways that they co-opt are poorly defined. APH0032 is an effector that is expressed and localizes to the AVM late during the infection cycle. Herein, the APH0032 domain that is essential for associating with host cell membranes was mapped. Immunofluorescent labeling of infected cells that had been differentially permeabilized confirmed that APH0032 is exposed on the AVM's cytosolic face, signifying its potential to interface with host cell processes. SUMOylation is the covalent attachment of a member of the small ubiquitin-like modifier (SUMO) family of proteins to lysines in target substrates. Previous work from our laboratory determined that SUMOylation is important for A. phagocytophilum survival and that SUMOylated proteins decorate the AVM. Algorithmic prediction analyses identified APH0032 as a candidate for SUMOylation. Endogenous APH0032 was precipitated from infected cells using a SUMO affinity matrix, confirming that the effector co-opts SUMOylation during infection. APH0032 pronouncedly colocalized with SUMO1, but not SUMO2/3 moieties on the AVM. Ectopic expression of APH0032 in A. phagocytophilum infected host cells significantly boosted the bacterial load. This study delineates the first domain of any Anaplasmataceae protein that is essential for associating with the pathogen-occupied vacuole membrane, demonstrates the importance of APH0032 to infection, and identifies it as the second A. phagocytophilum effector that co-opts SUMOylation, thus underscoring the relevance of this post-translational modification to infection