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1.
Life Sci Alliance ; 4(9)2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34226277

RESUMO

Here, we recorded serum proteome profiles of 33 severe COVID-19 patients admitted to respiratory and intensive care units because of respiratory failure. We received, for most patients, blood samples just after admission and at two more later time points. With the aim to predict treatment outcome, we focused on serum proteins different in abundance between the group of survivors and non-survivors. We observed that a small panel of about a dozen proteins were significantly different in abundance between these two groups. The four structurally and functionally related type-3 cystatins AHSG, FETUB, histidine-rich glycoprotein, and KNG1 were all more abundant in the survivors. The family of inter-α-trypsin inhibitors, ITIH1, ITIH2, ITIH3, and ITIH4, were all found to be differentially abundant in between survivors and non-survivors, whereby ITIH1 and ITIH2 were more abundant in the survivor group and ITIH3 and ITIH4 more abundant in the non-survivors. ITIH1/ITIH2 and ITIH3/ITIH4 also showed opposite trends in protein abundance during disease progression. We defined an optimal panel of nine proteins for mortality risk assessment. The prediction power of this mortality risk panel was evaluated against two recent COVID-19 serum proteomics studies on independent cohorts measured in other laboratories in different countries and observed to perform very well in predicting mortality also in these cohorts. This panel may not be unique for COVID-19 as some of the proteins in the panel have previously been annotated as mortality markers in aging and in other diseases caused by different pathogens, including bacteria.


Assuntos
COVID-19/sangue , COVID-19/mortalidade , Proteoma/metabolismo , Índice de Gravidade de Doença , Idoso , COVID-19/virologia , Estudos de Coortes , Feminino , Hospitalização , Humanos , Imunoglobulinas/sangue , Masculino , SARS-CoV-2/fisiologia , Sobreviventes
2.
J Clin Invest ; 130(9): 4637-4651, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32484803

RESUMO

γ9δ2T cells play a major role in cancer immune surveillance, yet the clinical translation of their in vitro promise remains challenging. To address limitations of previous clinical attempts using expanded γ9δ2T cells, we explored the clonal diversity of γ9δ2T cell repertoires and characterized their target. We demonstrated that only a fraction of expanded γ9δ2T cells was active against cancer cells and that activity of the parental clone, or functional avidity of selected γ9δ2 T cell receptors (γ9δ2TCRs), was not associated with clonal frequency. Furthermore, we analyzed the target-receptor interface and provided a 2-receptor, 3-ligand model. We found that activation was initiated by binding of the γ9δ2TCR to BTN2A1 through the regions between CDR2 and CDR3 of the TCR γ chain and modulated by the affinity of the CDR3 region of the TCRδ chain, which was phosphoantigen independent (pAg independent) and did not depend on CD277. CD277 was secondary, serving as a mandatory coactivating ligand. We found that binding of CD277 to its putative ligand did not depend on the presence of γ9δ2TCR, did depend on usage of the intracellular CD277, created pAg-dependent proximity to BTN2A1, enhanced cell-cell conjugate formation, and stabilized the immunological synapse (IS). This process critically depended on the affinity of the γ9δ2TCR and required membrane flexibility of the γ9δ2TCR and CD277, facilitating their polarization and high-density recruitment during IS formation.


Assuntos
Proliferação de Células , Ativação Linfocitária , Modelos Imunológicos , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Antígenos de Neoplasias/imunologia , Butirofilinas/imunologia , Humanos , Células Jurkat , Proteínas de Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/patologia
3.
Cell Mol Immunol ; 16(5): 460-472, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-29568119

