Ube4A maintains metabolic homeostasis and facilitates insulin signaling in vivo
Mukherjee S, Chakraborty M, Msengi EN, Haubner J, Zhang J, Jellinek MJ, Carlson HL, Pyles K, Ulmasov B, Lutkewitte AJ, Carpenter D, McCommis KS, Ford DA, Finck BN, Neuschwander-Tetri BA and Chakraborty A
Ube4A maintains metabolic homeostasis and facilitates insulin signaling in vivo
Mukherjee S, Chakraborty M, Msengi EN, Haubner J, Zhang J, Jellinek MJ, Carlson HL, Pyles K, Ulmasov B, Lutkewitte AJ, Carpenter D, McCommis KS, Ford DA, Finck BN, Neuschwander-Tetri BA and Chakraborty A
Defining the regulators of cell metabolism and signaling is essential to design new therapeutic strategies in obesity and NAFLD/NASH. E3 ubiquitin ligases control diverse cellular functions by ubiquitination-mediated regulation of protein targets, and thus their functional aberration is associated with many diseases. The E3 ligase Ube4A has been implicated in human obesity, inflammation, and cancer. However, its in vivo function is unknown, and no animal models are available to study this novel protein.
Homodimeric Granzyme A opsonizes Mycobacterium tuberculosis and inhibits its intracellular growth in human monocytes via TLR4 and CD14
Rasi V, Phelps KR, Paulson KR, Eickhoff CS, Chinnaraj M, Pozzi N, Di Gioia M, Zanoni I, Shakya S, Carlson HL, Ford DA, Kolar GR and Hoft DF
Homodimeric Granzyme A opsonizes Mycobacterium tuberculosis and inhibits its intracellular growth in human monocytes via TLR4 and CD14
Rasi V, Phelps KR, Paulson KR, Eickhoff CS, Chinnaraj M, Pozzi N, Di Gioia M, Zanoni I, Shakya S, Carlson HL, Ford DA, Kolar GR and Hoft DF
Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete GzmA protective against intracellular Mtb growth. However, GzmA enzymatic activity is unnecessary for pathogen inhibition and the mechanisms of GzmA-mediated protection remain unknown. We show GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes and subsequent pathogen clearance within the phagolysosome. While monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind CD14 and TLR4. Without access to surface expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1, was associated with inhibitory activity. Further, GzmA colocalized with and was regulated by protein disulfide isomerase (PDI)A1, which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis and elimination of Mtb in human monocytes, and highlights PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.
Hepatic pyruvate and alanine metabolism are critical and complementary for maintenance of antioxidant capacity and resistance to oxidative insult
Yiew NKH, Vazquez JH, Martino MR, Kennon-McGill S, Price JR, Allard FD, Yee EU, Layman AJ, James LP, McCommis KS, Finck BN and McGill MR
Hepatic pyruvate and alanine metabolism are critical and complementary for maintenance of antioxidant capacity and resistance to oxidative insult
Yiew NKH, Vazquez JH, Martino MR, Kennon-McGill S, Price JR, Allard FD, Yee EU, Layman AJ, James LP, McCommis KS, Finck BN and McGill MR
Mitochondrial pyruvate is a critical intermediary metabolite in gluconeogenesis, lipogenesis, and NADH production. As a result, the mitochondrial pyruvate carrier (MPC) complex has emerged as a promising therapeutic target in metabolic diseases. Clinical trials are currently underway. However, recent in vitro data indicate that MPC inhibition diverts glutamine/glutamate away from glutathione synthesis and toward glutaminolysis to compensate for loss of pyruvate oxidation, possibly sensitizing cells to oxidative insult. Here, we explored this in vivo using the clinically relevant acetaminophen (APAP) overdose model of acute liver injury, which is driven by oxidative stress.
