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1.
Proc Natl Acad Sci U S A ; 121(19): e2322164121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38687799

RESUMO

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during midembryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these two paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Animais , Humanos , Camundongos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Retículo Endoplasmático/metabolismo , Hepatócitos/metabolismo , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética
2.
J Virol ; 97(12): e0127623, 2023 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-37975674

RESUMO

ABSTRACT: Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain unclear. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top four genes identified in our screen encode components of the same type I interferon (IFN-I) signaling complex­IFNAR1, IFNAR2, JAK1, and TYK2. The fifth gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response in both Calu-3 cells and iPSC-derived type 2 alveolar epithelial cells. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.


Assuntos
COVID-19 , Células Epiteliais , Interferon Tipo I , Pulmão , Humanos , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Interferon Tipo I/imunologia , Pulmão/patologia , Pulmão/virologia , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Linhagem Celular , Proliferação de Células
3.
PLoS Pathog ; 18(3): e1010377, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35231079

RESUMO

SARS-CoV-2 infection is initiated by binding of the viral spike protein to its receptor, ACE2, on the surface of host cells. ACE2 expression is heterogeneous both in vivo and in immortalized cell lines, but the molecular pathways that govern ACE2 expression remain unclear. We now report high-throughput CRISPR screens for functional modifiers of ACE2 surface abundance. In liver-derived HuH7 cells, we identified 35 genes whose disruption was associated with a change in the surface abundance of ACE2. Enriched among these ACE2 regulators were established transcription factors, epigenetic regulators, and functional networks. We further characterized individual HuH7 cell lines with disruption of SMAD4, EP300, PIAS1, or BAMBI and found these genes to regulate ACE2 at the mRNA level and to influence cellular susceptibility to SARS-CoV-2 infection. Orthogonal screening of lung-derived Calu-3 cells revealed a distinct set of ACE2 modifiers comprised of ACE2, KDM6A, MOGS, GPAA1, and UGP2. Collectively, our findings clarify the host factors involved in SARS-CoV-2 entry, highlight the cell type specificity of ACE2 regulatory networks, and suggest potential targets for therapeutic development.


Assuntos
COVID-19 , Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
PLoS Genet ; 17(1): e1009285, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33513160

RESUMO

Hypercholesterolemia is a causal and modifiable risk factor for atherosclerotic cardiovascular disease. A critical pathway regulating cholesterol homeostasis involves the receptor-mediated endocytosis of low-density lipoproteins into hepatocytes, mediated by the LDL receptor. We applied genome-scale CRISPR screening to query the genetic determinants of cellular LDL uptake in HuH7 cells cultured under either lipoprotein-rich or lipoprotein-starved conditions. Candidate LDL uptake regulators were validated through the synthesis and secondary screening of a customized library of gRNA at greater depth of coverage. This secondary screen yielded significantly improved performance relative to the primary genome-wide screen, with better discrimination of internal positive controls, no identification of negative controls, and improved concordance between screen hits at both the gene and gRNA level. We then applied our customized gRNA library to orthogonal screens that tested for the specificity of each candidate regulator for LDL versus transferrin endocytosis, the presence or absence of genetic epistasis with LDLR deletion, the impact of each perturbation on LDLR expression and trafficking, and the generalizability of LDL uptake modifiers across multiple cell types. These findings identified several previously unrecognized genes with putative roles in LDL uptake and suggest mechanisms for their functional interaction with LDLR.


Assuntos
Aterosclerose/genética , Colesterol/genética , Lipoproteínas LDL/genética , Receptores de LDL/genética , Aterosclerose/patologia , Sistemas CRISPR-Cas/genética , Colesterol/metabolismo , Endocitose/genética , Regulação da Expressão Gênica/genética , Genoma Humano/genética , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Lipoproteínas LDL/metabolismo , RNA Guia de Cinetoplastídeos/genética
5.
J Proteome Res ; 22(11): 3439-3446, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37844105

RESUMO

Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles and tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells, whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif..


