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1.
Lancet Oncol ; 25(6): e260-e269, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38821100

RESUMO

Theranostics has become a major area of innovation and progress in cancer care over the last decade. In view of the introduction of approved therapeutics in neuroendocrine tumours and prostate cancer in the last 10 years, the ability to provide access to these treatments has emerged as a key factor in ensuring global benefits from this cancer therapy approach. In this Series paper we explore the issues that affect access to and availability of theranostic radiopharmaceuticals, including supply and regulatory issues that might affect the availability of theranostic treatments for patients with cancer.


Assuntos
Compostos Radiofarmacêuticos , Nanomedicina Teranóstica , Humanos , Compostos Radiofarmacêuticos/uso terapêutico , Neoplasias/terapia , Medicina de Precisão
2.
Am J Physiol Lung Cell Mol Physiol ; 327(1): L19-L39, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38712429

RESUMO

Mechanical ventilation can cause ventilation-induced lung injury (VILI). The concept of stress concentrations suggests that surfactant dysfunction-induced microatelectases might impose injurious stresses on adjacent, open alveoli and function as germinal centers for injury propagation. The aim of the present study was to quantify the histopathological pattern of VILI progression and to test the hypothesis that injury progresses at the interface between microatelectases and ventilated lung parenchyma during low-positive end-expiratory pressure (PEEP) ventilation. Bleomycin was used to induce lung injury with microatelectases in rats. Lungs were then mechanically ventilated for up to 6 h at PEEP = 1 cmH2O and compared with bleomycin-treated group ventilated protectively with PEEP = 5 cmH2O to minimize microatelectases. Lung mechanics were measured during ventilation. Afterward, lungs were fixed at end-inspiration or end-expiration for design-based stereology. Before VILI, bleomycin challenge reduced the number of open alveoli [N(alvair,par)] by 29%. No differences between end-inspiration and end-expiration were observed. Collapsed alveoli clustered in areas with a radius of up to 56 µm. After PEEP = 5 cmH2O ventilation for 6 h, N(alvair,par) remained stable while PEEP = 1 cmH2O ventilation led to an additional loss of aerated alveoli by 26%, mainly due to collapse, with a small fraction partly edema filled. Alveolar loss strongly correlated to worsening of tissue elastance, quasistatic compliance, and inspiratory capacity. The radius of areas of collapsed alveoli increased to 94 µm, suggesting growth of the microatelectases. These data provide evidence that alveoli become unstable in neighborhood of microatelectases, which most likely occurs due to stress concentration-induced local vascular leak and surfactant dysfunction.NEW & NOTEWORTHY Low-volume mechanical ventilation in the presence of high surface tension-induced microatelectases leads to the degradation of lung mechanical function via the progressive loss of alveoli. Microatelectases grow at the interfaces of collapsed and open alveoli. Here, stress concentrations might cause injury and alveolar instability. Accumulation of small amounts of alveolar edema can be found in a fraction of partly collapsed alveoli but, in this model, alveolar flooding is not a major driver for degradation of lung mechanics.


Assuntos
Respiração com Pressão Positiva , Alvéolos Pulmonares , Lesão Pulmonar Induzida por Ventilação Mecânica , Animais , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/fisiopatologia , Ratos , Masculino , Respiração com Pressão Positiva/métodos , Respiração com Pressão Positiva/efeitos adversos , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologia , Bleomicina/toxicidade , Bleomicina/efeitos adversos , Ratos Sprague-Dawley , Pulmão/patologia , Pulmão/fisiopatologia , Respiração Artificial/efeitos adversos , Respiração Artificial/métodos , Mecânica Respiratória , Atelectasia Pulmonar/patologia , Atelectasia Pulmonar/fisiopatologia
3.
Subcell Biochem ; 101: 247-291, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36520310