Disruption of Ordered Membrane Domains as a Mechanism Underlying Neuropathic Pain
Handlin LJ, Macchi NL, Dumaire N, Salih L, McCommis KS, Moutal A and Dai G
Disruption of Ordered Membrane Domains as a Mechanism Underlying Neuropathic Pain
Handlin LJ, Macchi NL, Dumaire N, Salih L, McCommis KS, Moutal A and Dai G
Cell membranes consist of heterogeneous lipid domains that influence key cellular processes, including signal transduction, endocytosis, and electrical excitability. The goal of this study was to assess the size of cholesterol-enriched ordered membrane domains (OMD) in various cell types. Multiple cell types were tested using fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), whereby small nociceptor DRG neurons and cardiac pacemaker cells displayed the highest FRET intensities. This implies that electrically active cells tend to have large OMDs. Treatment of cells with the cholesterol-extracting reagent β-cyclodextrin (β-CD) led to a decrease in FRET, indicating a reduction in the OMD size, whereas detergents known to promote domain coalescence in artificial membranes increased OMD size. In an fatty liver model, palmitate supplementation increased FRET whereas oleate supplementation decreased FRET in isolated primary murine hepatocytes, highlighting the importance of unsaturated lipid tails in lipid domain organization. Disruption of OMD using β-CD potentiated action potential firing in nociceptor DRG neurons and decreased the free energy needed for opening native hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. After disrupting the OMD, HCN channels exhibited an increased relative open probability at the resting membrane potential (RMP). A significant reduction in FRET was observed in both a chemotherapy-induced neuropathic pain model and a spared nerve injury model of neuropathic pain, consistent with disrupted or shrunken OMD in these models. Collectively, these findings show that disturbances in lipid domains may contribute to the progression of neuropathic pain, and they suggest new therapeutic strategies to achieve pain relief.
Mutations in atypical hemolytic uremic syndrome provide evidence for the role of calcium in complement factor I
Java A, Atkinson J, Hu Z and Pozzi N
Mutations in atypical hemolytic uremic syndrome provide evidence for the role of calcium in complement factor I
Java A, Atkinson J, Hu Z and Pozzi N
Atypical hemolytic uremic syndrome (aHUS) is a rare thrombotic microangiopathy. Genetic variants in complement proteins are found in ≈60% of patients. Of these patients, ≈15% carry mutations in complement factor I (CFI). Factor I (FI) is a multidomain serine protease that cleaves and thereby inactivates C3b and C4b in the presence of cofactor proteins. Crystal structures have shown that FI possesses 2 calcium-binding domains, low-density lipoprotein receptor class A (LDLRA) 1 and LDLRA2. Yet, the role of calcium in FI is unknown. We determined that 9 genetic variants identified in aHUS (N151S, G162D, G188A, V230E, A240G, G243R, C247G, A258T, and Q260D) cluster around the calcium-binding site of LDLRA1. Using site-directed mutagenesis, we established that the synthesis of all, except A258T, was impaired, implying defective protein folding, perhaps due to loss of calcium binding. To further explore this possibility, we generated 12 alanine mutants that coordinate with the calcium in LDLRA1 and LDLRA2 (K239A, D242A, I244A, D246A, D252A, E253A, Y276A, N279A, E281A, D283A, D289A, and D290A) and are expected to perturb calcium binding. Except for K239A and Y276A, none of the mutants was secreted. These observations suggest that calcium ions play key structural and functional roles in FI.
Structural analyses of β-glycoprotein I: is there a circular conformation?
Kumar S, Wulf J, Basore K and Pozzi N
Structural analyses of β-glycoprotein I: is there a circular conformation?
Kumar S, Wulf J, Basore K and Pozzi N
Antiphospholipid antibodies targeting β-glycoprotein I (βGPI) cause thrombosis and pregnancy morbidity in antiphospholipid syndrome (APS) patients. How these antibodies recognize βGPI remains controversial.
