Structure-Activity Relationships of Natural and Semisynthetic Plecomacrolides Suggest Distinct Pathways for HIV-1 Immune Evasion and Vacuolar ATPase-Dependent Lysosomal Acidification.

The human immunodeficiency virus (HIV)-encoded accessory protein Nef enhances pathogenicity by reducing major histocompatibility complex I (MHC-I) cell surface expression, protecting HIV-infected cells from immune recognition. Nef-dependent downmodulation of MHC-I can be reversed by subnanomolar concentrations of concanamycin A (1), a well-known inhibitor of vacuolar ATPase, at concentrations below those that interfere with lysosomal acidification or degradation. We conducted a structure-activity relationship study that assessed 76 compounds for Nef inhibition, 24 and 72 h viability, and lysosomal neutralization in Nef-expressing primary T cells. This analysis demonstrated that the most potent compounds were natural concanamycins and their derivatives. Comparison against a set of new, semisynthetic concanamycins revealed that substituents at C-8 and acylation of C-9 significantly affected Nef potency, target cell viability, and lysosomal neutralization. These findings provide important progress toward understanding the mechanism of action of these compounds and the identification of an advanced lead anti-HIV Nef inhibitory compound.

Role of the Beta and Gamma Isoforms of the Adapter Protein SH2B1 in Regulating Energy Balance.

Human variants of the adapter protein SH2B1 are associated with severe childhood obesity, hyperphagia, and insulin resistance-phenotypes mimicked by mice lacking Sh2b1. SH2B1ß and γ isoforms are expressed ubiquitously, whereas SH2B1α and δ isoforms are expressed primarily in the brain. Restoring SH2B1ß driven by the neuron-specific enolase promoter largely reverses the metabolic phenotype of Sh2b1-null mice, suggesting crucial roles for neuronal SH2B1ß in energy balance control. Here we test this hypothesis by using CRISPR/Cas9 gene editing to delete the ß and γ isoforms from the neurons of mice (SH2B1ßγ neuron-specific knockout [NKO] mice) or throughout the body (SH2B1ßγ knockout [KO] mice). While parameters of energy balance were normal in both male and female SH2B1ßγ NKO mice, food intake, body weight, and adiposity were increased in male (but not female) SH2B1ßγ KO mice. Analysis of long-read single-cell RNA seq data from wild-type mouse brain revealed that neurons express almost exclusively the α and δ isoforms, whereas neuroglial cells express almost exclusively the ß and γ isoforms. Our work suggests that neuronal SH2B1ß and γ are not primary regulators of energy balance. Rather, non-neuronal SH2B1ß and γ in combination with neuronal SH2B1α and δ suffice for body weight maintenance. While SH2B1ß/γ and SH2B1α/δ share some functionality, SH2B1ß/γ appears to play a larger role in promoting leanness.

The nucleolar δ isoform of adapter protein SH2B1 enhances morphological complexity and function of cultured neurons.

The adapter protein SH2B1 is recruited to neurotrophin receptors, including TrkB (also known as NTRK2), the receptor for brain-derived neurotrophic factor (BDNF). Herein, we demonstrate that the four alternatively spliced isoforms of SH2B1 (SH2B1α-SH2B1δ) are important determinants of neuronal architecture and neurotrophin-induced gene expression. Primary hippocampal neurons from Sh2b1-/- [knockout (KO)] mice exhibit decreased neurite complexity and length, and BDNF-induced expression of the synapse-related immediate early genes Egr1 and Arc. Reintroduction of each SH2B1 isoform into KO neurons increases neurite complexity; the brain-specific δ isoform also increases total neurite length. Human obesity-associated variants, when expressed in SH2B1δ, alter neurite complexity, suggesting that a decrease or increase in neurite branching may have deleterious effects that contribute to the severe childhood obesity and neurobehavioral abnormalities associated with these variants. Surprisingly, in contrast to SH2B1α, SH2B1ß and SH2B1γ, which localize primarily in the cytoplasm and plasma membrane, SH2B1δ resides primarily in nucleoli. Some SH2B1δ is also present in the plasma membrane and nucleus. Nucleolar localization, driven by two highly basic regions unique to SH2B1δ, is required for SH2B1δ to maximally increase neurite complexity and BDNF-induced expression of Egr1, Arc and FosL1.

Deletion of the Brain-Specific α and δ Isoforms of Adapter Protein SH2B1 Protects Mice From Obesity.