RESUMO

In mammalian cells, the rough endoplasmic reticulum (ER) plays central roles in the biogenesis of extracellular plus organellar proteins and in various signal transduction pathways. For these reasons, the ER comprises molecular chaperones, which are involved in import, folding, assembly, export, plus degradation of polypeptides, and signal transduction components, such as calcium channels, calcium pumps, and UPR transducers plus adenine nucleotide carriers/exchangers in the ER membrane. The calcium- and ATP-dependent ER lumenal Hsp70, termed immunoglobulin heavy-chain-binding protein or BiP, is the central player in all these activities and involves up to nine different Hsp40-type co-chaperones, i.e., ER membrane integrated as well as ER lumenal J-domain proteins, termed ERj or ERdj proteins, two nucleotide exchange factors or NEFs (Grp170 and Sil1), and NEF-antagonists, such as MANF. Here we summarize the current knowledge on the ER-resident BiP/ERj chaperone network and focus on the interaction of BiP with the polypeptide-conducting and calcium-permeable Sec61 channel of the ER membrane as an example for BiP action and how its functional cycle is linked to ER protein import and various calcium-dependent signal transduction pathways.


Assuntos
Cálcio , Retículo Endoplasmático , Animais , Humanos , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/metabolismo , Transporte Proteico , Chaperona BiP do Retículo Endoplasmático , Mamíferos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo
4.
Adv Exp Med Biol ; 1406: 79-102, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37016112

RESUMO

Technology has revolutionized the way dentists are able to treat their patients. These technological advances have paved the way for the creation of virtual patient models utilizing these 3-dimensional intra-oral patient models, cone bean computer tomography (CBCT) radiograph scans, extraoral 3-dimensional scans, and jaw motion tracings to create a patient-specific model. These models are advantageous in planning surgical treatments by providing 3-dimensional views of vital anatomical structures to accurately identify the location, size, and shape of a structure or defect in order to plan accordingly. Virtual augmentation of either hard tissue (bone) and/or soft tissue (i.e., gingiva) can also be accomplished.Technology has allowed the capture of the dynamic motions of the jaw and combined them with the virtual patient to develop permanent restorations in harmony with the patient's orofacial complex. With the introduction of new technology in the realm of digital dentistry, patient care is being brought to a new and higher level. This creates a level of more optimal care that a dentist can deliver to patients.


Assuntos
Tomografia Computadorizada de Feixe Cônico , Imageamento Tridimensional , Humanos , Imageamento Tridimensional/métodos , Tomografia Computadorizada de Feixe Cônico/métodos , Invenções , Odontologia
5.
Int J Mol Sci ; 24(18)2023 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-37762469

RESUMO

To elucidate the redundancy in the components for the targeting of membrane proteins to the endoplasmic reticulum (ER) and/or their insertion into the ER membrane under physiological conditions, we previously analyzed different human cells by label-free quantitative mass spectrometry. The HeLa and HEK293 cells had been depleted of a certain component by siRNA or CRISPR/Cas9 treatment or were deficient patient fibroblasts and compared to the respective control cells by differential protein abundance analysis. In addition to clients of the SRP and Sec61 complex, we identified membrane protein clients of components of the TRC/GET, SND, and PEX3 pathways for ER targeting, and Sec62, Sec63, TRAM1, and TRAP as putative auxiliary components of the Sec61 complex. Here, a comprehensive evaluation of these previously described differential protein abundance analyses, as well as similar analyses on the Sec61-co-operating EMC and the characteristics of the topogenic sequences of the various membrane protein clients, i.e., the client spectra of the components, are reported. As expected, the analysis characterized membrane protein precursors with cleavable amino-terminal signal peptides or amino-terminal transmembrane helices as predominant clients of SRP, as well as the Sec61 complex, while precursors with more central or even carboxy-terminal ones were found to dominate the client spectra of the SND and TRC/GET pathways for membrane targeting. For membrane protein insertion, the auxiliary Sec61 channel components indeed share the client spectra of the Sec61 complex to a large extent. However, we also detected some unexpected differences, particularly related to EMC, TRAP, and TRAM1. The possible mechanistic implications for membrane protein biogenesis at the human ER are discussed and can be expected to eventually advance our understanding of the mechanisms that are involved in the so-called Sec61-channelopathies, resulting from deficient ER protein import.


Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Humanos , Células HEK293 , Canais de Translocação SEC , Espectrometria de Massas
6.
J Biol Chem ; 296: 100295, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33460650

RESUMO

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone glucose-regulated protein 78 (GRP78). We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time that MANF promotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting several UPR pathways simultaneously. Interestingly, MANF does not affect naïve neurons. We hypothesize that MANF regulates UPR signaling toward a mode more compatible with neuronal survival. Screening of MANF interacting proteins from two mammalian cell lines revealed a conserved interactome of 15 proteins including several ER chaperones such as GRP78, GRP170, protein disulfide isomerase family A member 1, and protein disulfide isomerase family A member 6. Further characterization confirmed previously published finding that MANF is a cofactor of GRP78 interacting with its nucleotide binding domain. Using microscale thermophoresis and nuclear magnetic resonance spectroscopy, we discovered that MANF is an ATP binding protein and that ATP blocks the MANF-GRP78 interaction. Interestingly, functional analysis of the antiapoptotic properties of MANF mutants in cultured neurons revealed divergent roles of MANF as a GRP78 cofactor and as an antiapoptotic regulator of UPR. We conclude that the co-factor type interaction with GRP78 is dispensable for the survival-promoting activity of MANF in neurons.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Retículo Endoplasmático/genética , Proteínas de Choque Térmico/genética , Fatores de Crescimento Neural/genética , Resposta a Proteínas não Dobradas , Animais , Apoptose/genética , Sobrevivência Celular , Neurônios Dopaminérgicos/citologia , Embrião de Mamíferos , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Mesencéfalo/citologia , Mesencéfalo/metabolismo , Camundongos , Fatores de Crescimento Neural/metabolismo , Cultura Primária de Células , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Mapeamento de Interação de Proteínas , Transdução de Sinais
7.
Hum Mol Genet ; 29(1): 97-116, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31691803

RESUMO

Corneal endothelial cell (CEnC) loss is often associated with blinding endothelial corneal dystrophies: dominantly inherited, common (5%) Fuchs endothelial corneal dystrophy (FECD) and recessive, rare congenital hereditary endothelial dystrophy (CHED). Mutations of SLC4A11, an abundant corneal solute transporter, cause CHED and some cases of FECD. The link between defective SLC4A11 solute transport function and CEnC loss is, however, unclear. Cell adhesion assays using SLC4A11-transfected HEK293 cells and primary human CEnC revealed that SLC4A11 promotes adhesion to components of Descemet's membrane (DM), the basement membrane layer to which CEnC bind. An antibody against SLC4A11 extracellular loop 3 (EL3) suppressed cell adhesion, identifying EL3 as the DM-binding site. Earlier studies showed that some SLC4A11 mutations cause FECD and CHED by impairing solute transport activity or cell surface trafficking. Without affecting these functions, FECD-causing mutations in SLC4A11-EL3 compromised cell adhesion capacity. In an energy-minimized SLC4A11-EL3 three-dimensional model, these mutations cluster and are buried within the EL3 structure. A GST fusion protein of SLC4A11-EL3 interacts with principal DM protein, COL8A2, as identified by mass spectrometry. Engineered SLC4A11-EL3-containing protein, STIC (SLC4A11-EL3 Transmembrane-GPA Integrated Chimera), promotes cell adhesion in transfected HEK293 cells and primary human CEnC, confirming the cell adhesion role of EL3. Taken together, the data suggest that SLC4A11 directly binds DM to serve as a cell adhesion molecule (CAM). These data further suggest that cell adhesion defects contribute to FECD and CHED pathology. Observations with STIC point toward a new therapeutic direction in these diseases: replacement of lost cell adhesion capacity.


