RESUMEN

N-glycans act as quality control tags by recruiting lectin chaperones to assist protein maturation in the endoplasmic reticulum. The location and composition of N-glycans (glyco-code) are key to the chaperone-selection process. Serpins, a class of serine protease inhibitors, fold non-sequentially to achieve metastable active states. Here, the role of the glyco-code in assuring successful maturation and quality control of two human serpins, alpha-1 antitrypsin (AAT) and antithrombin III (ATIII), is described. We find that AAT, which has glycans near its N terminus, is assisted by early lectin chaperone binding. In contrast, ATIII, which has more C-terminal glycans, is initially helped by BiP and then later by lectin chaperones mediated by UGGT reglucosylation. UGGT action is increased for misfolding-prone disease variants, and these clients are preferentially glucosylated on their most C-terminal glycan. Our study illustrates how serpins utilize N-glycan presence, position, and composition to direct their proper folding, quality control, and trafficking.

Asunto(s)

Chaperonas Moleculares , Pliegue de Proteína , Humanos , Chaperonas Moleculares/metabolismo , Lectinas/metabolismo , Polisacáridos/química , Control de Calidad

RESUMEN

Iron-sulfur clusters are ubiquitous cofactors required for fundamental biological processes. Structural and spectroscopic analysis of Fe-S proteins is often limited by low cluster occupancy in recombinantly produced proteins. In this work, we report a systematic comparison of different maturation strategies for three well-established [4Fe-4S] proteins. Aconitase B, HMBPP reductase (IspH), and quinolinate synthase (NadA) were used as model proteins as they have previously been characterized. The protein production strategies include expression of the gene of interest in BL21(DE3) cells, maturation of the apo protein using chemical or semi-enzymatic reconstitution, co-expression with two different plasmids containing the iron-sulfur cluster (isc) or sulfur formation (suf) operon, a cell strain lacking IscR, the transcriptional regulator of the ISC machinery, and an engineered "SufFeScient" derivative of BL21(DE3). Our results show that co-expression of a Fe-S biogenesis pathway influences the protein yield and the cluster content of the proteins. The presence of the Fe-S cluster is contributing to correct folding and structural stability of the proteins. In vivo maturation reduces the formation of Fe-S aggregates, which occur frequently when performing chemical reconstitution. Furthermore, we show that the in vivo strategies can be extended to the radical SAM protein ThnB, which was previously only maturated by chemical reconstitution. Our results shed light on the differences of in vitro and in vivo Fe-S cluster maturation and points out the pitfalls of chemical reconstitution.

Asunto(s)

Proteínas de Escherichia coli , Proteínas Hierro-Azufre , Proteínas Hierro-Azufre/metabolismo , Proteínas de Escherichia coli/metabolismo , Oxidorreductasas/metabolismo , Hierro/metabolismo , Azufre/metabolismo

RESUMEN

The tyrosine kinase receptor encoded by the MET oncogene has been extensively studied. Surprisingly, one extracellular domain, PSI, evolutionary conserved between plexins, semaphorins, and integrins, has no established function. The MET PSI sequence contains two CXXC motifs, usually found in protein disulfide isomerases (PDI). Using a scrambled oxidized RNAse enzymatic activity assay in vitro, we show, for the first time, that the MET extracellular domain displays disulfide isomerase activity, abolished by PSI domain antibodies. PSI domain deletion or mutations of CXXC sites to AXXA or SXXS result in a significant impairment of the cleavage of the MET 175 kDa precursor protein, abolishing the maturation of α and ß chains, of, respectively, 50 kDa and 145 kDa, disulfide-linked. The uncleaved precursor is stuck in the Golgi apparatus and, interestingly, is constitutively phosphorylated. However, no signal transduction is observed as measured by AKT and MAPK phosphorylation. Consequently, biological responses to the MET ligand-hepatocyte growth factor (HGF)-such as growth and epithelial to mesenchymal transition, are hampered. These data show that the MET PSI domain is functional and is required for the maturation, surface expression, and biological functions of the MET oncogenic protein.