Assuntos
Proteínas de Transporte , Retículo Endoplasmático , Proteínas de Membrana , Humanos , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi , Proteínas de Membrana/metabolismo , Transporte Proteico
6.
Biochem J ; 479(7): 839-855, 2022 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-35322856

RESUMO

α1-antitrypsin (AAT) is a serine protease inhibitor synthesized in hepatocytes and protects the lung from damage by neutrophil elastase. AAT gene mutations result in AAT deficiency (AATD), which leads to lung and liver diseases. The AAT Z variant forms polymer within the endoplasmic reticulum (ER) of hepatocytes and results in reduction in AAT secretion and severe disease. Previous studies demonstrated a secretion defect of AAT in LMAN1 deficient cells, and mild decreases in AAT levels in male LMAN1 and MCFD2 deficient mice. LMAN1 is a transmembrane lectin that forms a complex with a small soluble protein MCFD2. The LMAN1-MCFD2 protein complex cycles between the ER and the Golgi. Here, we report that LMAN1 and MCFD2 knockout (KO) HepG2 and HEK293T cells display reduced AAT secretion and elevated intracellular AAT levels due to a delayed ER-to-Golgi transport of AAT. Secretion defects in KO cells were rescued by wild-type LMAN1 or MCFD2, but not by mutant proteins. Elimination of the second glycosylation site of AAT abolished LMAN1 dependent secretion. Co-immunoprecipitation experiment in MCFD2 KO cells suggested that AAT interaction with LMAN1 is independent of MCFD2. Furthermore, our results suggest that secretion of the Z variant, both monomers and polymers, is also LMAN1-dependent. Results provide direct evidence supporting that the LMAN1-MCFD2 complex is a cargo receptor for the ER-to-Golgi transport of AAT and that interactions of LMAN1 with an N-glycan of AAT is critical for this process. These results have implications in production of recombinant AAT and in developing treatments for AATD patients.


Assuntos
Fator VIII , Fator V , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Fator V/genética , Fator V/metabolismo , Fator VIII/genética , Células HEK293 , Humanos , Masculino , Lectinas de Ligação a Manose/genética , Camundongos , Proteínas de Transporte Vesicular/genética , alfa 1-Antitripsina/genética
7.
J Lipid Res ; 63(8): 100248, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35753407

RESUMO

The low-density lipoprotein receptor (LDLR) mediates the hepatic uptake of circulating low-density lipoproteins (LDLs), a process that modulates the development of atherosclerotic cardiovascular disease. We recently identified RAB10, encoding a small GTPase, as a positive regulator of LDL uptake in hepatocellular carcinoma cells (HuH7) in a genome-wide CRISPR screen, though the underlying molecular mechanism for this effect was unknown. We now report that RAB10 regulates hepatocyte LDL uptake by promoting the recycling of endocytosed LDLR from RAB11-positive endosomes to the plasma membrane. We also show that RAB10 similarly promotes the recycling of the transferrin receptor, which binds the transferrin protein that mediates the transport of iron in the blood, albeit from a distinct RAB4-positive compartment. Taken together, our findings suggest a model in which RAB10 regulates LDL and transferrin uptake by promoting both slow and rapid recycling routes for their respective receptor proteins.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Endocitose , Endossomos , Hepatócitos , Lipoproteínas LDL , Receptores de LDL , Receptores da Transferrina , Transferrina , Proteínas rab de Ligação ao GTP
8.
Annu Rev Med ; 71: 71-83, 2020 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-31469969

RESUMO

The modern genomic era has seen remarkable advancement in our understanding of the molecular basis for disease, yet translation of basic discoveries into new disease treatments has arguably lagged behind. Recently, breakthroughs in genome editing technologies have created hope for their potential to directly treat the genetic causes of disease. Like any therapeutic intervention, genome editing should be considered in light of its potential risks and benefits. In this review, we highlight the promise of genome editing therapies, as well as the conceptual and technical barriers to their clinical application, with a special emphasis on hematologic malignancies.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Terapia Genética/métodos , Neoplasias Hematológicas/genética , Feminino , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/terapia , Humanos , Masculino , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Prognóstico , Medição de Risco , Análise de Sobrevida , Resultado do Tratamento
9.
bioRxiv ; 2024 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-38766193