Assuntos
Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Adesão Celular/fisiologia , Distrofias Hereditárias da Córnea/metabolismo , Proteínas de Transporte de Ânions/genética , Antiporters/genética , Adesão Celular/genética , Células Cultivadas , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/patologia , Lâmina Limitante Posterior/metabolismo , Células HEK293 , Humanos , Mutação/genética
8.
Nature ; 540(7631): 134-138, 2016 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-27905431

RESUMO

In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Domínios Proteicos , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Ribossômicas/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo
9.
Biochem J ; 478(22): 4005-4024, 2021 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-34726690

RESUMO

The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon 'breathing' during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone's stabilisation of the latter underpins the phenotype observed.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Macrolídeos/farmacologia , Canais de Translocação SEC/metabolismo , Animais , Células HCT116 , Células HEK293 , Humanos , Camundongos , Células RAW 264.7
10.
Int J Mol Sci ; 23(10)2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35628123

RESUMO

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of approximately 10,000 different soluble and membrane proteins of human cells, which amounts to about 30% of the proteome [...].


Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Transporte Proteico
11.
BMC Biol ; 18(1): 19, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32101139

RESUMO

BACKGROUND: The lumen of the endoplasmic reticulum (ER) acts as a cellular Ca2+ store and a site for oxidative protein folding, which is controlled by the reduced glutathione (GSH) and glutathione-disulfide (GSSG) redox pair. Although depletion of luminal Ca2+ from the ER provokes a rapid and reversible shift towards a more reducing poise in the ER, the underlying molecular basis remains unclear. RESULTS: We found that Ca2+ mobilization-dependent ER luminal reduction was sensitive to inhibition of GSH synthesis or dilution of cytosolic GSH by selective permeabilization of the plasma membrane. A glutathione-centered mechanism was further indicated by increased ER luminal glutathione levels in response to Ca2+ efflux. Inducible reduction of the ER lumen by GSH flux was independent of the Ca2+-binding chaperone calreticulin, which has previously been implicated in this process. However, opening the translocon channel by puromycin or addition of cyclosporine A mimicked the GSH-related effect of Ca2+ mobilization. While the action of puromycin was ascribable to Ca2+ leakage from the ER, the mechanism of cyclosporine A-induced GSH flux was independent of calcineurin and cyclophilins A and B and remained unclear. CONCLUSIONS: Our data strongly suggest that ER influx of cytosolic GSH, rather than inhibition of local oxidoreductases, is responsible for the reductive shift upon Ca2+ mobilization. We postulate the existence of a Ca2+- and cyclosporine A-sensitive GSH transporter in the ER membrane. These findings have important implications for ER redox homeostasis under normal physiology and ER stress.


Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Glutationa/metabolismo , Calreticulina/metabolismo , Humanos , Ligação Proteica
12.
J Allergy Clin Immunol ; 146(5): 1180-1193, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32325141

RESUMO

BACKGROUND: The molecular cause of severe congenital neutropenia (SCN) is unknown in 30% to 50% of patients. SEC61A1 encodes the α-subunit of the Sec61 complex, which governs endoplasmic reticulum protein transport and passive calcium leakage. Recently, mutations in SEC61A1 were reported to be pathogenic in common variable immunodeficiency and glomerulocystic kidney disease. OBJECTIVE: Our aim was to expand the spectrum of SEC61A1-mediated disease to include autosomal dominant SCN. METHODS: Whole exome sequencing findings were validated, and reported mutations were compared by Western blotting, Ca2+ flux assays, differentiation of transduced HL-60 cells, in vitro differentiation of primary CD34 cells, quantitative PCR for unfolded protein response (UPR) genes, and single-cell RNA sequencing on whole bone marrow. RESULTS: We identified a novel de novo missense mutation in SEC61A1 (c.A275G;p.Q92R) in a patient with SCN who was born to nonconsanguineous Belgian parents. The mutation results in diminished protein expression, disturbed protein translocation, and an increase in calcium leakage from the endoplasmic reticulum. In vitro differentiation of CD34+ cells recapitulated the patient's clinical arrest in granulopoiesis. The impact of Q92R-Sec61α1 on neutrophil maturation was validated by using HL-60 cells, in which transduction reduced differentiation into CD11b+CD16+ cells. A potential mechanism for this defect is the uncontrolled initiation of the unfolded protein stress response, with single-cell analysis of primary bone marrow revealing perturbed UPR in myeloid precursors and in vitro differentiation of primary CD34+ cells revealing upregulation of CCAAT/enhancer-binding protein homologous protein and immunoglobulin heavy chain binding protein UPR-response genes. CONCLUSION: Specific mutations in SEC61A1 cause SCN through dysregulation of the UPR.


