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1.
Mol Metab ; 73: 101731, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37121509

RESUMO

OBJECTIVE: The metalloprotease ADAM17 (also called TACE) plays fundamental roles in homeostasis by shedding key signaling molecules from the cell surface. Although its importance for the immune system and epithelial tissues is well-documented, little is known about the role of ADAM17 in metabolic homeostasis. The purpose of this study was to determine the impact of ADAM17 expression, specifically in adipose tissues, on metabolic homeostasis. METHODS: We used histopathology, molecular, proteomic, transcriptomic, in vivo integrative physiological and ex vivo biochemical approaches to determine the impact of adipose tissue-specific deletion of ADAM17 upon adipocyte and whole organism metabolic physiology. RESULTS: ADAM17adipoq-creΔ/Δ mice exhibited a hypermetabolic phenotype characterized by elevated energy consumption and increased levels of adipocyte thermogenic gene expression. On a high fat diet, these mice were more thermogenic, while exhibiting elevated expression levels of genes associated with lipid oxidation and lipolysis. This hypermetabolic phenotype protected mutant mice from obesogenic challenge, limiting weight gain, hepatosteatosis and insulin resistance. Activation of beta-adrenoceptors by the neurotransmitter norepinephrine, a key regulator of adipocyte physiology, triggered the shedding of ADAM17 substrates, and regulated ADAM17 expression at the mRNA and protein levels, hence identifying a functional connection between thermogenic licensing and the regulation of ADAM17. Proteomic studies identified Semaphorin 4B (SEMA4B), as a novel ADAM17-shed adipokine, whose expression is regulated by physiological thermogenic cues, that acts to inhibit adipocyte differentiation and dampen thermogenic responses in adipocytes. Transcriptomic data showed that cleaved SEMA4B acts in an autocrine manner in brown adipocytes to repress the expression of genes involved in adipogenesis, thermogenesis, and lipid uptake, storage and catabolism. CONCLUSIONS: Our findings identify a novel ADAM17-dependent axis, regulated by beta-adrenoceptors and mediated by the ADAM17-cleaved form of SEMA4B, that modulates energy balance in adipocytes by inhibiting adipocyte differentiation, thermogenesis and lipid catabolism.


Assuntos
Adipocinas , Semaforinas , Animais , Camundongos , Adipócitos Marrons/metabolismo , Adipocinas/metabolismo , Diferenciação Celular , Lipídeos , Proteômica , Receptores Adrenérgicos beta/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Termogênese/fisiologia
2.
Life Sci Alliance ; 6(4)2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36720499

RESUMO

The metalloprotease ADAM17 is a sheddase of key molecules, including TNF and epidermal growth factor receptor ligands. ADAM17 exists within an assemblage, the "sheddase complex," containing a rhomboid pseudoprotease (iRhom1 or iRhom2). iRhoms control multiple aspects of ADAM17 biology. The FERM domain-containing protein iTAP/Frmd8 is an iRhom-binding protein that prevents the precocious shunting of ADAM17 and iRhom2 to lysosomes and their consequent degradation. As pathophysiological role(s) of iTAP/Frmd8 have not been addressed, we characterized the impact of iTAP/Frmd8 loss on ADAM17-associated phenotypes in mice. We show that iTAP/Frmd8 KO mice exhibit defects in inflammatory and intestinal epithelial barrier repair functions, but not the collateral defects associated with global ADAM17 loss. Furthermore, we show that iTAP/Frmd8 regulates cancer cell growth in a cell-autonomous manner and by modulating the tumor microenvironment. Our work suggests that pharmacological intervention at the level of iTAP/Frmd8 may be beneficial to target ADAM17 activity in specific compartments during chronic inflammatory diseases or cancer, while avoiding the collateral impact on the vital functions associated with the widespread inhibition of ADAM17.


Assuntos
Neoplasias , Animais , Camundongos , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Inflamação , Neoplasias/genética , Microambiente Tumoral
3.
FEBS J ; 289(22): 6822-6831, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36377590

RESUMO

The major criterion that distinguishes eukaryotes from prokaryotes is the presence of organelles in the former. Organelles provide a compartment in which biochemical processes are corralled within bespoke biophysical conditions and act as storage depots, powerhouses, waste storage/recycling units and innate immune signalling hubs. A key challenge faced by organelles is to define, and then retain, their identity; this is mediated by complex proteostasis mechanisms including the import of an organelle-specific proteome, the exclusion of non-organellar proteins and the removal of misfolded proteins via dedicated quality control mechanisms. This Special Issue on Organelle Homeostasis provides an engaging, eclectic, yet integrative, perspective on organelle homeostasis in a range of organelles including those from the secretory and endocytic pathways, mitochondria, the autophagy-lysosomal pathway and the nucleus and its sub-compartments. Some lesser-known organelles including migrasomes (organelles that are released by migrating cells) and GOMED (a Golgi-specific form of autophagy) are also introduced. In the spirit of the principles of organelle biology, we hope you find the reviews in this Issue both encapsulating and captivating, and we thank the authors for their excellent contributions.


