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1.
Mov Disord ; 39(2): 339-349, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38014556

RESUMO

BACKGROUND: Recent studies have advanced our understanding of the genetic drivers of Parkinson's disease (PD). Rare variants in more than 20 genes are considered causal for PD, and the latest PD genome-wide association study (GWAS) identified 90 independent risk loci. However, there remains a gap in our understanding of PD genetics outside of the European populations in which the vast majority of these studies were focused. OBJECTIVE: The aim was to identify genetic risk factors for PD in a South Asian population. METHODS: A total of 674 PD subjects predominantly with age of onset (AoO) ≤50 years (encompassing juvenile, young, or early-onset PD) were recruited from 10 specialty movement disorder centers across India over a 2-year period; 1376 control subjects were selected from the reference population GenomeAsia, Phase 2. We performed various case-only and case-control genetic analyses for PD diagnosis and AoO. RESULTS: A genome-wide significant signal for PD diagnosis was identified in the SNCA region, strongly colocalizing with SNCA region signal from European PD GWAS. PD cases with pathogenic mutations in PD genes exhibited, on average, lower PD polygenic risk scores than PD cases lacking any PD gene mutations. Gene burden studies of rare, predicted deleterious variants identified BSN, encoding the presynaptic protein Bassoon that has been previously associated with neurodegenerative disease. CONCLUSIONS: This study constitutes the largest genetic investigation of PD in a South Asian population to date. Future work should seek to expand sample numbers in this population to enable improved statistical power to detect PD genes in this understudied group. © 2023 Denali Therapeutics and The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.


Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Humanos , Pessoa de Meia-Idade , Doença de Parkinson/epidemiologia , Doença de Parkinson/genética , Doença de Parkinson/diagnóstico , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Mutação
2.
Dev Cell ; 58(18): 1782-1800.e10, 2023 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-37494933

RESUMO

Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism.


Assuntos
Sistemas CRISPR-Cas , Gotículas Lipídicas , Humanos , Perilipina-2/genética , Perilipina-2/metabolismo , Gotículas Lipídicas/metabolismo , Sistemas CRISPR-Cas/genética , Metabolismo dos Lipídeos/genética , Linhagem Celular
3.
Cell Chem Biol ; 30(9): 1090-1103.e7, 2023 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-37178691

RESUMO

Ferroptosis is a regulated form of cell death associated with the iron-dependent accumulation of phospholipid hydroperoxides. Inducing ferroptosis is a promising approach to treat therapy-resistant cancer. Ferroptosis suppressor protein 1 (FSP1) promotes ferroptosis resistance in cancer by generating the antioxidant form of coenzyme Q10 (CoQ). Despite the important role of FSP1, few molecular tools exist that target the CoQ-FSP1 pathway. Through a series of chemical screens, we identify several structurally diverse FSP1 inhibitors. The most potent of these compounds, ferroptosis sensitizer 1 (FSEN1), is an uncompetitive inhibitor that acts selectively through on-target inhibition of FSP1 to sensitize cancer cells to ferroptosis. Furthermore, a synthetic lethality screen reveals that FSEN1 synergizes with endoperoxide-containing ferroptosis inducers, including dihydroartemisinin, to trigger ferroptosis. These results provide new tools that catalyze the exploration of FSP1 as a therapeutic target and highlight the value of combinatorial therapeutic regimes targeting FSP1 and additional ferroptosis defense pathways.


Assuntos
Ferroptose , Neoplasias , Humanos , Antioxidantes/metabolismo , Peroxidação de Lipídeos , Neoplasias/tratamento farmacológico , Morte Celular
4.
STAR Protoc ; 4(2): 102201, 2023 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-37000620

RESUMO

Phenotypic screens involving pooled CRISPR-Cas9 libraries offer a powerful, rapid yet affordable approach to evaluate gene functions on a global scale. Here, we present a protocol for performing pooled CRISPR-Cas9 loss-of-function screens to identify genetic modifiers using either fluorescence-based or cell death phenotypic readouts. We describe steps for designing and amplifying the library and generating and screening cells. We then detail deep sequencing and statistical analysis using cas9 High Throughput maximum Likelihood Estimator. For complete details on the use and execution of this protocol, please refer to Bersuker et al. (2019),1 Li et al. (2022),2 and Roberts et al. (2022).3.