Assuntos
Síndrome Congênita de Insuficiência da Medula Óssea/genética , Mutação/genética , Neutropenia/congênito , Neutrófilos/fisiologia , Canais de Translocação SEC/genética , Antígenos CD34/metabolismo , Transtornos Cromossômicos , Feminino , Genes Dominantes , Células HL-60 , Humanos , Neutropenia/genética , Linhagem , Análise de Célula Única , Resposta a Proteínas não Dobradas/genética , Sequenciamento do Exoma , Adulto Jovem
13.
Int J Mol Sci ; 22(23)2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34884833

RESUMO

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co- or post-translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients.


Assuntos
Retículo Endoplasmático/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Peroxinas/metabolismo , Proteoma/análise , Proteômica/métodos , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Humanos , Lipoproteínas/antagonistas & inibidores , Lipoproteínas/genética , Espectrometria de Massas , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Peroxinas/antagonistas & inibidores , Peroxinas/genética , Peroxissomos/metabolismo , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Síndrome de Zellweger/metabolismo , Síndrome de Zellweger/patologia
14.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208277

RESUMO

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.


Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Transporte/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteoma/análise , Proteoma/metabolismo , RNA Mensageiro/genética
15.
Subcell Biochem ; 93: 83-141, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31939150

RESUMO

The membrane of the endoplasmic reticulum (ER) in human cells harbors the protein translocon, which facilitates membrane insertion and translocation of almost every newly synthesized polypeptide targeted to organelles of the secretory pathway. The translocon comprises the polypeptide-conducting Sec61 channel and several additional proteins, which are associated with the heterotrimeric Sec61 complex. This ensemble of proteins facilitates ER targeting of precursor polypeptides, Sec61 channel opening and closing, and modification of precursor polypeptides in transit through the Sec61 complex. Recently, cryoelectron tomography of translocons in native ER membranes has given unprecedented insights into the architecture and dynamics of the native, ribosome-associated translocon and the Sec61 channel. These structural data are discussed in light of different Sec61 channel activities including ribosome receptor function, membrane insertion or translocation of newly synthesized polypeptides as well as the possible roles of the Sec61 channel as a passive ER calcium leak channel and regulator of ATP/ADP exchange between cytosol and ER.


Assuntos
Proteínas de Membrana/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/química , Transporte Proteico , Canais de Translocação SEC/química , Canais de Translocação SEC/metabolismo
16.
Int J Mol Sci ; 21(5)2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32138345

RESUMO

Isoform one of the mammalian Na+/H+ exchanger is a plasma membrane protein that is ubiquitously present in humans. It regulates intracellular pH through the removal of one intracellular proton in exchange for a single extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, C-terminal tail. We examined amino acids of the C-terminal tail that are important in the targeting and activity of the protein. A previous study demonstrated that stop codon polymorphisms can result in decreased activity, expression, targeting and enhanced protein degradation. Here, we determine elements that are critical in these anomalies. A series of progressive deletions of the C-terminal tail demonstrated a progressive decrease in activity and targeting, though these remained until a final drop off with the deletion of amino acids 563-566. The deletion of the 562LIAGERS568 sequence or the alteration to the 562LAAAARS568 sequence caused the decreased protein expression, aberrant targeting, reduced activity and enhanced degradation of the Na+/H+ exchanger (NHE1) protein. The 562LIAGERS568 sequence bound to other regions of the C-terminal cytosolic domain. We suggest this region is necessary for the activity, targeting, stability, and expression of the NHE1 protein. The results define a new sequence that is important in maintenance of NHE1 protein levels and activity.


