Rescue of Familial Lecithin:Cholesterol Acyltranferase Deficiency Mutations with an Allosteric Activator.

Lecithin:cholesterol acyltransferase (LCAT) deficiencies represent severe disorders characterized by aberrant cholesterol esterification in plasma, leading to life-threatening conditions. This study investigates the efficacy of Compound 2, a piperidinyl pyrazolopyridine allosteric activator that binds the membrane-binding domain of LCAT, in rescuing the activity of LCAT variants associated with disease. The variants K218N, N228K, and G230R, all located in the cap and lid domains of LCAT, demonstrated notable activity restoration in response to Compound 2.

Structural Analysis of Membrane-associated Forms of Helicobacter pylori VacA Toxin.

Helicobacter pylori colonizes the stomach in about half of the human population, leading to an increased risk of peptic ulcer disease and gastric cancer. H. pylori secretes an 88 kDa VacA toxin that contributes to pathogenesis.

Comparative Biochemical Studies of Disease-Associated Human Dicer Mutations on Processing of a Pre-microRNA and snoRNA.

Dicer is an RNase III enzyme that is responsible for the maturation of small RNAs such as microRNAs. As Dicer's cleavage products play key roles in promoting cellular homeostasis through the fine-tuning of gene expression, dysregulation of Dicer activity can lead to several human diseases, including cancers. Mutations in Dicer have been found to induce tumorigenesis and lead to the development of a rare pleiotropic tumor predisposition syndrome found in children and young adults called DICER1 syndrome.

Structural analysis of the P132L disease mutation in caveolin-1 reveals its role in the assembly of oligomeric complexes.

Caveolin-1 (CAV1) is a membrane-sculpting protein that oligomerizes to generate flask-shaped invaginations of the plasma membrane known as caveolae. Mutations in CAV1 have been linked to multiple diseases in humans. Such mutations often interfere with oligomerization and the intracellular trafficking processes required for successful caveolae assembly, but the molecular mechanisms underlying these defects have not been structurally explained.

Cryo-EM reveals new species-specific proteins and symmetry elements in the Dot/Icm T4SS.

is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.

N-terminal Transmembrane-Helix Epitope Tag for X-ray Crystallography and Electron Microscopy of Small Membrane Proteins.

Structural studies of membrane proteins, especially small membrane proteins, are associated with well-known experimental challenges. Complexation with monoclonal antibody fragments is a common strategy to augment such proteins; however, generating antibody fragments that specifically bind a target protein is not trivial. Here we identify a helical epitope, from the membrane-proximal external region (MPER) of the gp41-transmembrane subunit of the HIV envelope protein, that is recognized by several well-characterized antibodies and that can be fused as a contiguous extension of the N-terminal transmembrane helix of a broad range of membrane proteins.

Lipid-based vaccine nanoparticles for induction of humoral immune responses against HIV-1 and SARS-CoV-2.

The current health crisis of corona virus disease 2019 (COVID-19) highlights the urgent need for vaccine systems that can generate potent and protective immune responses. Protein vaccines are safe, but conventional approaches for protein-based vaccines often fail to elicit potent and long-lasting immune responses. Nanoparticle vaccines designed to co-deliver protein antigens and adjuvants can promote their delivery to antigen-presenting cells and improve immunogenicity.

The δ-opioid receptor positive allosteric modulator BMS 986187 is a G-protein-biased allosteric agonist.

Background And Purpose: The δ-opioid receptor is an emerging target for the management of chronic pain and depression. Biased signalling, the preferential activation of one signalling pathway over another downstream of δ-receptors, may generate better therapeutic profiles. BMS 986187 is a positive allosteric modulator of δ-receptors.

Molecular basis for activation of lecithin:cholesterol acyltransferase by a compound that increases HDL cholesterol.

Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating compounds are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of human LCAT in complex with a potent piperidinylpyrazolopyridine activator and an acyl intermediate-like inhibitor, revealing LCAT in an active conformation. Unlike other LCAT activators, the piperidinylpyrazolopyridine activator binds exclusively to the membrane-binding domain (MBD).