RESUMO

An elevated level of lipoprotein(a), or Lp(a), in the bloodstream has been causally linked to the development of atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Steady state levels of circulating lipoproteins are modulated by their rate of clearance, but the identity of the Lp(a) uptake receptor(s) has been controversial. In this study, we performed a genome-scale CRISPR screen to functionally interrogate all potential Lp(a) uptake regulators in HuH7 cells. Strikingly, the top positive and negative regulators of Lp(a) uptake in our screen were LDLR and MYLIP, encoding the LDL receptor and its ubiquitin ligase IDOL, respectively. We also found a significant correlation for other genes with established roles in LDLR regulation. No other gene products, including those previously proposed as Lp(a) receptors, exhibited a significant effect on Lp(a) uptake in our screen. We validated the functional influence of LDLR expression on HuH7 Lp(a) uptake, confirmed in vitro binding between the LDLR extracellular domain and purified Lp(a), and detected an association between loss-of-function LDLR variants and increased circulating Lp(a) levels in the UK Biobank cohort. Together, our findings support a central role for the LDL receptor in mediating Lp(a) uptake by hepatocytes.

10.
bioRxiv ; 2024 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-38463989

RESUMO

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during mid-embryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these 2 paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.

11.
bioRxiv ; 2023 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-37066360

RESUMO

Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles/tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif.

12.
bioRxiv ; 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36909579

RESUMO

Disease progression during SARS-CoV-2 infection is tightly linked to the fate of lung epithelial cells, with severe cases of COVID-19 characterized by direct injury of the alveolar epithelium and an impairment in its regeneration from progenitor cells. The molecular pathways that govern respiratory epithelial cell death and proliferation during SARS-CoV-2 infection, however, remain poorly understood. We now report a high-throughput CRISPR screen for host genetic modifiers of the survival and proliferation of SARS-CoV-2-infected Calu-3 respiratory epithelial cells. The top 4 genes identified in our screen encode components of the same type I interferon signaling complex - IFNAR1, IFNAR2, JAK1, and TYK2. The 5th gene, ACE2, was an expected control encoding the SARS-CoV-2 viral receptor. Surprisingly, despite the antiviral properties of IFN-I signaling, its disruption in our screen was associated with an increase in Calu-3 cell fitness. We validated this effect and found that IFN-I signaling did not sensitize SARS-CoV-2-infected cultures to cell death but rather inhibited the proliferation of surviving cells after the early peak of viral replication and cytopathic effect. We also found that IFN-I signaling alone, in the absence of viral infection, was sufficient to induce this delayed antiproliferative response. Together, these findings highlight a cell autonomous antiproliferative response by respiratory epithelial cells to persistent IFN-I signaling during SARS-CoV-2 infection. This response may contribute to the deficient alveolar regeneration that has been associated with COVID-19 lung injury and represents a promising area for host-targeted therapeutic development.

13.
J Biol Chem ; 286(38): 33109-17, 2011 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-21784841

RESUMO

The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cílios/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Acilação/efeitos dos fármacos , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/química , Sequência Conservada , Detergentes/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Leishmania/efeitos dos fármacos , Leishmania/metabolismo , Lisina/metabolismo , Dados de Sequência Molecular , Mutação/genética , Ácido Mirístico/metabolismo , Palmitatos/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/química , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Trypanosoma cruzi/efeitos dos fármacos
14.
J Cell Sci ; 123(Pt 4): 529-36, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-20145001

RESUMO

Cilia are specialized surface regions of eukaryotic cells that serve a variety of functions, ranging from motility to sensation and to regulation of cell growth and differentiation. The discovery that a number of human diseases, collectively known as ciliopathies, result from defective cilium function has expanded interest in these structures. Among the many properties of cilia, motility and intraflagellar transport have been most extensively studied. The latter is the process by which multiprotein complexes associate with microtubule motors to transport structural subunits along the axoneme to and from the ciliary tip. By contrast, the mechanisms by which membrane proteins and lipids are specifically targeted to the cilium are still largely unknown. In this Commentary, we review the current knowledge of protein and lipid targeting to ciliary membranes and outline important issues for future study. We also integrate this information into a proposed model of how the cell specifically targets proteins and lipids to the specialized membrane of this unique organelle.


