Fetuin B in white adipose tissue induces inflammation and is associated with peripheral insulin resistance in mice and humans.

OBJECTIVE: Fetuin B is a steatosis-responsive hepatokine that causes glucose intolerance in mice, but the underlying mechanisms remain incompletely described. This study aimed to elucidate the mechanisms of action of fetuin B by investigating its putative effects on white adipose tissue metabolism. METHODS: First, fetuin B gene and protein expression was measured in multiple organs in mice and in cultured adipocytes. Next, the authors performed a hyperinsulinemic-euglycemic clamp in mice and in humans to examine the link between white adipose tissue fetuin B content and indices of insulin sensitivity. Finally, the effect of fetuin B on inflammation was investigated in cultured adipocytes by quantitative polymerase chain reaction and full RNA sequencing. RESULTS: This study demonstrated in adipocytes and mice that fetuin B was produced and secreted by the liver and taken up by adipocytes and adipose tissue. There was a strong negative correlation between white adipose tissue fetuin B content and peripheral insulin sensitivity in mice and in humans. RNA sequencing and polymerase chain reaction analysis revealed that fetuin B induced an inflammatory response in adipocytes. CONCLUSIONS: Fetuin B content in white adipose tissue strongly associated with peripheral insulin resistance in mice and humans. Furthermore, fetuin B induced a proinflammatory response in adipocytes, which might drive peripheral insulin resistance.

Hippo pathway regulation by phosphatidylinositol transfer protein and phosphoinositides.

The Hippo pathway plays a key role in development, organ size control and tissue homeostasis, and its dysregulation contributes to cancer. The LATS tumor suppressor kinases phosphorylate and inhibit the YAP/TAZ transcriptional co-activators to suppress gene expression and cell growth. Through a screen of marine natural products, we identified microcolin B (MCB) as a Hippo activator that preferentially kills YAP-dependent cancer cells. Structure-activity optimization yielded more potent MCB analogs, which led to the identification of phosphatidylinositol transfer proteins α and ß (PITPα/ß) as the direct molecular targets. We established a critical role of PITPα/ß in regulating LATS and YAP. Moreover, we showed that PITPα/ß influence the Hippo pathway via plasma membrane phosphatidylinositol-4-phosphate. This study uncovers a previously unrecognized role of PITPα/ß in Hippo pathway regulation and as potential cancer therapeutic targets.

Global profiling of phosphorylation-dependent changes in cysteine reactivity.

Proteomics has revealed that the ~20,000 human genes engender a far greater number of proteins, or proteoforms, that are diversified in large part by post-translational modifications (PTMs). How such PTMs affect protein structure and function is an active area of research but remains technically challenging to assess on a proteome-wide scale. Here, we describe a chemical proteomic method to quantitatively relate serine/threonine phosphorylation to changes in the reactivity of cysteine residues, a parameter that can affect the potential for cysteines to be post-translationally modified or engaged by covalent drugs. Leveraging the extensive high-stoichiometry phosphorylation occurring in mitotic cells, we discover numerous cysteines that exhibit phosphorylation-dependent changes in reactivity on diverse proteins enriched in cell cycle regulatory pathways. The discovery of bidirectional changes in cysteine reactivity often occurring in proximity to serine/threonine phosphorylation events points to the broad impact of phosphorylation on the chemical reactivity of proteins and the future potential to create small-molecule probes that differentially target proteoforms with PTMs.

An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells.

Electrophilic compounds originating from nature or chemical synthesis have profound effects on immune cells. These compounds are thought to act by cysteine modification to alter the functions of immune-relevant proteins; however, our understanding of electrophile-sensitive cysteines in the human immune proteome remains limited. Here, we present a global map of cysteines in primary human T cells that are susceptible to covalent modification by electrophilic small molecules. More than 3,000 covalently liganded cysteines were found on functionally and structurally diverse proteins, including many that play fundamental roles in immunology. We further show that electrophilic compounds can impair T cell activation by distinct mechanisms involving the direct functional perturbation and/or degradation of proteins. Our findings reveal a rich content of ligandable cysteines in human T cells and point to electrophilic small molecules as a fertile source for chemical probes and ultimately therapeutics that modulate immunological processes and their associated disorders.

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer.

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.