5.
Contact (Thousand Oaks) ; 5: 25152564221125613, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36147729

RESUMO

Lipid transfer proteins mediate the exchange of lipids between closely apposed membranes at organelle contact sites and play key roles in lipid metabolism, membrane homeostasis, and cellular signaling. A recently discovered novel family of lipid transfer proteins, which includes the VPS13 proteins (VPS13A-D), adopt a rod-like bridge conformation with an extended hydrophobic groove that enables the bulk transfer of membrane lipids for membrane growth. Loss of function mutations in VPS13A and VPS13C cause chorea acanthocytosis and Parkinson's disease, respectively. VPS13A and VPS13C localize to multiple organelle contact sites, including endoplasmic reticulum (ER) - lipid droplet (LD) contact sites, but the functional roles of these proteins in LD regulation remains mostly unexplored. Here we employ CRISPR-Cas9 genome editing to generate VPS13A and VPS13C knockout cell lines in U-2 OS cells via deletion of exon 2 and introduction of an early frameshift. Analysis of LD content in these cell lines revealed that loss of either VPS13A or VPS13C results in reduced LD abundance under oleate-stimulated conditions. These data implicate two lipid transfer proteins, VPS13A and VPS13C, in LD regulation.

6.
Nat Chem Biol ; 18(7): 751-761, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35637349

RESUMO

The selenoprotein glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid peroxides into nontoxic lipid alcohols. GPX4 has emerged as a promising therapeutic target for cancer treatment, but some cancer cells are resistant to ferroptosis triggered by GPX4 inhibition. Using a chemical-genetic screen, we identify LRP8 (also known as ApoER2) as a ferroptosis resistance factor that is upregulated in cancer. Loss of LRP8 decreases cellular selenium levels and the expression of a subset of selenoproteins. Counter to the canonical hierarchical selenoprotein regulatory program, GPX4 levels are strongly reduced due to impaired translation. Mechanistically, low selenium levels result in ribosome stalling at the inefficiently decoded GPX4 selenocysteine UGA codon, leading to ribosome collisions, early translation termination and proteasomal clearance of the N-terminal GPX4 fragment. These findings reveal rewiring of the selenoprotein hierarchy in cancer cells and identify ribosome stalling and collisions during GPX4 translation as ferroptosis vulnerabilities in cancer.


Assuntos
Ferroptose , Selênio , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Ribossomos/metabolismo , Selênio/metabolismo , Selênio/farmacologia , Selenoproteínas/genética
7.
Annu Rev Cell Dev Biol ; 36: 115-139, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33021827

RESUMO

Lipid droplets (LDs) are endoplasmic reticulum-derived organelles that consist of a core of neutral lipids encircled by a phospholipid monolayer decorated with proteins. As hubs of cellular lipid and energy metabolism, LDs are inherently involved in the etiology of prevalent metabolic diseases such as obesity and nonalcoholic fatty liver disease. The functions of LDs are regulated by a unique set of associated proteins, the LD proteome, which includes integral membrane and peripheral proteins. These proteins control key activities of LDs such as triacylglycerol synthesis and breakdown, nutrient sensing and signal integration, and interactions with other organelles. Here we review the mechanisms that regulate the composition of the LD proteome, such as pathways that mediate selective and bulk LD protein degradation and potential connections between LDs and cellular protein quality control.


Assuntos
Gotículas Lipídicas/metabolismo , Proteínas/metabolismo , Animais , Autofagia , Humanos , Proteólise , Proteoma/metabolismo , Ubiquitina/metabolismo
8.
Curr Biol ; 30(13): R770-R773, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32634419

RESUMO

Lipid droplets (LDs) are neutral lipid storage organelles assembled at the endoplasmic reticulum (ER). A new study reveals that the high membrane curvature of ER tubules catalyzes the nucleation of a neutral lipid lens, an early step in LD biogenesis.