Assuntos
Retículo Endoplasmático , Organelas , Retículo Endoplasmático/metabolismo , Organelas/metabolismo , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Mitocôndrias , Homeostase
4.
Science ; 375(6577): eabi4343, 2022 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-35025629

RESUMO

The outer mitochondrial membrane (OMM) is essential for cellular homeostasis. Yet little is known of the mechanisms that remodel it during natural stresses. We found that large "SPOTs" (structures positive for OMM) emerge during Toxoplasma gondii infection in mammalian cells. SPOTs mediated the depletion of the OMM proteins mitofusin 1 and 2, which restrict parasite growth. The formation of SPOTs depended on the parasite effector TgMAF1 and the host mitochondrial import receptor TOM70, which is required for optimal parasite proliferation. TOM70 enabled TgMAF1 to interact with the host OMM translocase SAM50. The ablation of SAM50 or the overexpression of an OMM-targeted protein promoted OMM remodeling independently of infection. Thus, Toxoplasma hijacks the formation of SPOTs, a cellular response to OMM stress, to promote its growth.


Assuntos
Membranas Mitocondriais/fisiologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Animais , Linhagem Celular , GTP Fosfo-Hidrolases/metabolismo , Humanos , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/metabolismo , Ligação Proteica , Estresse Fisiológico , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/ultraestrutura , Toxoplasmose/parasitologia , Vacúolos/fisiologia , Vacúolos/ultraestrutura
5.
Mol Metab ; 31: 67-84, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31918923

RESUMO

OBJECTIVE: Obesity is the result of positive energy balance. It can be caused by excessive energy consumption but also by decreased energy dissipation, which occurs under several conditions including when the development or activation of brown adipose tissue (BAT) is impaired. Here we evaluated whether iRhom2, the essential cofactor for the Tumour Necrosis Factor (TNF) sheddase ADAM17/TACE, plays a role in the pathophysiology of metabolic syndrome. METHODS: We challenged WT versus iRhom2 KO mice to positive energy balance by chronic exposure to a high fat diet and then compared their metabolic phenotypes. We also carried out ex vivo assays with primary and immortalized mouse brown adipocytes to establish the autonomy of the effect of loss of iRhom2 on thermogenesis and respiration. RESULTS: Deletion of iRhom2 protected mice from weight gain, dyslipidemia, adipose tissue inflammation, and hepatic steatosis and improved insulin sensitivity when challenged by a high fat diet. Crucially, the loss of iRhom2 promotes thermogenesis via BAT activation and beige adipocyte recruitment, enabling iRhom2 KO mice to dissipate excess energy more efficiently than WT animals. This effect on enhanced thermogenesis is cell-autonomous in brown adipocytes as iRhom2 KOs exhibit elevated UCP1 levels and increased mitochondrial proton leak. CONCLUSION: Our data suggest that iRhom2 is a negative regulator of thermogenesis and plays a role in the control of adipose tissue homeostasis during metabolic disease.


Assuntos
Proteínas de Transporte/metabolismo , Obesidade/metabolismo , Termogênese , Animais , Dieta Hiperlipídica/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/induzido quimicamente
6.
Elife ; 72018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29897333

RESUMO

The apical inflammatory cytokine TNF regulates numerous important biological processes including inflammation and cell death, and drives inflammatory diseases. TNF secretion requires TACE (also called ADAM17), which cleaves TNF from its transmembrane tether. The trafficking of TACE to the cell surface, and stimulation of its proteolytic activity, depends on membrane proteins, called iRhoms. To delineate how the TNF/TACE/iRhom axis is regulated, we performed an immunoprecipitation/mass spectrometry screen to identify iRhom-binding proteins. This identified a novel protein, that we name iTAP (iRhom Tail-Associated Protein) that binds to iRhoms, enhancing the cell surface stability of iRhoms and TACE, preventing their degradation in lysosomes. Depleting iTAP in primary human macrophages profoundly impaired TNF production and tissues from iTAP KO mice exhibit a pronounced depletion in active TACE levels. Our work identifies iTAP as a physiological regulator of TNF signalling and a novel target for the control of inflammation.