Structure-Activity Relationship Studies of the Aryl Acetamide Triazolopyridazines against Reveals Remarkable Role of Fluorine
Schubert TJ, Oboh E, Peek H, Philo E, Teixeira JE, Stebbins EE, Miller P, Oliva J, Sverdrup FM, Griggs DW, Huston CD and Meyers MJ
Structure-Activity Relationship Studies of the Aryl Acetamide Triazolopyridazines against Reveals Remarkable Role of Fluorine
Schubert TJ, Oboh E, Peek H, Philo E, Teixeira JE, Stebbins EE, Miller P, Oliva J, Sverdrup FM, Griggs DW, Huston CD and Meyers MJ
Our previous work identified compound (SLU-2633) as a potent lead compound toward the identification of a novel treatment for cryptosporidiosis, caused by the parasite (EC = 0.17 μM). While this compound is potent and orally efficacious, the mechanism of action and biological target(s) of this series are currently unknown. In this study, we synthesized 70 compounds to develop phenotypic structure-activity relationships around the aryl "tail" group. In this process, we found that 2-substituted compounds are inactive, confirmed that electron withdrawing groups are preferred over electron donating groups, and that fluorine plays a remarkable role in the potency of these compounds. The most potent compound resulting from this work is SLU-10482 (, EC = 0.07 μΜ), which was found to be orally efficacious with an ED < 5 mg/kg BID in a -infection mouse model, superior to SLU-2633.
Hepatic hydrogen sulfide levels are reduced in mouse model of Hutchinson-Gilford progeria syndrome
Wilkie SE, Marcu DE, Carter RN, Morton NM, Gonzalo S and Selman C
Hepatic hydrogen sulfide levels are reduced in mouse model of Hutchinson-Gilford progeria syndrome
Wilkie SE, Marcu DE, Carter RN, Morton NM, Gonzalo S and Selman C
Hutchinson-Gilford progeria syndrome (HGPS) is a rare human disease characterised by accelerated biological ageing. Current treatments are limited, and most patients die before 15 years of age. Hydrogen sulfide (HS) is an important gaseous signalling molecule that it central to multiple cellular homeostasis mechanisms. Dysregulation of tissue HS levels is thought to contribute to an ageing phenotype in many tissues across animal models. Whether HS is altered in HGPS is unknown. We investigated hepatic HS production capacity and transcript, protein and enzymatic activity of proteins that regulate hepatic HS production and disposal in a mouse model of HGPS (G609G mice, mutated Lmna gene equivalent to a causative mutation in HGPS patients). G609G mice were maintained on either regular chow (RC) or high fat diet (HFD), as HFD has been previously shown to significantly extend lifespan of G609G mice, and compared to wild type (WT) mice maintained on RC. RC fed G609G mice had significantly reduced hepatic HS production capacity relative to WT mice, with a compensatory elevation in mRNA transcripts associated with several HS production enzymes, including cystathionine-γ-lyase (CSE). HS levels and CSE protein were partially rescued in HFD fed G609G mice. As current treatments for patients with HGPS have failed to confer significant improvements to symptoms or longevity, the need for novel therapeutic targets is acute and the regulation of HS through dietary or pharmacological means may be a promising new avenue for research.
Enhancing Hepatic MBOAT7 Expression in Mice With Nonalcoholic Steatohepatitis
Sharpe MC, Pyles KD, Hallcox T, Kamm DR, Piechowski M, Fisk B, Albert CJ, Carpenter DH, Ulmasov B, Ford DA, Neuschwander-Tetri BA and McCommis KS
Enhancing Hepatic MBOAT7 Expression in Mice With Nonalcoholic Steatohepatitis
Sharpe MC, Pyles KD, Hallcox T, Kamm DR, Piechowski M, Fisk B, Albert CJ, Carpenter DH, Ulmasov B, Ford DA, Neuschwander-Tetri BA and McCommis KS
Polymorphisms near the membrane bound O-acyltransferase domain containing 7 (MBOAT7) genes are associated with worsened nonalcoholic fatty liver (NASH), and nonalcoholic fatty liver disease (NAFLD)/NASH may decrease MBOAT7 expression independent of these polymorphisms. We hypothesized that enhancing MBOAT7 function would improve NASH.
Editorial: Diabetes and non-alcoholic fatty liver disease: points of physiological and mechanistic intersection and current co-therapeutic approaches
Giannoukakis N and McCommis KS
Editorial: Diabetes and non-alcoholic fatty liver disease: points of physiological and mechanistic intersection and current co-therapeutic approaches
Giannoukakis N and McCommis KS