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1.
Biochem J ; 478(6): 1303-1307, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33755101

RESUMO

Signaling pathways play critical roles in regulating the activation of T cells. Recognition of foreign peptide presented by MHC to the T cell receptor (TCR) triggers a signaling cascade of proximal kinases and adapter molecules that lead to the activation of Effector kinase pathways. These effector kinase pathways play pivotal roles in T cell activation, differentiation, and proliferation. RNA sequencing-based methods have provided insights into the gene expression programs that support the above-mentioned cell biological responses. The proteome is often overlooked. A recent study by Damasio et al. [Biochem. J. (2021) 478, 79-98. doi:10.1042/BCJ20200661] focuses on characterizing the effect of extracellular signal-regulated kinase (ERK) on the remodeling of the proteome of activated CD8+ T cells using Mass spectrometric analysis. Surprisingly, the Effector kinase ERK pathway is responsible for only a select proportion of the proteome that restructures during T cell activation. The primary targets of ERK signaling are transcription factors, cytokines, and cytokine receptors. In this commentary, we discuss the recent findings by Damasio et al. [Biochem. J. (2021) 478, 79-98. doi:10.1042/BCJ20200661] in the context of different Effector kinase pathways in activated T cells.


Assuntos
Receptores de Antígenos de Linfócitos T , Linfócitos T , Citocinas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo
2.
J Cell Sci ; 130(14): 2359-2370, 2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-28596240

RESUMO

Mitochondria play essential roles in cellular energy processes, including ATP production, control of reactive oxygen species (ROS) and apoptosis. While mitochondrial function is regulated by the dynamics of fusion and fission, mitochondrial homeostasis remains incompletely understood. Recent studies implicate dynamin-2 and dynamin-related protein-1 (Drp1, also known as DNM1L), as GTPases involved in mitochondrial fission. Here, we identify the ATPase and endocytic protein EHD1 as a novel regulator of mitochondrial fission. EHD1 depletion induces a static and elongated network of mitochondria in the cell. However, unlike dynamin-2 and Drp1, whose depletion protects cells from staurosporine-induced mitochondrial fragmentation, EHD1-depleted cells remain sensitive to staurosporine, suggesting a different mechanism for EHD1 function. Recent studies have demonstrated that VPS35 and the retromer complex influence mitochondrial homeostasis either by Mul1-mediated ubiquitylation and degradation of the fusion protein Mfn2, or by removal of inactive Drp1 from the mitochondrial membrane. We demonstrate that EHD1 and its interaction partner rabankyrin-5 interact with the retromer complex to influence mitochondrial dynamics, likely by inducing VPS35-mediated removal of inactive Drp1 from mitochondrial membranes. Our study sheds light on mitochondrial dynamics, expanding a new paradigm of endocytic protein regulation of mitochondrial homeostasis.


Assuntos
Homeostase/fisiologia , Mitocôndrias/metabolismo , Proteínas de Transporte Vesicular/genética , Endocitose/fisiologia , Humanos , Proteínas de Transporte Vesicular/metabolismo
3.
J Biol Chem ; 291(26): 13465-78, 2016 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-27189942