Assuntos
Cílios/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Animais , Axonema/metabolismo , Axonema/ultraestrutura , Cílios/ultraestrutura , Complexo de Golgi/metabolismo , Humanos , Fusão de Membrana , Modelos Biológicos , Movimento , Transporte Proteico
16.
Eukaryot Cell ; 10(3): 455-63, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21193548

RESUMO

Many eukaryotic proteins are posttranslationally modified by the esterification of cysteine thiols to long-chain fatty acids. This modification, protein palmitoylation, is catalyzed by a large family of palmitoyl acyltransferases that share an Asp-His-His-Cys Cys-rich domain but differ in their subcellular localizations and substrate specificities. In Trypanosoma brucei, the flagellated protozoan parasite that causes African sleeping sickness, protein palmitoylation has been observed for a few proteins, but the extent and consequences of this modification are largely unknown. We undertook the present study to investigate T. brucei protein palmitoylation at both the enzyme and substrate levels. Treatment of parasites with an inhibitor of total protein palmitoylation caused potent growth inhibition, yet there was no effect on growth by the separate, selective inhibition of each of the 12 individual T. brucei palmitoyl acyltransferases. This suggested either that T. brucei evolved functional redundancy for the palmitoylation of essential palmitoyl proteins or that palmitoylation of some proteins is catalyzed by a noncanonical transferase. To identify the palmitoylated proteins in T. brucei, we performed acyl biotin exchange chemistry on parasite lysates, followed by streptavidin chromatography, two-dimensional liquid chromatography-tandem mass spectrometry protein identification, and QSpec statistical analysis. A total of 124 palmitoylated proteins were identified, with an estimated false discovery rate of 1.0%. This palmitoyl proteome includes all of the known palmitoyl proteins in procyclic-stage T. brucei as well as several proteins whose homologues are palmitoylated in other organisms. Their sequences demonstrate the variety of substrate motifs that support palmitoylation, and their identities illustrate the range of cellular processes affected by palmitoylation in these important pathogens.


Assuntos
Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/parasitologia , Sequência de Aminoácidos , Humanos , Lipoilação , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Alinhamento de Sequência , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento
17.
Elife ; 112022 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-36193893

RESUMO

PCSK9 negatively regulates low-density lipoprotein receptor (LDLR) abundance on the cell surface, leading to decreased hepatic clearance of LDL particles and increased levels of plasma cholesterol. We previously identified SURF4 as a cargo receptor that facilitates PCSK9 secretion in HEK293T cells (Emmer et al., 2018). Here, we generated hepatic SURF4-deficient mice (Surf4fl/fl Alb-Cre+) to investigate the physiologic role of SURF4 in vivo. Surf4fl/fl Alb-Cre+ mice exhibited normal viability, gross development, and fertility. Plasma PCSK9 levels were reduced by ~60% in Surf4fl/fl Alb-Cre+ mice, with a corresponding ~50% increase in steady state LDLR protein abundance in the liver, consistent with SURF4 functioning as a cargo receptor for PCSK9. Surprisingly, these mice exhibited a marked reduction in plasma cholesterol and triglyceride levels out of proportion to the partial increase in hepatic LDLR abundance. Detailed characterization of lipoprotein metabolism in these mice instead revealed a severe defect in hepatic lipoprotein secretion, consistent with prior reports of SURF4 also promoting the secretion of apolipoprotein B (APOB). Despite a small increase in liver mass and lipid content, histologic evaluation revealed no evidence of steatohepatitis or fibrosis in Surf4fl/fl Alb-Cre+ mice. Acute depletion of hepatic SURF4 by CRISPR/Cas9 or liver-targeted siRNA in adult mice confirms these findings. Together, these data support the physiologic significance of SURF4 in the hepatic secretion of PCSK9 and APOB-containing lipoproteins and its potential as a therapeutic target in atherosclerotic cardiovascular diseases.


Assuntos
Pró-Proteína Convertase 9 , Receptores de LDL , Camundongos , Humanos , Animais , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , RNA Interferente Pequeno/metabolismo , Células HEK293 , Camundongos Endogâmicos C57BL , Receptores de LDL/genética , Receptores de LDL/metabolismo , Fígado/metabolismo , Apolipoproteínas B/metabolismo , Lipoproteínas/metabolismo , Triglicerídeos/metabolismo , Colesterol/metabolismo , Lipoproteínas LDL/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
18.
Eukaryot Cell ; 9(6): 934-42, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20418379