Mice lacking SH2B1 and humans with variants of SH2B1 display severe obesity and insulin resistance. SH2B1 is an adapter protein that is recruited to the receptors of multiple hormones and neurotrophic factors. Of the four known alternatively spliced SH2B1 isoforms, SH2B1ß and SH2B1γ exhibit ubiquitous expression, whereas SH2B1α and SH2B1δ are essentially restricted to the brain. To understand the roles for SH2B1α and SH2B1δ in energy balance and glucose metabolism, we generated mice lacking these brain-specific isoforms (αδ knockout [αδKO] mice). αδKO mice exhibit decreased food intake, protection from weight gain on standard and high-fat diets, and an adiposity-dependent improvement in glucose homeostasis. SH2B1 has been suggested to impact energy balance via the modulation of leptin action. However, αδKO mice exhibit leptin sensitivity that is similar to that of wild-type mice by multiple measures. Thus, decreasing the abundance of SH2B1α and/or SH2B1δ relative to the other SH2B1 isoforms likely shifts energy balance toward a lean phenotype via a primarily leptin-independent mechanism. Our findings suggest that the different alternatively spliced isoforms of SH2B1 perform different functions in vivo.

Crucial Role of the SH2B1 PH Domain for the Control of Energy Balance.

Disruption of the adaptor protein SH2B1 (SH2-B, PSM) is associated with severe obesity, insulin resistance, and neurobehavioral abnormalities in mice and humans. Here, we identify 15 SH2B1 variants in severely obese children. Four obesity-associated human SH2B1 variants lie in the Pleckstrin homology (PH) domain, suggesting that the PH domain is essential for SH2B1's function. We generated a mouse model of a human variant in this domain (P322S). P322S/P322S mice exhibited substantial prenatal lethality. Examination of the P322S/+ metabolic phenotype revealed late-onset glucose intolerance. To circumvent P322S/P322S lethality, mice containing a two-amino acid deletion within the SH2B1 PH domain (ΔP317, R318 [ΔPR]) were studied. Mice homozygous for ΔPR were born at the expected Mendelian ratio and exhibited obesity plus insulin resistance and glucose intolerance beyond that attributable to their increased adiposity. These studies demonstrate that the PH domain plays a crucial role in how SH2B1 controls energy balance and glucose homeostasis.

Phosphorylation of the Unique C-Terminal Tail of the Alpha Isoform of the Scaffold Protein SH2B1 Controls the Ability of SH2B1α To Enhance Nerve Growth Factor Function.

The scaffold protein SH2B1, a major regulator of body weight, is recruited to the receptors of multiple cytokines and growth factors, including nerve growth factor (NGF). The ß isoform but not the α isoform of SH2B1 greatly enhances NGF-dependent neurite outgrowth of PC12 cells. Here, we asked how the unique C-terminal tails of the α and ß isoforms modulate SH2B1 function. We compared the actions of SH2B1α and SH2B1ß to those of the N-terminal 631 amino acids shared by both isoforms. In contrast to the ß tail, the α tail inhibited the ability of SH2B1 to both cycle through the nucleus and enhance NGF-mediated neurite outgrowth, gene expression, phosphorylation of Akt and phospholipase C-gamma (PLC-γ), and autophosphorylation of the NGF receptor TrkA. These functions were restored when Tyr753 in the α tail was mutated to phenylalanine. We provide evidence that TrkA phosphorylates Tyr753 in SH2B1α, as well as tyrosines 439 and 55 in both SH2B1α and SH2B1ß. Finally, coexpression of SH2B1α but not SH2B1α with a mutation of Y to F at position 753 (Y753F) inhibited the ability of SH2B1ß to enhance neurite outgrowth. These results suggest that the C-terminal tails of SH2B1 isoforms are key determinants of the cellular role of SH2B1. Furthermore, the function of SH2B1α is regulated by phosphorylation of the α tail.

Functional characterization of obesity-associated variants involving the α and ß isoforms of human SH2B1.

We have previously reported rare variants in sarcoma (Src) homology 2 (SH2) B adaptor protein 1 (SH2B1) in individuals with obesity, insulin resistance, and maladaptive behavior. Here, we identify 4 additional SH2B1 variants by sequencing 500 individuals with severe early-onset obesity. SH2B1 has 4 alternatively spliced isoforms. One variant (T546A) lies within the N-terminal region common to all isoforms. As shown for past variants in this region, T546A impairs SH2B1ß enhancement of nerve growth factor-induced neurite outgrowth, and the individual with the T546A variant exhibits mild developmental delay. The other 3 variants (A663V, V695M, and A723V) lie in the C-terminal tail of SH2B1α. SH2B1α variant carriers were hyperinsulinemic but did not exhibit the behavioral phenotype observed in individuals with SH2B1 variants that disrupt all isoforms. In in vitro assays, SH2B1α, like SH2B1ß, enhances insulin- and leptin-induced insulin receptor substrate 2 (IRS2) phosphorylation and GH-induced cell motility. None of the variants affect SH2B1α enhancement of insulin- and leptin-induced IRS2 phosphorylation. However, T546A, A663V, and A723V all impair the ability of SH2B1α to enhance GH-induced cell motility. In contrast to SH2B1ß, SH2B1α does not enhance nerve growth factor-induced neurite outgrowth. These studies suggest that genetic variants that disrupt isoforms other than SH2B1ß may be functionally significant. Further studies are needed to understand the mechanism by which the individual isoforms regulate energy homeostasis and behavior.