Assuntos
Gotículas Lipídicas , Biogênese de Organelas , Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Lipídeos
9.
Nature ; 575(7784): 688-692, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31634900

RESUMO

Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, which suggests that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR-Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis-resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ) (also known as ubiquinone-10), which acts as a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumour xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutic agents.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Ferroptose/genética , Proteínas Mitocondriais/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Ubiquinona/análogos & derivados , Animais , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Regulação Enzimológica da Expressão Gênica , Xenoenxertos , Humanos , Peróxidos Lipídicos/metabolismo , Masculino , Camundongos , Camundongos SCID , Proteínas Mitocondriais/genética , Ubiquinona/metabolismo
10.
Mol Biol Cell ; 28(2): 270-284, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-27881664

RESUMO

The endoplasmic reticulum (ER) mediates the folding, maturation, and deployment of the secretory proteome. Proteins that fail to achieve their native conformation are retained in the ER and targeted for clearance by ER-associated degradation (ERAD), a sophisticated process that mediates the ubiquitin-dependent delivery of substrates to the 26S proteasome for proteolysis. Recent findings indicate that inhibition of long-chain acyl-CoA synthetases with triacsin C, a fatty acid analogue, impairs lipid droplet (LD) biogenesis and ERAD, suggesting a role for LDs in ERAD. However, whether LDs are involved in the ERAD process remains an outstanding question. Using chemical and genetic approaches to disrupt diacylglycerol acyltransferase (DGAT)-dependent LD biogenesis, we provide evidence that LDs are dispensable for ERAD in mammalian cells. Instead, our results suggest that triacsin C causes global alterations in the cellular lipid landscape that disrupt ER proteostasis by interfering with the glycan trimming and dislocation steps of ERAD. Prolonged triacsin C treatment activates both the IRE1 and PERK branches of the unfolded protein response and ultimately leads to IRE1-dependent cell death. These findings identify an intimate relationship between fatty acid metabolism and ER proteostasis that influences cell viability.


Assuntos
Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/fisiologia , Animais , Linhagem Celular , Diacilglicerol O-Aciltransferase , Retículo Endoplasmático/fisiologia , Degradação Associada com o Retículo Endoplasmático/fisiologia , Humanos , Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteólise , Triazenos , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Resposta a Proteínas não Dobradas/fisiologia
11.
Lymphat Res Biol ; 10(3): 122-8, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22984909

RESUMO

Lymphatic dysfunction in lymphedema results in chronic accumulation of interstitial fluid and life-long tissue swelling. In the absence of restored lymphatic drainage via adequate lymphangiogenesis, the interstitial environment can remodel in ways that decrease the elevated interstitial stress. Presently, relatively little is known about the glycosaminoglycans (GAGs) that become upregulated in the interstitium during lymphedema. We employed a mouse tail model of acute lymphedema that reproduces important features of the chronic human condition to establish a relationship between hyaluronan (HA) and sulfated GAG concentration with tissue swelling. We found that HA was upregulated by tissue injury at day 5 and became upregulated again by skin swelling (HA content increasing by 27% relative to controls at days 15 and 20). Surprisingly, the second phase of HA expression was associated with the declining phase of the tail skin swelling (tail diameter significantly decreasing by 17% from day 10 peak to day 20), demonstrating that HA is upregulated by tissue swelling and may help to counteract the edema in the mouse tail. This finding was confirmed by intradermal injection of an HA degrading enzyme (hyaluronidase) to the swollen tail, which was found to worsen the tail swelling. Sulfated GAGs, including chondroitin sulfate (CS), were not regulated by tissue swelling. The results demonstrate that HA, but not sulfated GAGs, is upregulated in the interstitium by acute tissue swelling. We speculate that HA expression during lymphedema may be part of a natural adaptive mechanism of the interstitial environment to reduce capillary filtration and increase interstitial fluid outflow following lymphatic obstruction and fluid accumulation.


Assuntos
Ácido Hialurônico/metabolismo , Linfedema/metabolismo , Linfedema/patologia , Doença Aguda , Animais , Sulfatos de Condroitina/metabolismo , Modelos Animais de Doenças , Glicosaminoglicanos/metabolismo , Humanos , Hialuronoglucosaminidase/metabolismo , Hialuronoglucosaminidase/farmacologia , Camundongos , Cauda/efeitos dos fármacos , Cauda/metabolismo , Cauda/patologia , Fatores de Tempo
12.
Am J Physiol Heart Circ Physiol ; 299(1): H46-54, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20207821