Asunto(s)

Factor de Crecimiento de Hepatocito , Semaforinas , Factor de Crecimiento de Hepatocito/metabolismo , Proteína Disulfuro Isomerasas/genética , Ligandos , Transición Epitelial-Mesenquimal , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Semaforinas/genética , Oncogenes , Disulfuros , Integrinas/genética , Ribonucleasas/genética

RESUMEN

Opsin3 (OPN3) is a photoreceptor membrane protein with a typical seven-alpha helical transmembrane structure that belongs to the G-protein-coupled receptor (GPCR) superfamily and is widely expressed in brain. In recent years, it has been reported that OPN3 is also highly expressed in adipose tissue, and the protein is associated with the production of skin melanin. We found that the N82 site is the glycosylation site of OPN3. SNAP-tagTM has diverse functions and can be applied to a variety of different studies. By constructing a SNAP-tagged OPN3 recombinant protein, the distribution position of SNAP-OPN3 in cells can be clearly observed by fluorescence confocal microscopy using SNAP-Surface® 549 and SNAP-Cell® OregonGreen®, which provides a new method for studying the function of OPN3. It also shows that SNAP-tag does not affect the function of OPN3. Using the SNAP tag we found that OPN3 cannot be taken up to the cell membrane after glycosylation site mutation.

Asunto(s)

Melaninas , Piel , Membrana Celular , Glicosilación , Proteínas de la Membrana

RESUMEN

Opsin3 (OPN3) is a photoreceptor membrane protein with a typical seven-alpha helical transmembrane structure that belongs to the G-protein-coupled receptor (GPCR) superfamily and is widely expressed in brain. In recent years, it has been reported that OPN3 is also highly expressed in adipose tissue, and the protein is associated with the production of skin melanin. We found that the N82 site is the glycosylation site of OPN3. SNAP-tagTM has diverse functions and can be applied to a variety of different studies. By constructing a SNAP-tagged OPN3 recombinant protein, the distribution position of SNAP-OPN3 in cells can be clearly observed by fluorescence confocal microscopy using SNAP-Surface® 549 and SNAP-Cell® OregonGreen®, which provides a new method for studying the function of OPN3. It also shows that SNAP-tag does not affect the function of OPN3. Using the SNAP tag we found that OPN3 cannot be taken up to the cell membrane after glycosylation site mutation.

Asunto(s)

Membrana Celular , Glicosilación , Melaninas , Proteínas de la Membrana , Piel

RESUMEN

The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. Molecular dynamics simulations indicate that this unique architecture generates specificity for SPs based on the length of their hydrophobic segments.

Asunto(s)

Retículo Endoplásmico/enzimología , Péptido Hidrolasas/metabolismo , Señales de Clasificación de Proteína , Serina Endopeptidasas/metabolismo , Células A549 , Dominio Catalítico , Microscopía por Crioelectrón , Células HEK293 , Células Hep G2 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Simulación de Dinámica Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Proteómica , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Relación Estructura-Actividad , Especificidad por Sustrato , Espectrometría de Masas en Tándem , Células U937

RESUMEN

Upon T-cell receptor stimulation, the Programmed cell Death-1 receptor (PD-1) expressed on T-cells can interact with its ligand PD-L1 expressed at the surface of cancer cells or antigen-presenting cells. Monoclonal antibodies targeting PD-1 or PD-L1 are routinely used for the treatment of cancers, but their clinical efficacy varies largely across the variety of tumor types. A part of the variability is linked to the existence of several forms of PD-L1, either expressed on the plasma membrane (mPD-L1), at the surface of secreted cellular exosomes (exoPD-L1), in cell nuclei (nPD-L1), or as a circulating, soluble protein (sPD-L1). Here, we have reviewed the different origins and roles of sPD-L1 in humans to highlight the biochemical and functional heterogeneity of the soluble protein. sPD-L1 isoforms can be generated essentially by two non-exclusive processes: (i) proteolysis of m/exoPD-L1 by metalloproteases, such as metalloproteinases (MMP) and A disintegrin and metalloproteases (ADAM), which are capable of shedding membrane PD-L1 to release an active soluble form, and (ii) the alternative splicing of PD-L1 pre-mRNA, leading in some cases to the release of sPD-L1 protein isoforms lacking the transmembrane domain. The expression and secretion of sPD-L1 have been observed in a large variety of pathologies, well beyond cancer, notably in different pulmonary diseases, chronic inflammatory and autoimmune disorders, and viral diseases. The expression and role of sPD-L1 during pregnancy are also evoked. The structural heterogeneity of sPD-L1 proteins, and associated functional/cellular plurality, should be kept in mind when considering sPD-L1 as a biomarker or as a drug target. The membrane, exosomal and soluble forms of PD-L1 are all integral parts of the highly dynamic PD-1/PD-L1 signaling pathway, essential for immune-tolerance or immune-escape.