Human SH2B1 mutations are associated with maladaptive behaviors and obesity.

Src homology 2 B adapter protein 1 (SH2B1) modulates signaling by a variety of ligands that bind to receptor tyrosine kinases or JAK-associated cytokine receptors, including leptin, insulin, growth hormone (GH), and nerve growth factor (NGF). Targeted deletion of Sh2b1 in mice results in increased food intake, obesity, and insulin resistance, with an intermediate phenotype seen in heterozygous null mice on a high-fat diet. We identified SH2B1 loss-of-function mutations in a large cohort of patients with severe early-onset obesity. Mutation carriers exhibited hyperphagia, childhood-onset obesity, disproportionate insulin resistance, and reduced final height as adults. Unexpectedly, mutation carriers exhibited a spectrum of behavioral abnormalities that were not reported in controls, including social isolation and aggression. We conclude that SH2B1 plays a critical role in the control of human food intake and body weight and is implicated in maladaptive human behavior.

Tyrosines 868, 966, and 972 in the kinase domain of JAK2 are autophosphorylated and required for maximal JAK2 kinase activity.

Janus kinase 2 (JAK2) is activated by a majority of cytokine family receptors including receptors for GH, leptin, and erythropoietin. To identify novel JAK2-regulatory and/or -binding sites, we set out to identify autophosphorylation sites in the kinase domain of JAK2. Two-dimensional phosphopeptide mapping of in vitro autophosphorylated JAK2 identified tyrosines 868, 966, and 972 as sites of autophosphorylation. Phosphorylated tyrosines 868 and 972 were also identified by mass spectrometry analysis of JAK2 activated by an erythropoietin-bound chimeric erythropoietin receptor/leptin receptor. Phosphospecific antibodies suggest that the phosphorylation of all three tyrosines increases in response to GH. Compared with wild-type JAK2, which is constitutively active when overexpressed, JAK2 lacking tyrosine 868, 966, or 972 has substantially reduced activity. Coexpression with GH receptor and protein tyrosine phosphatase1B allowed us to investigate GH-dependent activation of these mutated JAK2s in human embryonic kidney 293T cells. All three mutated JAK2s are activated by GH, although to a lesser extent than wild-type JAK2. The three mutated JAK2s also mediate GH activation of signal transducer and activator of transcription 3 (Stat3), signal transducer and activator of transcription 5b (Stat5b) and ERK1, but at reduced levels. Coexpression with Src-homology 2B1beta (SH2B1beta), like coexpression with GH-bound GH receptor, partially restores the activity of all three JAK2 mutants. Based on these results and the crystal structure of the JAK2 kinase domain, we hypothesize that small changes in the conformation of the regions of JAK2 surrounding tyrosines 868, 966, and 972 due to e.g. phosphorylation, binding to a ligand-bound cytokine receptor, and/or binding to Src-homology 2B1, may be essential for JAK2 to assume a maximally active conformation.

Phosphorylation of JAK2 at serine 523: a negative regulator of JAK2 that is stimulated by growth hormone and epidermal growth factor.

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling for multiple receptor tyrosine kinases and several G-protein-coupled receptors. In this study, phosphopeptide affinity enrichment and mass spectrometry identified serine 523 (Ser523) in JAK2 as a site of phosphorylation. A phosphoserine 523 antibody revealed that Ser523 is rapidly but transiently phosphorylated in response to growth hormone (GH). MEK1 inhibitor UO126 suppresses GH-dependent phosphorylation of Ser523, suggesting that extracellular signal-regulated kinases (ERKs) 1 and/or 2 or another kinase downstream of MEK1 phosphorylate Ser523 in response to GH. Other ERK activators, phorbol 12-myristate 13-acetate and epidermal growth factor, also stimulate phosphorylation of Ser523. When Ser523 in JAK2 was mutated, JAK2 kinase activity as well as GH-dependent tyrosyl phosphorylation of JAK2 and Stat5 was enhanced, suggesting that phosphorylation of Ser523 inhibits JAK2 kinase activity. We hypothesize that phosphorylation of Ser523 in JAK2 by ERKs 1 and/or 2 or other as-yet-unidentified kinases acts in a negative feedback manner to dampen activation of JAK2 in response to GH and provides a mechanism by which prior exposure to environmental factors that regulate Ser523 phosphorylation might modulate the cell's response to GH.