RESUMO

The triggering receptor expressed on myeloid cells-1 (TREM-1) is a receptor expressed on innate immune cells. By promoting the amplification of inflammatory signals that are initially triggered by Toll-like receptors (TLRs), TREM-1 has been characterized as a major player in the pathophysiology of acute and chronic inflammatory diseases, such as septic shock, myocardial infarction, atherosclerosis, and inflammatory bowel diseases. However, the molecular events leading to the activation of TREM-1 in innate immune cells remain unknown. Here, we show that TREM-1 is activated by multimerization and that the levels of intracellular Ca2+ release, reactive oxygen species, and cytokine production correlate with the degree of TREM-1 aggregation. TREM-1 activation on primary human monocytes by LPS required a two-step process consisting of upregulation followed by clustering of TREM-1 at the cell surface, in contrast to primary human neutrophils, where LPS induced a rapid cell membrane reorganization of TREM-1, which confirmed that TREM-1 is regulated differently in primary human neutrophils and monocytes. In addition, we show that the ectodomain of TREM-1 is able to homooligomerize in a concentration-dependent manner, which suggests that the clustering of TREM-1 on the membrane promotes its oligomerization. We further show that the adapter protein DAP12 stabilizes TREM-1 surface expression and multimerization. TREM-1 multimerization at the cell surface is also mediated by its endogenous ligand, a conclusion supported by the ability of the TREM-1 inhibitor LR12 to limit TREM-1 multimerization. These results provide evidence for ligand-induced, receptor-mediated dimerization of TREM-1. Collectively, our findings uncover the mechanisms necessary for TREM-1 activation in monocytes and neutrophils.


Assuntos
Membrana Celular/metabolismo , Inflamação/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sinalização do Cálcio , Humanos , Imunidade Inata , Lipopolissacarídeos , Proteínas de Membrana/metabolismo , Cultura Primária de Células , Multimerização Proteica , Espécies Reativas de Oxigênio/metabolismo , Agregação de Receptores , Receptor Gatilho 1 Expresso em Células Mieloides/imunologia , Células U937
4.
Parasitology ; 146(1): 33-41, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29871709

RESUMO

Apicomplexan parasites have unconventional actins that play a central role in important cellular processes such as apicoplast replication, motility of dense granules, endocytic trafficking and force generation for motility and host cell invasion. In this study, we investigated the actin of the apicomplexan Neospora caninum - a parasite associated with infectious abortion and neonatal mortality in livestock. Neospora caninum actin was detected and identified in two bands by one-dimensional (1D) western blot and in nine spots by the 2D technique. The mass spectrometry data indicated that N. caninum has at least nine different actin isoforms, possibly caused by post-translational modifications. In addition, the C4 pan-actin antibody detected specifically actin in N. caninum cellular extract. Extracellular N. caninum tachyzoites were treated with toxins that act on actin, jasplakinolide and cytochalasin D. Both substances altered the peripheric cytoplasmic localization of actin on tachyzoites. Our findings add complexity to the study of the apicomplexan actin in cellular processes, since the multiple functions of this important protein might be regulated by mechanisms involving post-translational modifications.


Assuntos
Aborto Séptico/veterinária , Actinas/química , Coccidiose/veterinária , Neospora/química , Aborto Séptico/mortalidade , Actinas/isolamento & purificação , Animais , Animais Recém-Nascidos , Western Blotting , Chlorocebus aethiops , Coccidiose/mortalidade , Simulação por Computador , Eletroforese em Gel Bidimensional , Feminino , Imunofluorescência , Cromatografia Gasosa-Espectrometria de Massas , Gado , Gravidez , Isoformas de Proteínas , Proteômica/métodos , Alinhamento de Sequência , Células Vero
5.
Elife ; 62017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28290984

RESUMO

Microtubules are dynamic polymers that in cells can grow, shrink or pause, but the factors that promote pausing are poorly understood. Here, we show that the mammalian kinesin-4 KIF21B is a processive motor that can accumulate at microtubule plus ends and induce pausing. A few KIF21B molecules are sufficient to induce strong growth inhibition of a microtubule plus end in vitro. This property depends on non-motor microtubule-binding domains located in the stalk region and the C-terminal WD40 domain. The WD40-containing KIF21B tail displays preference for a GTP-type over a GDP-type microtubule lattice and contributes to the interaction of KIF21B with microtubule plus ends. KIF21B also contains a motor-inhibiting domain that does not fully block the interaction of the protein with microtubules, but rather enhances its pause-inducing activity by preventing KIF21B detachment from microtubule tips. Thus, KIF21B combines microtubule-binding and regulatory activities that together constitute an autonomous microtubule pausing factor.