RESUMO

Vascular endothelial growth factor (VEGF)-C is necessary for lymphangiogenesis, and excess VEGF-C has been shown to be ameliorative for edema produced by lymphatic obstruction in experimental models. However, it has recently been shown that edema can resolve in the mouse tail even in the complete absence of capillary lymphangiogenesis when distal lymph fluid crosses the regenerating wound site interstitially. This finding has raised questions about the action of VEGF-C/VEGF receptor (VEGFR) signaling during the resolution of experimental edema. Here, the roles of VEGFR-2 and VEGFR-3 signaling in edema resolution were explored. It was found that edema resolved following neutralization of either VEGFR-2 or VEGFR-3 in the mouse tail skin, which inhibited lymphangiogenesis. Neutralization of either VEGFR-2 or VEGFR-3 reduced angiogenesis at the site of obstruction at day 10 (9.2 +/- 1.2% and 11.5 +/- 1.0% blood capillary coverage, respectively) relative to controls (14.3 +/- 1.5% blood capillary coverage). Combined VEGFR-2/-3 neutralization more strongly inhibited angiogenesis (6.9 +/- 1.5% blood capillary coverage), leading to a reduced wound repair of the lymphatic obstruction and extended edema in the tail skin. In contrast, improved tissue repair of the obstruction site increased edema resolution. Macrophages in the swollen tissue were excluded as contributing factors in the VEGFR-dependent extended edema. These results support a role for VEGFR-2/-3-combined signaling in the resolution of experimental edema that is lymphangiogenesis independent.


Assuntos
Linfangiogênese , Linfedema/fisiopatologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cicatrização , Animais , Anticorpos Monoclonais/administração & dosagem , Capilares/metabolismo , Capilares/fisiopatologia , Modelos Animais de Doenças , Feminino , Linfa/metabolismo , Linfedema/metabolismo , Linfografia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica , Transdução de Sinais , Fatores de Tempo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores
13.
Am J Physiol Heart Circ Physiol ; 294(3): H1326-34, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18203849

RESUMO

Lymphangiogenesis is considered a promising approach for increasing fluid drainage during secondary lymphedema. However, organization of lymphatics into functional capillaries may be dependent upon interstitial flow (IF). The present study was undertaken to determine the importance of lymphangiogenesis for lymphedema resolution. We created a lymphatic obstruction that produces lymphedema in mouse tail skin. The relatively scar-free skin regeneration that occurred across the obstruction allowed the progression of lymphangiogenesis to be observed and compared with the evolution of lymphedema. The role of vascular endothelial growth factor-C (VEGF-C)/VEGF receptor (VEGFR)-3 signaling in lymphedema resolution was investigated by exogenous administration of VEGF-C or neutralizing antibodies against VEGFR-3. VEGF-C protein improved lymphedema at 15 days [reducing dermal thickness from 742 +/- 105 to 559 +/- 141 microm with 95% confidence intervals (CIs), P < 0.05] without increasing lymphatic capillary coverage (11.6 +/- 6.4% following VEGF-C treatment relative to 9.6 +/- 6.2% with 95% CIs, P > 0.50). Blocking VEGFR-3 signaling did not inhibit lymphedema resolution at 25 days (dermal thickness of 462 +/- 127 microm following VEGFR-3 inhibition relative to 502 +/- 87 microm with 95% CIs) or inhibit IF, although VEGFR-3 blocking prevented lymphangiogenesis (reducing lymphatic coverage to 0.2 +/- 0.7% relative to 8.7 +/- 7.3% with 95% CIs, P < 0.005). A second mouse tail lymphedema model was employed to investigate the ability of VEGF-C to increase fluid drainage across a scar. We found that neither neutralization of VEGFR-3 nor administration of VEGF-C affected the course of skin swelling over 25 days. These findings suggest that resolution of lymphedema in the mouse tail skin may be more dependent upon IF and regeneration of the extracellular matrix across the obstruction than lymphatic capillary regeneration.


Assuntos
Líquido Extracelular/fisiologia , Linfedema/fisiopatologia , Pele/fisiopatologia , Animais , Anticorpos Bloqueadores/farmacologia , Feminino , Imunofluorescência , Imuno-Histoquímica , Linfangiogênese/fisiologia , Linfedema/patologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Regeneração , Pele/patologia , Cauda , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia
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