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1.
Curr Protoc ; 3(4): e739, 2023 Apr.
Article in English | MEDLINE | ID: mdl-37078364

ABSTRACT

Inflammation is the body's response to injury and harmful stimuli and contributes to a range of infectious and noninfectious diseases. Inflammation occurs through a series of well-defined leukocyte-endothelial cell interactions, including rolling, activation, adhesion, transmigration, and subsequent migration through the extracellular matrix. Being able to visualize the stages of inflammation is important for a better understanding of its role in diseases processes. Detailed in this article are protocols for imaging immune cell infiltration and transendothelial migration in vascular tissue beds, including those in the mouse ear, cremaster muscle, brain, lung, and retina. Also described are protocols for inducing inflammation and quantifying leukocytes with FIJI imaging software. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of croton oil dermatitis Alternate Protocol 1: Induction of croton oil dermatitis using genetically fluorescent mice Basic Protocol 2: Intravital microscopy of the mouse cremaster muscle Support Protocol: Making a silicone stage Basic Protocol 3: Wide-field microscopy of the mouse brain Basic Protocol 4: Imaging the lungs (ex vivo) Alternate Protocol 2: Inflating the lungs without tracheostomy Basic Protocol 5: Inducing, imaging, and quantifying infiltration of leukocytes in mouse retina.


Subject(s)
Dermatitis , Transendothelial and Transepithelial Migration , Mice , Animals , Croton Oil , Leukocytes/physiology , Inflammation/diagnostic imaging
2.
J Cell Biol ; 221(3)2022 03 07.
Article in English | MEDLINE | ID: mdl-35157000

ABSTRACT

ADP-ribosylation is a reversible post-translational modification where an ADP-ribose moiety is covalently attached to target proteins by ADP-ribosyltransferases (ARTs). Although best known for its nuclear roles, ADP-ribosylation is increasingly recognized as a key regulatory strategy across cellular compartments. ADP-ribosylation of mitochondrial proteins has been widely reported, but the exact nature of mitochondrial ART enzymes is debated. We have identified neuralized-like protein 4 (NEURL4) as a mitochondrial ART enzyme and show that most ART activity associated with mitochondria is lost in the absence of NEURL4. The NEURL4-dependent ADP-ribosylome in mitochondrial extracts from HeLa cells includes numerous mitochondrial proteins previously shown to be ADP-ribosylated. In particular, we show that NEURL4 is required for the regulation of mtDNA integrity via poly-ADP-ribosylation of mtLIG3, the rate-limiting enzyme for base excision repair (BER). Collectively, our studies reveal that NEURL4 acts as the main mitochondrial ART enzyme under physiological conditions and provide novel insights in the regulation of mitochondria homeostasis through ADP-ribosylation.


Subject(s)
ADP-Ribosylation , Mitochondrial Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , DNA, Mitochondrial/metabolism , HeLa Cells , Homeostasis , Humans , Protein Domains , Ubiquitin-Protein Ligases/chemistry
3.
Mol Metab ; 6(1): 125-137, 2017 01.
Article in English | MEDLINE | ID: mdl-28123943

ABSTRACT

OBJECTIVE: Insulin signaling plays a unique role in the regulation of energy homeostasis and the impairment of insulin action is associated with altered lipid metabolism, obesity, and Type 2 Diabetes. The main aim of this study was to provide further insight into the regulatory mechanisms governing the insulin signaling pathway by investigating the role of non-proteolytic ubiquitination in insulin-mediated activation of AKT. METHODS: The molecular mechanism of AKT regulation through ubiquitination is first dissected in vitro in 3T3-L1 preadipocytes and then validated in vivo using mice with adipo-specific deletion of GPS2, an endogenous inhibitor of Ubc13 activity (GPS2-AKO mice). RESULTS: Our results indicate that K63 ubiquitination is a critical component of AKT activation in the insulin signaling pathway and that counter-regulation of this step is provided by GPS2 preventing AKT ubiquitination through inhibition of Ubc13 enzymatic activity. Removal of this negative checkpoint, through GPS2 downregulation or genetic deletion, results in sustained activation of insulin signaling both in vitro and in vivo. As a result, the balance between lipid accumulation and utilization is shifted toward storage in the adipose tissue and GPS2-AKO mice become obese under normal laboratory chow diet. However, the adipose tissue of GPS2-AKO mice is not inflamed, the levels of circulating adiponectin are elevated, and systemic insulin sensitivity is overall improved. CONCLUSIONS: Our findings characterize a novel layer of regulation of the insulin signaling pathway based on non-proteolytic ubiquitination of AKT and define GPS2 as a previously unrecognized component of the insulin signaling cascade. In accordance with this role, we have shown that GPS2 presence in adipocytes modulates systemic metabolism by restricting the activation of insulin signaling during the fasted state, whereas in absence of GPS2, the adipose tissue is more efficient at lipid storage, and obesity becomes uncoupled from inflammation and insulin resistance.


Subject(s)
Adipose Tissue/metabolism , Insulin/metabolism , Intracellular Signaling Peptides and Proteins/physiology , 3T3 Cells , Adipocytes/metabolism , Animals , Diabetes Mellitus, Type 2/metabolism , Inflammation/genetics , Insulin/genetics , Insulin/physiology , Insulin Resistance/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Obesity/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Ubiquitin-Conjugating Enzymes/antagonists & inhibitors , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitination
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