Design, synthesis, and biological activity of substituted 2-amino-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid derivatives as inhibitors of the inflammatory kinases TBK1 and IKKε for the treatment of obesity.

The non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa-B kinase ε (IKKε) play a key role in insulin-independent pathways that promote energy storage and block adaptive energy expenditure during obesity. Utilizing docking calculations and the x-ray structure of TBK1 bound to amlexanox, an inhibitor of these kinases with modest potency, a series of analogues was synthesized to develop a structure activity relationship (SAR) around the A- and C-rings of the core scaffold. A strategy was developed wherein R and R A-ring substituents were incorporated late in the synthetic sequence by utilizing palladium-catalyzed cross-coupling reactions on appropriate bromo precursors.

Carboxylic Acid Derivatives of Amlexanox Display Enhanced Potency toward TBK1 and IKK and Reveal Mechanisms for Selective Inhibition.

Chronic low-grade inflammation is a hallmark of obesity, which is a risk factor for the development of type 2 diabetes. The drug amlexanox inhibits IB kinase (IKK) and TANK binding kinase 1 (TBK1) to promote energy expenditure and improve insulin sensitivity. Clinical studies have demonstrated efficacy in a subset of diabetic patients with underlying adipose tissue inflammation, albeit with moderate potency, necessitating the need for improved analogs.

A retractable lid in lecithin:cholesterol acyltransferase provides a structural mechanism for activation by apolipoprotein A-I.

Lecithin:cholesterol acyltransferase (LCAT) plays a key role in reverse cholesterol transport by transferring an acyl group from phosphatidylcholine to cholesterol, promoting the maturation of high-density lipoproteins (HDL) from discoidal to spherical particles. LCAT is activated through an unknown mechanism by apolipoprotein A-I (apoA-I) and other mimetic peptides that form a belt around HDL. Here, we report the crystal structure of LCAT with an extended lid that blocks access to the active site, consistent with an inactive conformation.

Phosphorylation of the exocyst protein Exo84 by TBK1 promotes insulin-stimulated GLUT4 trafficking.

Insulin stimulates glucose uptake through the translocation of the glucose transporter GLUT4 to the plasma membrane. The exocyst complex tethers GLUT4-containing vesicles to the plasma membrane, a process that requires the binding of the G protein (heterotrimeric guanine nucleotide-binding protein) RalA to the exocyst complex. We report that upon activation of RalA, the protein kinase TBK1 phosphorylated the exocyst subunit Exo84.

A subcutaneous adipose tissue-liver signalling axis controls hepatic gluconeogenesis.

The search for effective treatments for obesity and its comorbidities is of prime importance. We previously identified IKK-ε and TBK1 as promising therapeutic targets for the treatment of obesity and associated insulin resistance. Here we show that acute inhibition of IKK-ε and TBK1 with amlexanox treatment increases cAMP levels in subcutaneous adipose depots of obese mice, promoting the synthesis and secretion of the cytokine IL-6 from adipocytes and preadipocytes, but not from macrophages.

Metabolic crosstalk: molecular links between glycogen and lipid metabolism in obesity.

Glycogen and lipids are major storage forms of energy that are tightly regulated by hormones and metabolic signals. We demonstrate that feeding mice a high-fat diet (HFD) increases hepatic glycogen due to increased expression of the glycogenic scaffolding protein PTG/R5. PTG promoter activity was increased and glycogen levels were augmented in mice and cells after activation of the mechanistic target of rapamycin complex 1 (mTORC1) and its downstream target SREBP1.

Inflammation produces catecholamine resistance in obesity via activation of PDE3B by the protein kinases IKKε and TBK1.

Obesity produces a chronic inflammatory state involving the NFκB pathway, resulting in persistent elevation of the noncanonical IκB kinases IKKε and TBK1. In this study, we report that these kinases attenuate β-adrenergic signaling in white adipose tissue. Treatment of 3T3-L1 adipocytes with specific inhibitors of these kinases restored β-adrenergic signaling and lipolysis attenuated by TNFα and Poly (I:C).

An inhibitor of the protein kinases TBK1 and IKK-ɛ improves obesity-related metabolic dysfunctions in mice.