RESUMO

African trypanosomes express a family of dually acylated, EF-hand calcium-binding proteins called the calflagins. These proteins associate with lipid raft microdomains in the flagellar membrane, where they putatively function as calcium signaling proteins. Here we show that these proteins bind calcium with high affinity and that their expression is regulated during the life cycle stage of the parasite, with protein levels approximately 10-fold higher in the mammalian bloodstream form than in the insect vector procyclic stage. We also demonstrate a role for the calflagins in mammalian infection, as inhibition of the entire calflagin family by RNA interference dramatically increased host survival and attenuated parasitemia in a mouse model of sleeping sickness. In contrast to infection with parental wild-type parasites, which demonstrated an unremitting parasitemia and death within 6 to 10 days, infection with calflagin-depleted parasites demonstrated prolonged survival associated with a sudden decrease in parasitemia at approximately 8 days postinfection. Subsequent relapsing and remitting waves of parasitemia thereafter were associated with alternate expression of the variant surface glycoprotein, suggesting that initial clearance was antigen specific. Interestingly, despite the notable in vivo phenotype and flagellar localization of the calflagins, in vitro analysis of the calflagin-deficient parasites demonstrated normal proliferation, flagellar motility, and morphology. Further analysis of the kinetics of surface antibody clearance also did not demonstrate a deficit in the calflagin-deficient parasites; thus, the molecular basis for the altered course of infection is independent of an effect on parasite cell cycle progression, motility, or degradation of surface-bound antibodies.


Assuntos
Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Parasitemia/parasitologia , Proteínas de Protozoários/antagonistas & inibidores , Trypanosoma brucei brucei , Tripanossomíase Africana/parasitologia , Animais , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNA
19.
bioRxiv ; 2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33791703

RESUMO

The membrane protein angiotensin-converting enzyme 2 (ACE2) is a physiologic regulator of the renin-angiotensin system and the cellular receptor for the SARS-CoV-2 virus. Prior studies of ACE2 expression have primarily focused on mRNA abundance, with investigation at the protein level limited by uncertain specificity of commercial ACE2 antibodies. Here, we report our development of a sensitive and specific flow cytometry-based assay for cellular ACE2 protein abundance. Application of this approach to multiple cell lines revealed an unexpected degree of cellular heterogeneity, with detectable ACE2 protein in only a subset of cells in each isogenic population. This heterogeneity was mediated at the mRNA level by transcripts predominantly initiated from the ACE2 proximal promoter. ACE2 expression was heritable but not fixed over multiple generations of daughter cells, with gradual drift toward the original heterogeneous background. RNA-seq profiling identified distinct transcriptomes of ACE2-expressing relative cells to non-expressing cells, with enrichment in functionally related genes and transcription factor target sets. Our findings provide a validated approach for the specific detection of ACE2 protein at the surface of single cells, support an epigenetic mechanism ACE2 gene regulation, and identify specific pathways associated with ACE2 expression in HuH7 cells.

20.
Sci Rep ; 11(1): 15900, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354120

RESUMO

The membrane protein angiotensin-converting enzyme 2 (ACE2) is a physiologic regulator of the renin-angiotensin system and the cellular receptor for the SARS-CoV-2 virus. Prior studies of ACE2 expression have primarily focused on mRNA abundance, with investigation at the protein level limited by uncertain specificity of commercial ACE2 antibodies. Here, we report our development of a sensitive and specific flow cytometry-based assay for cellular ACE2 protein abundance. Application of this approach to multiple cell lines revealed an unexpected degree of cellular heterogeneity, with detectable ACE2 protein in only a subset of cells in each isogenic population. This heterogeneity was mediated at the mRNA level by transcripts predominantly initiated from the ACE2 proximal promoter. ACE2 expression was heritable but not fixed over multiple generations of daughter cells, with gradual drift toward the original heterogeneous background. RNA-seq profiling identified distinct transcriptomes of ACE2-expressing relative cells to non-expressing cells, with enrichment in functionally related genes and transcription factor target sets. Our findings provide a validated approach for the specific detection of ACE2 protein at the surface of single cells, support an epigenetic mechanism of ACE2 gene regulation, and identify specific pathways associated with ACE2 expression in HuH7 cells.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/genética , Transcriptoma , Enzima de Conversão de Angiotensina 2/análise , Linhagem Celular , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , RNA Mensageiro/genética , Receptores Virais/análise , Receptores Virais/genética , SARS-CoV-2/isolamento & purificação
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