RESUMEN

The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na+ and K+ ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a ß-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways. The maturation and trafficking of the Na,K-ATPase include co- and post-translational processing of the enzyme in the endoplasmic reticulum (ER) and the Golgi apparatus and subsequent delivery to the plasma membrane (PM). The ER folding of the enzyme is considered as the rate-limiting step in the membrane delivery of the protein. It has been demonstrated that only assembled Na,K-ATPase α:ß-complexes may exit the organelle, whereas unassembled, misfolded or unfolded subunits are retained in the ER and are subsequently degraded. Loss of function of the Na,K-ATPase has been associated with lung, heart, kidney and neurological disorders. Recently, it has been shown that ER dysfunction, in particular, alterations in the homeostasis of the organelle, as well as impaired ER-resident chaperone activity may impede folding of Na,K-ATPase subunits, thus decreasing the abundance and function of the enzyme at the PM. Here, we summarize our current understanding on maturation and subsequent processing of the Na,K-ATPase in the ER under physiological and pathophysiological conditions.

Asunto(s)

Retículo Endoplásmico , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Iones/metabolismo , Pliegue de Proteína , ATPasa Intercambiadora de Sodio-Potasio/metabolismo

RESUMEN

Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully-active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, kcat , and the rate constant of productive binding, kon , of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, implying that inactive monomer units of ALP are deficient in correct disulfide bond formation and zinc ion coordination. These findings provide basis for further study on molecular mechanism and biogenesis of ALP, and also offer the way to prepare homogenous and active populations of ALP for highly quantitative and sensitive bioassays with ALP.

Asunto(s)

Fosfatasa Alcalina , Bioensayo/métodos , Sistema Libre de Células/enzimología , Imagen Individual de Molécula/métodos , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/química , Fosfatasa Alcalina/metabolismo , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Microscopía Fluorescente

RESUMEN

Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. Several EOGT mutations that may affect putative N-glycosylation consensus sites are recorded in the cancer database, but the presence and function of N-glycans in EOGT have not yet been characterized. Here, we identified N-glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predicted N-glycosylation consensus sequences on EOGT are highly conserved among mammalian species. Within these sites, we found that Asn-263 and Asn-354, but not Asn-493, are modified with N-glycans. Lectin blotting, endoglycosidase H digestion, and MS analysis revealed that both residues are modified with oligomannose N-glycans. Loss of an individual N-glycan on EOGT did not affect its endoplasmic reticulum (ER) localization, enzyme activity, and ability to O-GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of both N-glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting that N-glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease in O-GlcNAc stoichiometry was observed in Notch1 co-expressed with an N263Q/N354Q variant compared with WT EOGT. Moreover, the N263Q/N354Q variant exhibited altered subcellular distribution within the ER in HEK293T cells, indicating that N-glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles of N-glycans in sustaining O-GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.

Asunto(s)

Retículo Endoplásmico/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Sistemas CRISPR-Cas/genética , Línea Celular , Cromatografía Líquida de Alta Presión , Estrés del Retículo Endoplásmico/efectos de los fármacos , Edición Génica , Glicopéptidos/análisis , Glicosilación , Humanos , Ratones , Mutagénesis Sitio-Dirigida , N-Acetilglucosaminiltransferasas/deficiencia , N-Acetilglucosaminiltransferasas/genética , Receptor Notch1/genética , Receptor Notch1/metabolismo , Alineación de Secuencia , Espectrometría de Masas en Tándem , Tunicamicina/farmacología

RESUMEN

Although the last 30years have witnessed the mapping of the wiring diagrams of the gene regulatory networks that dictate cell fate and animal body plans, specific understanding building on such network diagrams that shows how DNA regulatory regions control gene expression lags far behind. These networks have yet to yield the predictive power necessary to, for example, calculate how the concentration dynamics of input transcription factors and DNA regulatory sequence prescribes output patterns of gene expression that, in turn, determine body plans themselves. Here, we argue that reaching a predictive understanding of developmental decision-making calls for an interplay between theory and experiment aimed at revealing how the regulation of the processes of the central dogma dictate network connections and how network topology guides cells toward their ultimate developmental fate. To make this possible, it is crucial to break free from the snapshot-based understanding of embryonic development facilitated by fixed-tissue approaches and embrace new technologies that capture the dynamics of developmental decision-making at the single cell level, in living embryos.