Assuntos
Cinesina/metabolismo , Microtúbulos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Multimerização Proteica
6.
EMBO J ; 35(24): 2634-2657, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27797822

RESUMO

The emergence of proteomics has led to major technological advances in mass spectrometry (MS). These advancements not only benefitted MS-based high-throughput proteomics but also increased the impact of mass spectrometry on the field of structural and molecular biology. Here, we review how state-of-the-art MS methods, including native MS, top-down protein sequencing, cross-linking-MS, and hydrogen-deuterium exchange-MS, nowadays enable the characterization of biomolecular structures, functions, and interactions. In particular, we focus on the role of mass spectrometry in integrated structural and molecular biology investigations of biological macromolecular complexes and cellular machineries, highlighting work on CRISPR-Cas systems and eukaryotic transcription complexes.


Assuntos
Espectrometria de Massas/métodos , Biologia Molecular/tendências , Substâncias Macromoleculares/química
7.
Elife ; 52016 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-27410476

RESUMO

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5ß and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.


Assuntos
Adesões Focais/metabolismo , Microtúbulos/metabolismo , Talina/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas do Citoesqueleto , Células HEK293 , Células HeLa , Humanos
8.
Curr Opin Chem Biol ; 30: 14-20, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26590485

RESUMO

Both genomics and proteomics technologies have matured in the last decade to a level where they are able to deliver system-wide data on the qualitative and quantitative abundance of their respective molecular entities, that is DNA/RNA and proteins. A next logical step is the collective use of these technologies, ideally gathering data on matching samples. The first large scale so-called proteogenomics studies are emerging, and display the benefits each of these layers of analysis has on the other layers to together generate more meaningful insight into the connection between the phenotype/physiology and genotype of the system under study. Here we review a selected number of these studies, highlighting what they can uniquely deliver. We also discuss the future potential and remaining challenges, from a somewhat proteome biased perspective.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteômica/métodos , Animais , Humanos
9.
Proteome Sci ; 7: 46, 2009 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-20034391

RESUMO

BACKGROUND: Progression through the cell cycle is accompanied by tightly controlled regulation of transcription. On one hand, a subset of genes is expressed in a cell cycle-dependent manner. On the other hand, a general inhibition of transcription occurs during mitosis. Genetic and genome-wide studies suggest cell cycle regulation at the level of transcription initiation by protein complexes containing the common DNA-binding subunit TATA binding protein (TBP). TBP is a key player in regulating transcription by all three nuclear RNA polymerases. It forms at least four distinct protein complexes with TBP-associated factors (TAFs): SL1, B-TFIID, TFIID, and TFIIIB. Some TAFs are known to remain associated with TBP during the cell cycle. Here we analyze all TAFs and their phosphorylation status during the cell cycle using a quantitative mass spectrometry approach. RESULTS: TBP protein complexes present in human cells at the G2/M and G1/S transitions were analyzed by combining affinity purification with quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC). Phosphorylations were mapped and quantified after enrichment of tryptic peptides by titanium dioxide. This revealed that subunit stoichiometries of TBP complexes remained intact, but their relative abundances in nuclear extracts changed during the cell cycle. Several novel phosphorylations were detected on subunits of the TBP complexes TFIID and SL1. G2/M-specific phosphorylations were detected on TAF1, TAF4, TAF7, and TAFI41/TAF1D, and G1/S-specific dephosphorylations were detected on TAF3. Many phosphorylated residues were evolutionary conserved from human to zebrafish and/or drosophila, and were present in conserved regions suggesting important regulatory functions. CONCLUSIONS: This study provides the first quantitative proteomic analysis of human TBP containing protein complexes at the G2/M and G1/S transitions, and identifies new cell cycle-dependent phosphorylations on TAFs present in their protein complex. We speculate that phosphorylation of complex-specific subunits may be involved in regulating the activities of TBP protein complexes during the cell cycle.

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