RESUMO

An elaborate network of dynamic lipid membranes, termed tubular recycling endosomes (TRE), coordinates the process of endocytic recycling in mammalian cells. The C-terminal Eps15 homology domain (EHD)-containing proteins have been implicated in the bending and fission of TRE, thus regulating endocytic recycling. EHD proteins have an EH domain that interacts with proteins containing an NPF motif. We found that NPF-containing EHD1 interaction partners such as molecules interacting with CasL-like1 (MICAL-L1) and Syndapin2 are essential for TRE biogenesis. Also crucial for TRE biogenesis is the generation of phosphatidic acid, an essential lipid component of TRE that serves as a docking point for MICAL-L1 and Syndapin2. EHD1 and EHD3 have 86% amino acid identity; they homo- and heterodimerize and partially co-localize to TRE. Despite their remarkable identity, they have distinct mechanistic functions. EHD1 induces membrane vesiculation, whereas EHD3 supports TRE biogenesis and/or stabilization by an unknown mechanism. While using phospholipase D inhibitors (which block the conversion of glycerophospholipids to phosphatidic acid) to deplete cellular TRE, we observed that, upon inhibitor washout, there was a rapid and dramatic regeneration of MICAL-L1-marked TRE. Using this "synchronized" TRE biogenesis system, we determined that EHD3 is involved in the stabilization of TRE rather than in their biogenesis. Moreover, we identify the residues Ala-519/Asp-520 of EHD1 and Asn-519/Glu-520 of EHD3 as defining the selectivity of these two paralogs for NPF-containing binding partners, and we present a model to explain the atomic mechanism and provide new insight for their differential roles in vesiculation and tubulation, respectively.


Assuntos
Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Lipídeos de Membrana/metabolismo , Microtúbulos/metabolismo , Peptídeos/metabolismo , Asparagina/genética , Asparagina/metabolismo , Proteínas de Transporte/genética , Endossomos/genética , Glutamina/genética , Glutamina/metabolismo , Células HeLa , Humanos , Lipídeos de Membrana/genética , Microtúbulos/genética , Peptídeos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
4.
J Biol Chem ; 288(42): 30172-30180, 2013 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-24019528

RESUMO

Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane. Recent studies have led to the hypothesis that the C-terminal Eps15 homology domain (EHD) ATPase proteins are involved in the vesiculation process. Here, we address the functional roles of the four EHD proteins. We developed a novel semipermeabilized cell system in which addition of purified EHD proteins to reconstitute vesiculation allows us to assess the ability of each protein to vesiculate MICAL-L1-decorated tubular recycling endosomes (TREs). Using this assay, we show that EHD1 vesiculates membranes, consistent with enhanced TRE generation observed upon EHD1 depletion. EHD4 serves a role similar to that of EHD1 in TRE vesiculation, whereas EHD2, despite being capable of vesiculating TREs in the semipermeabilized cells, fails to do so in vivo. Surprisingly, the addition of EHD3 causes tubulation of endocytic membranes in our semipermeabilized cell system, consistent with the lack of tubulation observed upon EHD3 depletion. Our novel vesiculation assay and in vitro electron microscopy analysis, combined with in vivo data, provide evidence that the functions of both EHD1 and EHD4 are primarily in TRE membrane vesiculation, whereas EHD3 is a membrane-tubulating protein.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endossomos/química , Endossomos/genética , Endossomos/ultraestrutura , Células HeLa , Humanos , Membranas Intracelulares/química , Proteínas com Domínio LIM/química , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Proteínas dos Microfilamentos , Oxigenases de Função Mista , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
5.
bioRxiv ; 2023 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-38187679

RESUMO

Normal hematopoiesis requires constant prolific production of different blood cell lineages by multipotent hematopoietic stem cells (HSC). Stem- and progenitor- cells need to balance dormancy with proliferation. How genetic alterations impact frequency, lineage potential, and metabolism of HSC is largely unknown. Here, we compared induced expression of KRAS G12D or RasGRP1 to normal hematopoiesis. At low-resolution, both Ras pathway lesions result in skewing towards myeloid lineages. Single-cell resolution CyTOF proteomics unmasked an expansion of HSC- and progenitor- compartments for RasGRP1, contrasted by a depletion for KRAS G12D . SCENITH™ quantitates protein synthesis with single-cell precision and corroborated that immature cells display low metabolic SCENITH™ rates. Both RasGRP1 and KRAS G12D elevated mean SCENITH™ signals in immature cells. However, RasGRP1-overexpressing stem cells retain a metabolically quiescent cell-fraction, whereas this fraction diminishes for KRAS G12D . Our temporal single cell proteomics and metabolomics datasets provide a resource of mechanistic insights into altered hematopoiesis at single cell resolution.