Assuntos
Proteína ADAM17/metabolismo , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Fator de Necrose Tumoral alfa/genética , Proteína ADAM17/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Linhagem Celular , Proteínas do Citoesqueleto/genética , Endossomos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/citologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Ligação Proteica , Proteólise , Células RAW 264.7 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
7.
Cell Rep ; 21(3): 745-757, 2017 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-29045841

RESUMO

Cell surface metalloproteases coordinate signaling during development, tissue homeostasis, and disease. TACE (TNF-α-converting enzyme), is responsible for cleavage ("shedding") of membrane-tethered signaling molecules, including the cytokine TNF, and activating ligands of the EGFR. The trafficking of TACE within the secretory pathway requires its binding to iRhom2, which mediates the exit of TACE from the endoplasmic reticulum. An important, but mechanistically unclear, feature of TACE biology is its ability to be stimulated rapidly on the cell surface by numerous inflammatory and growth-promoting agents. Here, we report a role for iRhom2 in TACE stimulation on the cell surface. TACE shedding stimuli trigger MAP kinase-dependent phosphorylation of iRhom2 N-terminal cytoplasmic tail. This recruits 14-3-3 proteins, enforcing the dissociation of TACE from complexes with iRhom2, promoting the cleavage of TACE substrates. Our data reveal that iRhom2 controls multiple aspects of TACE biology, including stimulated shedding on the cell surface.


Assuntos
Proteína ADAM17/metabolismo , Proteínas de Transporte/metabolismo , Proteólise , Proteínas 14-3-3/metabolismo , Animais , Proteínas de Transporte/química , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Fosfosserina/metabolismo , Transdução de Sinais , Especificidade por Substrato , Receptores Toll-Like/metabolismo
8.
Sci Rep ; 7(1): 7283, 2017 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-28779096

RESUMO

Rhomboids are intramembrane serine proteases conserved in all kingdoms of life. They regulate epidermal growth factor receptor signalling in Drosophila by releasing signalling ligands from their transmembrane tethers. Their functions in mammals are poorly understood, in part because of the lack of endogenous substrates identified thus far. We used a quantitative proteomics approach to investigate the substrate repertoire of rhomboid protease RHBDL2 in human cells. We reveal a range of novel substrates that are specifically cleaved by RHBDL2, including the interleukin-6 receptor (IL6R), cell surface protease inhibitor Spint-1, the collagen receptor tyrosine kinase DDR1, N-Cadherin, CLCP1/DCBLD2, KIRREL, BCAM and others. We further demonstrate that these substrates can be shed by endogenously expressed RHBDL2 and that a subset of them is resistant to shedding by cell surface metalloproteases. The expression profiles and identity of the substrates implicate RHBDL2 in physiological or pathological processes affecting epithelial homeostasis.


Assuntos
Epitélio/metabolismo , Homeostase , Proteoma , Proteômica , Serina Proteases/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Células Epiteliais/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteômica/métodos , Serina Endopeptidases , Serina Proteases/genética , Especificidade por Substrato
10.
Plant Physiol Biochem ; 43(12): 1067-73, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16386428

RESUMO

The physiological role of class III peroxidases (EC 1.11.1.7) in controlling plant growth and development has been investigated by over-expression of both native and heterologous peroxidases. However, it has remained an enigma as to why the phenotypes of different peroxidase over-expressing transgenics vary. In order to resolve the conflicting information about the consequences of peroxidase over-expression, we have explored the role of the subcellular targeting of HRP-C in controlling stem growth, root development, axillary branching and abiotic stress tolerance in tobacco (Nicotiana tabacum L.). Altering the sub-cellular targeting of vacuolar HRP-C, such that over-expressed peroxidase accumulates in the cytoplasm and cell wall, induced phenotypic changes that are typically associated with altered auxin homeostasis, and over-expression of cell wall located peroxidases. We conclude that sub-cellular targeting is a determinant of the phenotype of peroxidase over-expressing plants.


Assuntos
Genes Sintéticos , Peroxidase do Rábano Silvestre/biossíntese , Nicotiana/enzimologia , Folhas de Planta/enzimologia , Plantas Geneticamente Modificadas/enzimologia , Parede Celular/fisiologia , Citoplasma/fisiologia , Genes de Plantas , Peroxidase do Rábano Silvestre/genética , Ácidos Indolacéticos/metabolismo , Lignina/biossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Nicotiana/genética , Nicotiana/crescimento & desenvolvimento , Raios Ultravioleta
11.
Ann Bot ; 93(3): 303-10, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14749254

RESUMO

BACKGROUND AND AIMS: Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. METHODS: Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. KEY RESULTS: Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. CONCLUSIONS: There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity. These will be valuable for exploring the role of these enzymes in stress amelioration and plant development.


Assuntos
Armoracia/enzimologia , Nicotiana/genética , Peroxidase/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Sequência de Aminoácidos , Armoracia/genética , Northern Blotting , Western Blotting , Fracionamento Celular/métodos , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Peroxidase/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Frações Subcelulares , Nicotiana/enzimologia
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