Asunto(s)

Tipificación del Cuerpo , Biología Evolutiva , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Modelos Teóricos , Biología Sintética

RESUMEN

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80-2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

Asunto(s)

Fucosiltransferasas/química , Guanosina Difosfato/química , Simulación de Dinámica Molecular , Animales , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Humanos , Ratones

RESUMEN

Helicobacter pylori infection always induces gastritis, which may progress to ulcer disease or cancer. The mechanisms underlying mucosal injury by the bacteria are incompletely understood. Here, we identify a novel pathway for H. pylori-induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase. Na-K-ATPase comprises α- and ß-subunits that assemble in the endoplasmic reticulum (ER) before trafficking to the plasma membrane. Attachment of H. pylori to gastric epithelial cells increased Na-K-ATPase ubiquitylation, decreased its surface and total levels, and impaired ion balance. H. pylori did not alter degradation of plasmalemma-resident Na-K-ATPase subunits or their mRNA levels. Infection decreased association of α- and ß-subunits with ER chaperone BiP and impaired assembly of α/ß-heterodimers, as was revealed by quantitative mass spectrometry and immunoblotting of immunoprecipitated complexes. The total level of BiP was not altered, and the decrease in interaction with BiP was not observed for other BiP client proteins. The H. pylori-induced decrease in Na-K-ATPase was prevented by BiP overexpression, stopping protein synthesis, or inhibiting proteasomal, but not lysosomal, protein degradation. The results indicate that H. pylori impairs chaperone-assisted maturation of newly made Na-K-ATPase subunits in the ER independently of a generalized ER stress and induces their ubiquitylation and proteasomal degradation. The decrease in Na-K-ATPase levels is also seen in vivo in the stomachs of gerbils and chronically infected children. Further understanding of H. pylori-induced Na-K-ATPase degradation will provide insights for protection against advanced disease.NEW & NOTEWORTHY This work provides evidence that Helicobacter pylori decreases levels of Na-K-ATPase, a vital transport enzyme, in gastric epithelia, both in acutely infected cultured cells and in chronically infected patients and animals. The bacteria interfere with BiP-assisted folding of newly-made Na-K-ATPase subunits in the endoplasmic reticulum, accelerating their ubiquitylation and proteasomal degradation and decreasing efficiency of the assembly of native enzyme. Decreased Na-K-ATPase expression contributes to H. pylori-induced gastric injury.

Asunto(s)

Retículo Endoplásmico/enzimología , Células Epiteliales/enzimología , Mucosa Gástrica/enzimología , Gastritis/enzimología , Proteínas de Choque Térmico/metabolismo , Infecciones por Helicobacter/enzimología , Helicobacter pylori/patogenicidad , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Células Cultivadas , Retículo Endoplásmico/microbiología , Chaperón BiP del Retículo Endoplásmico , Estabilidad de Enzimas , Células Epiteliales/microbiología , Mucosa Gástrica/microbiología , Gastritis/genética , Gastritis/microbiología , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Interacciones Huésped-Patógeno , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína , Proteolisis , ATPasa Intercambiadora de Sodio-Potasio/genética , Ubiquitinación

RESUMEN

Many cancers occur from locations of inflammation due to chronic irritation and/or infection. Tumor microenvironment contains various different inflammatory cells and mediators that orchestrate diverse neoplastic processes, including proliferation, survival, adhesion and migration. In parallel, tumor cells have adapted some of the signaling molecules used by inflammatory cells, such as selectins and chemokines as well as their receptors for invasion, extravasation and subsequently metastasis. Expression and/or activation of the majority of these molecules is mediated by the proprotein convertases (PCs); proteases expressed by both tumor cells and inflammatory cells. This review analyzes the potential role of these enzymatic system in inflammation-associated cancer impacting on the malignant and metastatic potential of cancer cells, describing the possible use of PCs as a new anti-inflammatory therapeutic approach to tumor progression and metastasis.