Assuntos
Isoformas de Proteínas/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Plasmídeos/genética , Isoformas de Proteínas/genética , Estabilidade Proteica , Proteólise , Trocador 1 de Sódio-Hidrogênio/genética
17.
Int J Mol Sci ; 21(9)2020 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-32397251

RESUMO

The plasma membrane transporter SOS1 (SALT-OVERLY SENSITIVE1) is vital for plant survival under salt stress. SOS1 activity is tightly regulated, but little is known about the underlying mechanism. SOS1 contains a cytosolic, autoinhibitory C-terminal tail (abbreviated as SOS1 C-term), which is targeted by the protein kinase SOS2 to trigger its transport activity. Here, to identify additional binding proteins that regulate SOS1 activity, we synthesized the SOS1 C-term domain and used it as bait to probe Arabidopsis thaliana cell extracts. Several 14-3-3 proteins, which function in plant salt tolerance, specifically bound to and interacted with the SOS1 C-term. Compared to wild-type plants, when exposed to salt stress, Arabidopsis plants overexpressing SOS1 C-term showed improved salt tolerance, significantly reduced Na+ accumulation in leaves, reduced induction of the salt-responsive gene WRKY25, decreased soluble sugar, starch, and proline levels, less impaired inflorescence formation and increased biomass. It appears that overexpressing SOS1 C-term leads to the sequestration of inhibitory 14-3-3 proteins, allowing SOS1 to be more readily activated and leading to increased salt tolerance. We propose that the SOS1 C-term binds to previously unknown proteins such as 14-3-3 isoforms, thereby regulating salt tolerance. This finding uncovers another regulatory layer of the plant salt tolerance program.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Tolerância ao Sal/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Biomassa , Citosol/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Folhas de Planta/metabolismo , Prolina/metabolismo , Ligação Proteica , Domínios Proteicos/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Amido/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
18.
J Allergy Clin Immunol ; 141(4): 1427-1438, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28782633

RESUMO

BACKGROUND: Primary antibody deficiencies (PADs) are the most frequent primary immunodeficiencies in human subjects. The genetic causes of PADs are largely unknown. Sec61 translocon alpha 1 subunit (SEC61A1) is the major subunit of the Sec61 complex, which is the main polypeptide-conducting channel in the endoplasmic reticulum membrane. SEC61A1 is a target gene of spliced X-box binding protein 1 and strongly induced during plasma cell (PC) differentiation. OBJECTIVE: We identified a novel genetic defect and studied its pathologic mechanism in 11 patients from 2 unrelated families with PADs. METHODS: Whole-exome and targeted sequencing were conducted to identify novel genetic mutations. Functional studies were carried out ex vivo in primary cells of patients and in vitro in different cell lines to assess the effect of SEC61A1 mutations on B-cell differentiation and survival. RESULTS: We investigated 2 families with patients with hypogammaglobulinemia, severe recurrent respiratory tract infections, and normal peripheral B- and T-cell subpopulations. On in vitro stimulation, B cells showed an intrinsic deficiency to develop into PCs. Genetic analysis and targeted sequencing identified novel heterozygous missense (c.254T>A, p.V85D) and nonsense (c.1325G>T, p.E381*) mutations in SEC61A1, segregating with the disease phenotype. SEC61A1-V85D was deficient in cotranslational protein translocation, and it disturbed the cellular calcium homeostasis in HeLa cells. Moreover, SEC61A1-V85D triggered the terminal unfolded protein response in multiple myeloma cell lines. CONCLUSION: We describe a monogenic defect leading to a specific PC deficiency in human subjects, expanding our knowledge about the pathogenesis of antibody deficiencies.