6.
Stem Cell Reports ; 18(3): 636-653, 2023 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-36827975

RESUMO

Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy.


Assuntos
COVID-19 , Vírus da Influenza A Subtipo H1N1 , Humanos , SARS-CoV-2 , Organoides , Tetraspaninas/genética
7.
bioRxiv ; 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34100012

RESUMO

SARS coronavirus-2 (SARS-CoV-2) is causing a global pandemic with large variation in COVID-19 disease spectrum. SARS-CoV-2 infection requires host receptor ACE2 on lung epithelium, but epithelial underpinnings of variation are largely unknown. We capitalized on comprehensive organoid assays to report remarkable variation in SARS-CoV-2 infection rates of lung organoids from different subjects. Tropism is highest for TUBA- and MUC5AC-positive organoid cells, but levels of TUBA-, MUC5A-, or ACE2- positive cells do not predict infection rate. We identify surface molecule Tetraspanin 8 (TSPAN8) as novel mediator of SARS-CoV-2 infection, which is not downregulated by this specific virus. TSPAN8 levels, prior to infection, strongly correlate with infection rate and TSPAN8-blocking antibodies diminish SARS-CoV-2 infection. We propose TSPAN8 as novel functional biomarker and potential therapeutic target for COVID-19.

8.
Elife ; 92020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32510327

RESUMO

Dynamic trafficking of G protein-coupled receptors (GPCRs) out of cilia is mediated by the BBSome. In concert with its membrane recruitment factor, the small GTPase ARL6/BBS3, the BBSome ferries GPCRs across the transition zone, a diffusion barrier at the base of cilia. Here, we present the near-atomic structures of the BBSome by itself and in complex with ARL6GTP, and we describe the changes in BBSome conformation induced by ARL6GTP binding. Modeling the interactions of the BBSome with membranes and the GPCR Smoothened (SMO) reveals that SMO, and likely also other GPCR cargoes, must release their amphipathic helix 8 from the membrane to be recognized by the BBSome.


Assuntos
Proteínas de Transporte/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Bovinos , Microscopia Crioeletrônica , Regulação da Expressão Gênica , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes
9.
Mol Biol Cell ; 29(22): 2622-2631, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30188792

RESUMO

Centrosomes are the major microtubule-nucleating and microtubule-organizing centers of cells and play crucial roles in microtubule anchoring, organelle positioning, and ciliogenesis. At the centrosome core lies a tightly associated or "engaged" mother-daughter centriole pair.  During mitotic exit, removal of centrosomal proteins pericentrin and Cep215 promotes "disengagement" by the dissolution of intercentriolar linkers, ensuring a single centriole duplication event per cell cycle.  Herein, we explore a new mechanism involving vesicular trafficking for the removal of centrosomal Cep215. Using small interfering RNA and CRISPR/Cas9 gene-edited cells, we show that the endocytic protein EHD1 regulates Cep215 transport from centrosomes to the spindle midbody, thus facilitating disengagement and duplication. We demonstrate that EHD1 and Cep215 interact and show that Cep215 displays increased localization to vesicles containing EHD1 during mitosis. Moreover, Cep215-containing vesicles are positive for internalized transferrin, demonstrating their endocytic origin. Thus, we describe a novel relationship between endocytic trafficking and the centrosome cycle, whereby vesicles of endocytic origin are used to remove key regulatory proteins from centrosomes to control centriole duplication.


