Biology subjects

Obeid, L. M.

Publications and source records attributed to Obeid, L. M..

3 recordsLinked to original sources

bioRxiv2020

Neutral sphingomyelinase 2 regulates inflammatory responses in monocytes/macrophages induced by TNF-α

Obesity is associated with elevated levels of TNF- and proinflammatory CD11c monocytes /macrophages. TNF- mediated dysregulation in the plasticity of monocytes/macrophages is concomitant with pathogenesis of several inflammatory diseases, including metabolic syndrome, but the underlying mechanisms are incompletely understood. Since neutral sphingomyelinase 2 (nSMase2; product of the sphingomyelin phosphodiesterase 3 gene, SMPD3) is a key enzyme for ceramide production involved in inflammation, we investigated whether nSMase2 contributed to the inflammatory changes in the monocytes/macrophages induced by TNF-. In this study, we demonstrate that the disruption of nSMase activity in monocytes/macrophages either by chemical inhibitor GW4869 or small interfering RNA (siRNA) against SMPD3 results in defects in the TNF- mediated expression of CD11c. Furthermore, blockage of nSMase in monocytes/macrophages inhibited the secretion of inflammatory mediators IL-1b and MCP-1. In contrast, inhibition of acid SMase (aSMase) activity did not attenuate CD11c expression or secretion of IL-1b and MCP-1. TNF--induced phosphorylation of JNK, p38 and NF-{kappa}B was also attenuated by the inhibition of nSMase2. Moreover, NF-kB/AP-1 activity was blocked by the inhibition of nSMase2. SMPD3 was elevated in PBMCs from obese individuals and positively corelated with TNF- gene expression. These findings indicate that nSMase2 acts, at least in part, as a master switch in the TNF- mediated inflammatory responses in monocytes/macrophages.

immunology↗

bioRxiv2020

Regulated compartmentalization of enzymes in Golgi by GRASP55 controls cellular glycosphingolipid profile and function

Glycans are important regulators of cell and organismal physiology. This requires that the glycan biosynthesis be controlled to achieve specific cellular glycan profiles. Glycans are assembled in the Golgi apparatus on secretory cargoes that traverse it. The mechanisms by which the Golgi apparatus ensures cell- and cargo-specific glycosylation remain obscure. We investigated how the Golgi apparatus regulates glycosylation by studying biosynthesis of glycosphingolipids, glycosylated lipids with critical roles in signalling and differentiation. We identified the Golgi matrix protein GRASP55 as a controller of sphingolipid glycosylation by regulating the compartmentalized localization of key sphingolipid biosynthetic enzymes in the Golgi. GRASP55 controls the localization of the enzymes by binding to them and regulating their entry into peri-Golgi vesicles. Impairing GRASP55-enzyme interaction decompartmentalizes these enzymes, changes the substrate flux across competing glycosylation pathways that results in alteration of the cellular glycosphingolipid profile. This GRASP55 regulated pathway of enzyme compartmentalization allows cells to make cell density-dependent adaptations in glycosphingolipid biosynthesis to suit cell growth needs. Thus, the Golgi apparatus controls the cellular glycan (glycosphingolipid) profile by governing competition between biosynthetic reactions through regulated changes in enzyme compartmentalization.

cell biology↗

bioRxiv2018

Alkaline ceramidase catalyzes the hydrolysis of ceramides via a catalytic mechanism shared by Zn2+ dependent amidases

Human alkaline ceramidase 3 (ACER3) is one of three alkaline ceramidases (ACERs) that catalyze the conversion of ceramide to sphingosine. ACERs are the members of the CREST superfamily of integral-membrane lipid hydrolases, including the adiponectin receptors which play roles in energy metabolism. All CREST members conserve a set of three Histidine, one Aspartate, and one Serine residue. However, the structural and catalytic roles for these residues are unclear. Here, we use ACER3 as a prototype enzyme to gain insight into this unique class of enzymes. Recombinant ACER3 was expressed in yeast cells that lack endogenous ceramidase activity, and microsomes were used for biochemical characterization. Six point mutantions of the conserved CREST motif were developed that are predicted to form a Zn-dependent active site based on homology with the human adiponectin receptors, whose crystal structures were recently determined. Five mutations completely lost their activity, except for S77A, which showed a 600-fold decrease compared with the wild-type enzyme. The activity of S77C mutation was pH sensitive, with neutral pH partially recovering ACER3 activity. This suggested a role for S77 in stabilizing the oxyanion of the transition state and differs from the proposed role in Zinc coordination for the adiponectin receptors (Vasiliauskaite-Brooks et. al., Nature, 2017). Together, these data suggest ACER3 is a Zn2+-dependent amidase that uses a catalytic mechanism for ceramide hydrolysis that is similar to other soluble Zn-based amidases. Consistent with this mechanism, ACER3 was specifically inhibited by trichostatin A, an HDAC inhibitor, which is a strong chelator of Zinc.

biochemistry↗