👤 Ashmita Kc

TF (transcription factor) Prdm16 (positive regulatory domain-containing protein 16) regulates hematopoietic and neuronal stem cell homeostasis, adipose differentiation, and cardiac development. Its role in the circulatory system extends beyond the heart, as Prdm16 loss in arterial endothelial cells (ECs) impairs arterial reperfusion of ischemic mouse limbs due to endothelial dysfunction, and Zebrafish were used to analyze vascular development, arteriovenous endothelial specification, and the emergence of arteriovenous malformations in the absence or presence of Prdm16 or Notch signaling. Lentiviral-mediated Prdm16 overexpression in human endothelial (progenitor) cells was coupled to qRT-PCR (real-time quantitative polymerase chain reaction), Western blot, and transcriptional profiling to document Prdm16's importance for arterial lineage specification. Coimmunoprecipitation in HEK293 (human embryonic kidney 293) cells was performed to assess physical interaction between Prdm16 and the Notch pathway. Existing mouse and human data sets were reanalyzed to evaluate Prdm16 expression in mammalian arteriovenous malformations. Prdm16 actively promotes arterial EC identity while suppressing venous fate. Like in mice, Prdm16 is expressed by arterial ECs early during vascular development in zebrafish, where it synergistically coordinates arterial development together with canonical notch signaling, as their combined loss in zebrafish leads to arteriovenous malformations. PRDM16's arterializing effect on human ECs is dependent on canonical Notch activity, as it is blunted in the presence of canonical Notch inhibitors and potentiated in the presence of delta-like ligand 4. Mechanistically, Prdm16 does not increase the protein levels of the cleaved intracellular domain of Notch receptors (notch intracellular domain) but rather potentiates the effect of the latter via physical and functional interaction. Prdm16 further finetunes Notch signaling and arterial development by complexing with Hey2 (Hes-related family bHLH TF with YRPW motif 2), the basic helix-loop-helix TF acting downstream of canonical Notch during arterial lineage specification and development. Together, our data demonstrate an intricate interplay between Prdm16 and Notch in ECs and indicate that Prdm16 signaling may constitute a novel therapeutic target for arteriovenous malformations. Show less

The mechanisms governing adipose tissue macrophage (ATM) metabolic adaptation during diet-induced obesity (DIO) are poorly understood. In obese adipose tissue, ATMs are exposed to lipid fluxes, which can influence the activation of specific inflammatory and metabolic programs and contribute to the development of obesity-associated insulin resistance and other metabolic disorders. In the present study, we demonstrate that the membrane ATP-binding cassette g1 (Abcg1) transporter controls the ATM functional response to fatty acids (FAs) carried by triglyceride-rich lipoproteins, which are abundant in high-energy diets. Mice genetically lacking Abcg1 in the myeloid lineage presented an ameliorated inflammatory status in adipose tissue and reduced insulin resistance. Abcg1-deficient ATMs exhibited a less inflammatory phenotype accompanied by a low bioenergetic profile and modified FA metabolism. A closer look at the ATM lipidome revealed a shift in the handling of FA pools, including a redirection of saturated FAs from membrane phospholipids to lipid droplets, leading to a reduction in membrane rigidity and neutralization of proinflammatory FAs. ATMs from human individuals with obesity presented the same reciprocal relationship between Show less

The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius. Show less