Emerging evidence suggests that inflammation provides a link between obesity and insulin resistance. The noncanonical IκB kinases IKK-ɛ and TANK-binding kinase 1 (TBK1) are induced in liver and fat by NF-κB activation upon high-fat diet feeding and in turn initiate a program of counterinflammation that preserves energy storage. Here we report that amlexanox, an approved small-molecule therapeutic presently used in the clinic to treat aphthous ulcers and asthma, is an inhibitor of these kinases.

TC10 is regulated by caveolin in 3T3-L1 adipocytes.

Background: TC10 is a small GTPase found in lipid raft microdomains of adipocytes. The protein undergoes activation in response to insulin, and plays a key role in the regulation of glucose uptake by the hormone.

Methodology/principal Findings: TC10 requires high concentrations of magnesium in order to stabilize guanine nucleotide binding.

Munc18c interaction with syntaxin 4 monomers and SNARE complex intermediates in GLUT4 vesicle trafficking.

In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins.

Gapex-5, a Rab31 guanine nucleotide exchange factor that regulates Glut4 trafficking in adipocytes.

Insulin stimulates glucose uptake by promoting translocation of the Glut4 glucose transporter from intracellular storage compartments to the plasma membrane. In the absence of insulin, Glut4 is retained intracellularly; the mechanism underlying this process remains uncertain. Using the TC10-interacting protein CIP4 as bait in a yeast two-hybrid screen, we cloned a RasGAP and VPS9 domain-containing protein, Gapex-5/RME-6.

TC10alpha is required for insulin-stimulated glucose uptake in adipocytes.

Previous studies have suggested that activation of the Rho family member GTPase TC10 is necessary but not sufficient for the stimulation of glucose transport by insulin. We show here that endogenous TC10alpha is rapidly activated in response to insulin in 3T3L1 adipocytes in a phosphatidylinositol 3-kinase-independent manner, whereas platelet-derived growth factor was without effect. Knockdown of TC10alpha but not TC10beta by RNA interference inhibited insulin-stimulated glucose uptake as well as the translocation of the insulin-sensitive glucose transporter GLUT4 from intracellular sites to the plasma membrane.

Compartmentalization of the exocyst complex in lipid rafts controls Glut4 vesicle tethering.

Lipid raft microdomains act as organizing centers for signal transduction. We report here that the exocyst complex, consisting of Exo70, Sec6, and Sec8, regulates the compartmentalization of Glut4-containing vesicles at lipid raft domains in adipocytes. Exo70 is recruited by the G protein TC10 after activation by insulin and brings with it Sec6 and Sec8.

TC10 and insulin-stimulated glucose transport.

Insulin stimulates glucose uptake in insulin-responsive tissues by means of the translocation of the glucose transporter GLUT4 from intracellular sites to the plasma membrane. Two pathways are required, one involving activation of a phosphatidylinositol 3-kinase (PI 3-kinase) and downstream protein kinases, and one involving activation of the Rho-family GTPase TC10. TC10 activation by insulin is catalyzed by the exchange factor C3G, which is translocated to lipid rafts along with its binding partner CrkII as a consequence of Cbl tyrosine phosphorylation by the insulin receptor.

Insulin signaling and the regulation of glucose transport.

Gaps remain in our understanding of the precise molecular mechanisms by which insulin regulates glucose uptake in fat and muscle cells. Recent evidence suggests that insulin action involves multiple pathways, each compartmentalized in discrete domains. Upon activation, the receptor catalyzes the tyrosine phosphorylation of a number of substrates.

Incorporating the fourth year of medical school into an internal medicine residency: effect of an accelerated program on performance outcomes and career choice.

Background: To counter declining student interest in general internal medicine, New York Medical College and Saint Vincent's Catholic Medical Centers of New York implemented an experimental accelerated internal medicine curriculum that was provisionally approved by the American Board of Internal Medicine.

Description: This accelerated program allowed selected 4th-year medical students to obtain credit for their 1st year of an internal medicine residency while completing requirements for the MD degree.

Evaluation: Career and performance outcomes for trainees in the accelerated and traditional internal medicine residency programs were compared for 6 classes of graduates.