Assuntos
Centríolos/metabolismo , Vesículas Citoplasmáticas/metabolismo , Antígenos/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Citocinese , Endocitose , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico , Transferrina/metabolismo , Proteínas de Transporte Vesicular/metabolismo
10.
Mol Biol Cell ; 27(1): 108-26, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26510502

RESUMO

The endocytic recycling compartment (ERC) is a series of perinuclear tubular and vesicular membranes that regulates recycling to the plasma membrane. Despite evidence that cargo is sorted at the early/sorting endosome (SE), whether cargo mixes downstream at the ERC or remains segregated is an unanswered question. Here we use three-dimensional (3D) structured illumination microscopy and dual-channel and 3D direct stochastic optical reconstruction microscopy (dSTORM) to obtain new information about ERC morphology and cargo segregation. We show that cargo internalized either via clathrin-mediated endocytosis (CME) or independently of clathrin (CIE) remains segregated in the ERC, likely on distinct carriers. This suggests that no further sorting occurs upon cargo exit from SE. Moreover, 3D dSTORM data support a model in which some but not all ERC vesicles are tethered by contiguous "membrane bridges." Furthermore, tubular recycling endosomes preferentially traffic CIE cargo and may originate from SE membranes. These findings support a significantly altered model for endocytic recycling in mammalian cells in which sorting occurs in peripheral endosomes and segregation is maintained at the ERC.


Assuntos
Endossomos/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Células HeLa , Humanos , Microscopia/métodos , Transporte Proteico/fisiologia , Transferrina/metabolismo
11.
PLoS One ; 10(4): e0123710, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25875965

RESUMO

The C-terminal Eps 15 Homology Domain proteins (EHD1-4) play important roles in regulating endocytic trafficking. EHD2 is the only family member whose crystal structure has been solved, and it contains an unstructured loop consisting of two proline-phenylalanine (PF) motifs: KPFRKLNPF. In contrast, despite EHD2 having nearly 70% amino acid identity with its paralogs, EHD1, EHD3 and EHD4, the latter proteins contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to define the precise role of each PF motif in EHD2's homo-dimerization, binding with the protein partners, and subcellular localization. To test the role of the NPF motif, we generated an EHD2 NPF-to-NAF mutant to mimic the homologous sequences of EHD1 and EHD3. We demonstrated that this mutant lost both its ability to dimerize and bind to Syndapin2. However, it continued to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants displayed normal dimerization and Syndapin2 binding, but exhibited markedly increased nuclear localization and reduced association with the plasma membrane. We then hypothesized that the single PF motif of EHD1 (that aligns with the KPF of EHD2) might be responsible for both binding and localization functions of EHD1. Indeed, the EHD1 RPF motif was required for dimerization, interaction with MICAL-L1 and Syndapin2, as well as localization to tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. Overall, our data suggest that the EHD2 NPF phenylalanine residue is crucial for EHD2 localization to the plasma membrane, whereas the proline residue is essential for EHD2 dimerization and binding. These studies support the recently proposed model in which the EHD2 N-terminal region may regulate the availability of the unstructured loop for interactions with neighboring EHD2 dimers, thus promoting oligomerization.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia Confocal , Modelos Moleculares , Mutação , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Prolina/química , Prolina/genética , Prolina/metabolismo , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido
12.
Biomark Insights ; 7: 27-37, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22493560

RESUMO

BACKGROUND: Early stages of esophageal cancer lack a specific symptom, a reliable biomarker and accurate non-invasive diagnostic modalities prompting the pressing need for identification of a marker for early diagnosis of this disease. METHODS: In the present study we investigated the levels of circulating and tissue mRNAs of Oct-3/4, Sox-2, Nanog and Bmi-1 in esophageal cancer patients using Reverse-Transcription Polymerase Chain Reaction (RT-PCR) with the aim of evaluating their potential as minimally invasive diagnostic markers. RESULT: Increased transcript levels of Oct-4, Sox-2, Bmi-1 and Nanog were detected in (92%), (95%), (75%) and (67%) of the esophageal cancer tissues, respectively as compared with the matched distant normals. CONCLUSION: Interestingly, most of the preneoplastic tissues exhibited increased transcript levels of these stemness markers suggesting their role in early stages of esophageal tumorigenesis. Furthermore, the detection of elevated levels of circulating mRNAs of Oct-4 and Nanog in sera of esophageal cancer patients emphasizes their potential as minimally invasive diagnostic markers for esophageal cancer.

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