Asunto(s)

Carcinogénesis/inmunología , Inflamación/tratamiento farmacológico , Metástasis de la Neoplasia/inmunología , Neoplasias/inmunología , Proproteína Convertasas/metabolismo , Animales , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Carcinogénesis/efectos de los fármacos , Quimiocinas/inmunología , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Inflamación/inmunología , Inflamación/patología , Metástasis de la Neoplasia/prevención & control , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proproteína Convertasas/antagonistas & inhibidores , Selectinas/inmunología , Selectinas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología

RESUMEN

Vacuolar processing enzyme (VPE) is a cysteine-type endopeptidase that has a substrate-specificity for asparagine or aspartic acid residues and cleaves peptide bonds at their carboxyl-terminal side. Various vacuolar proteins are synthesized as larger proprotein precursors, and VPE is an important initiator of maturation and activation of these proteins. It mediates programmed cell death (PCD) by provoking vacuolar rupture and initiating the proteolytic cascade leading to PCD. Vacuolar processing enzyme also possesses a peptide ligation activity, which is responsible for producing cyclic peptides in several plant species. These unique functions of VPE support developmental and environmental responses in plants. The number of VPE homologues is higher in angiosperm species, indicating that there has been differentiation and specialization of VPE function over the course of evolution. Angiosperm VPEs are separated into two major types: the γ-type VPEs, which are expressed mainly in vegetative organs, and the ß-type VPEs, whose expression occurs mainly in storage organs; in eudicots, the δ-type VPEs are further separated within γ-type VPEs. This review also considers the importance of processing and peptide ligation by VPE in vacuolar protein maturation.

Asunto(s)

Cisteína Endopeptidasas , Vacuolas , Animales , Estadios del Ciclo de Vida , Proteínas de Plantas , Plantas

RESUMEN

The protein quality control machinery of the endoplasmic reticulum (ERQC) ensures that client proteins are properly folded. ERQC substrates may be recognized as nonnative by the presence of exposed hydrophobic surfaces, free thiols, or processed N-glycans. How these features dictate which ERQC pathways engage a given substrate is poorly understood. Here, using metabolic labeling, immunoprecipitations, various biochemical assays, and the human serpin antithrombin III (ATIII) as a model, we explored the role of ERQC systems in mammalian cells. Although ATIII has N-glycans and a hydrophobic core, we found that its quality control depended solely on free thiol content. Mutagenesis of all six Cys residues in ATIII to Ala resulted in its efficient secretion even though the product was not natively folded. ATIII variants with free thiols were retained in the endoplasmic reticulum but not degraded. These results provide insight into the hierarchy of ERQC systems and reveal a fundamental vulnerability of ERQC in a case of reliance on the thiol-dependent quality control pathway.

Asunto(s)

Antitrombina III/metabolismo , Control de Calidad , Serpinas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Animales , Células CHO , Células Cultivadas , Cricetulus , Retículo Endoplásmico/metabolismo , Humanos

RESUMEN

Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis, we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter PX or PM from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying PM also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (∼115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (∼8-45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.

RESUMEN

Several membrane-bound proteins with a single transmembrane domain are subjected to limited proteolysis at the cell surface. This cleavage leads to the release of their biologically active ectodomains, which can trigger different signalling pathways. In many cases, this ectodomain shedding is mediated by members of the family of a disintegrins and metalloproteinases (ADAMs). ADAM17 in particular is responsible for the cleavage of several proinflammatory mediators, growth factors, receptors and adhesion molecules. Due to its direct involvement in the release of these signalling molecules, ADAM17 can be positively and negatively involved in various physiological processes as well as in inflammatory, fibrotic and malignant pathologies. This central role of ADAM17 in a variety of processes requires strict multi-level regulation, including phosphorylation, various conformational changes and endogenous inhibitors. Recent research has shown that an early, crucial control mechanism is interaction with certain adapter proteins identified as iRhom1 and iRhom2, which are pseudoproteases of the rhomboid superfamily. Thus, iRhoms have also a decisive influence on physiological and pathophysiological signalling processes regulated by ADAM17. Their characteristic gene expression profiles, the specific consequences of gene knockouts and finally the occurrence of disease-associated mutations suggest that iRhom1 and iRhom2 undergo different gene regulation in order to fulfil their function in different cell types and are therefore only partially redundant. Therefore, there is not only interest in ADAM17, but also in iRhoms as therapeutic targets. However, to exploit the therapeutic potential, the regulation of ADAM17 activity and in particular its interaction with iRhoms must be well understood.