Assuntos
Síndromes de Imunodeficiência/genética , Mutação/genética , Plasmócitos/patologia , Canais de Translocação SEC/genética , Agamaglobulinemia/genética , Agamaglobulinemia/metabolismo , Agamaglobulinemia/patologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Cálcio/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Exoma/genética , Células HEK293 , Células HeLa , Heterozigoto , Humanos , Síndromes de Imunodeficiência/metabolismo , Plasmócitos/metabolismo , Transporte Proteico/genética , Infecções Respiratórias/genética , Infecções Respiratórias/metabolismo , Infecções Respiratórias/patologia , Linfócitos T/metabolismo , Linfócitos T/patologia , Resposta a Proteínas não Dobradas/genética
19.
J Biol Chem ; 292(52): 21383-21396, 2017 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-29084847

RESUMO

About one-quarter to nearly one-third of the proteins synthesized in the cytosol of eukaryotic cells are integrated into the plasma membrane or are secreted. Translocation of secretory proteins into the lumen of the endoplasmic reticulum or the periplasm of bacteria is mediated by a highly conserved heterotrimeric membrane protein complex denoted Sec61 in eukaryotes and SecYEG in bacteria. To evaluate a possible modulation of the translocation efficiency by secondary structures of the nascent peptide chain, we performed a comparative analysis in bacteria, yeast, and mammalian cells. Strikingly, neither the bacterial SecY nor the eukaryotic Sec61 translocon was able to efficiently transport proteins entirely composed of intrinsically disordered domains (IDDs) or ß-strands. However, translocation could be restored by α-helical domains in a position- and organism-dependent manner. In bacteria, we found that the α-helical domains have to precede the IDD or ß-strands, whereas in mammalian cells, C-terminally located α-helical domains are sufficient to promote translocation. Our study reveals an evolutionarily conserved deficiency of the Sec61/SecY complex to translocate IDDs and ß-strands in the absence of α-helical domains. Moreover, our results may suggest that adaptive pathways co-evolved with the expansion of IDDs in the proteome of eukaryotic cells to increase the transport capacity of the Sec61 translocon.


Assuntos
Canais de Translocação SEC/metabolismo , Canais de Translocação SEC/fisiologia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Canais de Translocação SEC/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Biochim Biophys Acta ; 1860(10): 2122-9, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27373685

RESUMO

BACKGROUND: In eukaryotic cells, many proteins have to be transported across or inserted into the endoplasmic reticulum membrane during their biogenesis on the ribosome. This process is facilitated by the protein translocon, a highly dynamic multi-subunit membrane protein complex. SCOPE OF REVIEW: The aim of this review is to summarize the current structural knowledge about protein translocon components in mammals. MAJOR CONCLUSIONS: Various structural biology approaches have been used in synergy to characterize the translocon in recent years. X-ray crystallography and cryoelectron microscopy single particle analysis have yielded highly detailed insights into the structure and functional mechanism of the protein-conducting channel Sec61, which constitutes the functional core of the translocon. Cryoelectron tomography and subtomogram analysis have advanced our understanding of the overall structure, molecular organization and compositional heterogeneity of the translocon in a native membrane environment. Tomography densities at subnanometer resolution revealed an intricate network of interactions between the ribosome, Sec61 and accessory translocon components that assist in protein transport, membrane insertion and maturation. GENERAL SIGNIFICANCE: The protein translocon is a gateway for approximately one third of all synthesized proteins and numerous human diseases are associated with malfunctioning of its components. Thus, detailed insights into the structure and molecular organization of the translocon will not only advance our understanding of membrane protein biogenesis in general, but they can potentially pave the way for novel therapeutic approaches against human diseases.


Assuntos
Retículo Endoplasmático/metabolismo , Transporte Proteico/genética , Ribossomos/genética , Canais de Translocação SEC/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Cristalografia por Raios X , Retículo Endoplasmático/genética , Humanos , Ribossomos/ultraestrutura , Canais de Translocação SEC/metabolismo , Tomografia
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