Asunto(s)

Proteína ADAM17/química , Proteína ADAM17/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Técnicas de Inactivación de Genes , Predisposición Genética a la Enfermedad/genética , Humanos , Inflamación , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Metaloproteasas , Ratones , Mutación , Fosforilación , Conformación Proteica , Transducción de Señal , Transcriptoma

RESUMEN

Ionotropic glycine receptors (GlyRs) enable fast synaptic neurotransmission in the adult spinal cord and brainstem. The inhibitory GlyR is a transmembrane glycine-gated chloride channel. The immature GlyR protein undergoes various processing steps, e.g., folding, assembly, and maturation while traveling from the endoplasmic reticulum to and through the Golgi apparatus, where post-translational modifications, e.g., glycosylation occur. The mature receptors are forward transported via microtubules to the cellular surface and inserted into neuronal membranes followed by synaptic clustering. The normal life cycle of a receptor protein includes further processes like internalization, recycling, and degradation. Defects in GlyR life cycle, e.g., impaired protein maturation and degradation have been demonstrated to underlie pathological mechanisms of various neurological diseases. The neurological disorder startle disease is caused by glycinergic dysfunction mainly due to missense mutations in genes encoding GlyR subunits (GLRA1 and GLRB). In vitro studies have shown that most recessive forms of startle disease are associated with impaired receptor biogenesis. Another neurological disease with a phenotype similar to startle disease is a special form of stiff-person syndrome (SPS), which is most probably due to the development of GlyR autoantibodies. Binding of GlyR autoantibodies leads to enhanced receptor internalization. Here we focus on the normal life cycle of GlyRs concentrating on assembly and maturation, receptor trafficking, post-synaptic integration and clustering, and GlyR internalization/recycling/degradation. Furthermore, this review highlights findings on impairment of these processes under disease conditions such as disturbed neuronal ER-Golgi trafficking as the major pathomechanism for recessive forms of human startle disease. In SPS, enhanced receptor internalization upon autoantibody binding to the GlyR has been shown to underlie the human pathology. In addition, we discuss how the existing mouse models of startle disease increased our current knowledge of GlyR trafficking routes and function. This review further illuminates receptor trafficking of GlyR variants originally identified in startle disease patients and explains changes in the life cycle of GlyRs in patients with SPS with respect to structural and functional consequences at the receptor level.

RESUMEN

Förster resonance energy transfer (FRET)-based sensors are a valuable tool to quantify cell biology, yet it remains necessary to identify and prevent potential artifacts in order to exploit their full potential. We show here that artifacts arising from slow donor mCerulean3 maturation can be substantially diminished by constitutive expression in both prokaryotic and eukaryotic cells, which can also be achieved by incorporation of faster-maturing FRET donors. We developed an improved version of the donor mTurquoise2 that matures faster than the parent protein. Our analysis shows that using equal maturing fluorophores in FRET-based sensors or using constitutive low expression conditions helps to reduce maturation-induced artifacts, without the need of additional noise-inducing spectral corrections. In general, we show that monitoring and controlling the maturation of fluorescent proteins in living cells is important and should be addressed in in vivo applications of genetically encoded FRET sensors.

Asunto(s)

Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas Fluorescentes Verdes/metabolismo , Secuencia de Bases , Fenómenos Fisiológicos Celulares , Cloranfenicol/farmacología , Escherichia coli/citología , Escherichia coli/genética , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas de Unión a Maltosa/metabolismo , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Modelos Biológicos , Mutación , Regiones Promotoras Genéticas , Inhibidores de la Síntesis de la Proteína/farmacología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética