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1.
ACS Chem Biol ; 18(12): 2506-2515, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37990966

RESUMO

Cells sense extracellular stimuli through membrane receptors and process information through an intracellular signaling network. Protein translocation triggers intracellular signaling, and techniques such as chemically induced dimerization (CID) have been used to manipulate signaling pathways by altering the subcellular localization of signaling molecules. However, in the fission yeast Schizosaccharomyces pombe, the commonly used FKBP-FRB system has technical limitations, and therefore, perturbation tools with low cytotoxicity and high temporal resolution are needed. We here applied our recently developed self-localizing ligand-induced protein translocation (SLIPT) system to S. pombe and successfully perturbed several cell cycle-related proteins. The SLIPT system utilizes self-localizing ligands to recruit binding partners to specific subcellular compartments such as the plasma membrane or nucleus. We optimized the self-localizing ligands to maintain the long-term recruitment of target molecules to the plasma membrane. By knocking in genes encoding the binding partners for self-localizing ligands, we observed changes in the localization of several endogenous molecules and found perturbations in the cell cycle and associated phenotypes. This study demonstrates the effectiveness of the SLIPT system as a chemogenetic tool for rapid perturbation of endogenous molecules in S. pombe, providing a valuable approach for studying intracellular signaling and cell cycle regulation with an improved temporal resolution.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Ligantes , Transporte Proteico , Proteínas de Ciclo Celular/metabolismo , Sistemas de Translocação de Proteínas/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
2.
Nucleic Acids Res ; 51(1): e1, 2023 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-36268868

RESUMO

The development of novel strategies to program cellular behaviors is a central goal in synthetic biology, and post-translational control mediated by engineered protein circuits is a particularly attractive approach to achieve rapid protein secretion on demand. We have developed a programmable protease-mediated post-translational switch (POSH) control platform composed of a chimeric protein unit that consists of a protein of interest fused via a transmembrane domain to a cleavable ER-retention signal, together with two cytosolic inducer-sensitive split protease components. The protease components combine in the presence of the specific inducer to generate active protease, which cleaves the ER-retention signal, releasing the transmembrane-domain-linked protein for trafficking to the trans-Golgi region. A furin site placed downstream of the protein ensures cleavage and subsequent secretion of the desired protein. We show that stimuli ranging from plant-derived, clinically compatible chemicals to remotely controllable inducers such as light and electrostimulation can program protein secretion in various POSH-engineered designer mammalian cells. As proof-of-concept, an all-in-one POSH control plasmid encoding insulin and abscisic acid-activatable split protease units was hydrodynamically transfected into the liver of type-1 diabetic mice. Induction with abscisic acid attenuated glycemic excursions in glucose-tolerance tests. Increased blood levels of insulin were maintained for 12 days.


Assuntos
Peptídeo Hidrolases , Processamento de Proteína Pós-Traducional , Biologia Sintética , Animais , Camundongos , Ácido Abscísico , Diabetes Mellitus Experimental , Endopeptidases/metabolismo , Insulina/genética , Insulina/metabolismo , Mamíferos/metabolismo , Peptídeo Hidrolases/metabolismo , Sistemas de Translocação de Proteínas , Biologia Sintética/métodos
3.
Cell Rep ; 40(11): 111340, 2022 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-36103818

RESUMO

Predatory Myxobacteria employ a multilayered predation strategy to kill and lyse soil microorganisms. Aiming to dissect the mechanism of contact-dependent killing of bacteria, we analyze four protein secretion systems in Myxococcus xanthus and investigate the predation of mutant strains on different timescales. We find that a Tad-like and a type 3-like secretion system (Tad and T3SS∗) fulfill distinct functions during contact-dependent prey killing: the Tad-like system is necessary to induce prey cell death, while the needle-less T3SS∗ initiates prey lysis. Fluorescence microscopy reveals that components of both systems interdependently localize to the predator-prey contact site prior to killing. Swarm expansion assays show that both Tad and T3SS∗ are required to handle live prey and that nutrient extraction from prey bacteria is sufficient to power M. xanthus motility. In conclusion, our observations indicate the functional interplay of two types of secretion systems for killing and lysis of bacterial cells.


Assuntos
Myxococcus xanthus , Animais , Myxococcus xanthus/fisiologia , Comportamento Predatório , Sistemas de Translocação de Proteínas , Solo
4.
Microbiology (Reading) ; 168(5)2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35536734

RESUMO

Protein trafficking across the bacterial envelope is a process that contributes to the organisation and integrity of the cell. It is the foundation for establishing contact and exchange between the environment and the cytosol. It helps cells to communicate with one another, whether they establish symbiotic or competitive behaviours. It is instrumental for pathogenesis and for bacteria to subvert the host immune response. Understanding the formation of envelope conduits and the manifold strategies employed for moving macromolecules across these channels is a fascinating playground. The diversity of the nanomachines involved in this process logically resulted in an attempt to classify them, which is where the protein secretion system types emerged. As our knowledge grew, so did the number of types, and their rightful nomenclature started to be questioned. While this may seem a semantic or philosophical issue, it also reflects scientific rigour when it comes to assimilating findings into textbooks and science history. Here I give an overview on bacterial protein secretion systems, their history, their nomenclature and why it can be misleading for newcomers in the field. Note that I do not try to suggest a new nomenclature. Instead, I explore the reasons why naming could have escaped our control and I try to reiterate basic concepts that underlie protein trafficking cross membranes.


Assuntos
Sistemas de Secreção Bacterianos , Sistemas de Translocação de Proteínas , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Sistemas de Translocação de Proteínas/metabolismo , Transporte Proteico
5.
Biomed Pharmacother ; 145: 112471, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34852990

RESUMO

NF-κB contributes to the biosynthesis of various chemokines, cytokines, and enzymes. It plays many crucial roles in the upstream neuroinflammatory pathways. Briefly, the inhibitory IkB subunit is cleaved and phosphorylated by the IKK-α/ß enzyme. It leads to the activation and translocation of the NF-κB (p50/p65) complex into the nucleus. Subsequently, the activated NF-κB interacts with the genomic DNA and contributes to expressing various proinflammatory cytokines. In the present study, we developed a novel NF-κB inhibitor encoded (D5) and investigated the efficacy of our druggable compound through several in silico, in vitro, and in situ analysis. The results demonstrated that D5 not only inhibited the mRNA expression of the IKK-α/ß enzyme (around 86-96% suppression rate for both cell lines at 12 and 24 h time frames) but also by interacting to the active site of the mentioned kinase (dock score -6.14 and binding energy -23.60 kcal/mol) reduced the level of phosphorylated IkB-α in the cytosol around 96-99% and p65 subunit in the nucleus around 73-90% (among all groups in 12 and 24 h time points). Additionally, the results indicated that D5 suppressed the NF-κB target mRNA levels of TNF-α and IL-6 in a total average of around 92%. Overall, The results demonstrated that D5 in a considerably lower concentration than Dis (0.71 µM vs. 52.73 µM) showed significantly higher inhibitory efficacy on NF-κB translocation approx. 200-300%. The results suggested D5 as a potent NF-κB silencer, but further investigations are required to validate our outcomes.


Assuntos
Quinase I-kappa B , NF-kappa B/metabolismo , Doenças Neuroinflamatórias , Sistemas de Translocação de Proteínas , Alcaloides/farmacologia , Benzodioxóis/farmacologia , Linhagem Celular , Inibidores das Enzimas do Citocromo P-450/farmacologia , Desenvolvimento de Medicamentos/métodos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Fosforilação/efeitos dos fármacos , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Sistemas de Translocação de Proteínas/efeitos dos fármacos , Sistemas de Translocação de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo
6.
FEBS J ; 289(16): 4704-4717, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-34092034

RESUMO

Equipped with a plethora of secreted toxic effectors, protein secretion systems are essential for bacteria to interact with and manipulate their neighboring environment to survive in host microbiota and other highly competitive communities. While effectors have received spotlight attention in secretion system studies, many require accessory chaperone and adaptor proteins for proper folding/unfolding and stability throughout the secretion process. Here, we review the functions of chaperones and adaptors of three protein secretions systems, type 3 secretion system (T3SS), type 4 secretion system (T4SS), and type 6 secretion system (T6SS), which are employed by many Gram-negative bacterial pathogens to deliver toxins to bacterial, plant, and mammalian host cells through direct contact. Since chaperone and adaptor functions of the T3SS and the T4SS are relatively well studied, we discuss in detail the methods of chaperone-facilitated effector secretion by the T6SS and highlight commonalities between the effector chaperone/adaptor proteins of these diverse secretion systems. While the chaperones and adaptors are generally referred to as accessory proteins as they are not directly involved in toxicities to target cells, they are nonetheless vital for the biological functions of the secretion systems. Future research on biochemical and structural properties of these chaperones will not only elucidate the mechanisms of chaperone-effector binding and release process but also facilitate custom design of cargo effectors to be translocated by these widespread secretion systems for biotechnological applications.


Assuntos
Proteínas de Bactérias , Sistemas de Translocação de Proteínas , Animais , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Bactérias Gram-Negativas/metabolismo , Mamíferos/metabolismo , Chaperonas Moleculares/metabolismo , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
7.
J Bacteriol ; 204(2): e0050421, 2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-34898262

RESUMO

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multiprotein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however, little is known regarding their function, regulation, and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence, and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of nonpilin proteins, we developed a NanoLuc (NLuc)-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. strain PCC 6803. The NLuc reporter presented a wide dynamic range with at least 1 order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to exploring protein secretion in cyanobacteria further. IMPORTANCE Protein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here, we introduce an NLuc-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge of cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.


Assuntos
Bioensaio/métodos , Luciferases/metabolismo , Sistemas de Translocação de Proteínas/metabolismo , Synechocystis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fímbrias Bacterianas , Sistemas de Translocação de Proteínas/genética , Transporte Proteico , Synechocystis/genética
8.
Int J Mol Sci ; 22(22)2021 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-34830459

RESUMO

Bacteria of genus Pectobacterium are Gram-negative rods of the family Pectobacteriaceae. They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are believed to play an important role in the pathogenicity and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of Pectobacterium. The results indicate that the morphology and MVs production depend on growth medium composition. In polygalacturonic acid (PGA) supplemented media, Pectobacterium produces large MVs (100-300 nm) and small vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in the MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to degrade pectates. What is more, the pathogenicity test indicated that the MVs derived from Pectobacterium were able to induce maceration of Zantedeschia sp. leaves. We also show that the MVs of ß-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that the MVs of Pectobacterium play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that the MVs production in Pectobacterium strains, which overexpress a green fluorescence protein (GFP), is higher than in wild-type strains. Moreover, proteomic analysis revealed that the GFP was present in the MVs. Therefore, it is possible that protein sequestration into MVs might not be strictly limited to periplasmic proteins. Our research highlights the importance of MVs production as a mechanism of cargo delivery in Pectobacterium and an effective secretion system.


Assuntos
Vesículas Extracelulares/genética , Interações Hospedeiro-Patógeno/genética , Pectobacterium/genética , Sistemas de Translocação de Proteínas/genética , Membrana Celular/genética , Membrana Celular/ultraestrutura , Vesículas Extracelulares/ultraestrutura , Pectobacterium/ultraestrutura , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Sistemas de Translocação de Proteínas/ultraestrutura , Transporte Proteico/genética , Virulência/genética
9.
Microb Cell Fact ; 20(1): 176, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34488755

RESUMO

Monomeric autotransporters have been used extensively to transport recombinant proteins or protein domains to the cell surface of Gram-negative bacteria amongst others for antigen display. Genetic fusion of such antigens into autotransporters has yielded chimeras that can be used for vaccination purposes. However, not every fusion construct is transported efficiently across the cell envelope. Problems occur in particular when the fused antigen attains a relatively complex structure in the periplasm, prior to its translocation across the outer membrane. The latter step requires the interaction with periplasmic chaperones and the BAM (ß-barrel assembly machinery) complex in the outer membrane. This complex catalyzes insertion and folding of ß-barrel outer membrane proteins, including the ß-barrel domain of autotransporters. Here, we investigated whether the availability of periplasmic chaperones or the BAM complex is a limiting factor for the surface localization of difficult-to-secrete chimeric autotransporter constructs. Indeed, we found that overproduction of in particular the BAM complex, increases surface display of difficult-to-secrete chimeras. Importantly, this beneficial effect appeared to be generic not only for a number of monomeric autotransporter fusions but also for fusions to trimeric autotransporters. Therefore, overproduction of BAM might be an attractive strategy to improve the production of recombinant autotransporter constructs.


Assuntos
Membrana Celular/metabolismo , Escherichia coli/metabolismo , Sistemas de Translocação de Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Escherichia coli/genética , Transporte Proteico
10.
J Microbiol ; 59(10): 920-930, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34554453

RESUMO

The pathogenesis of Staphylococcus aureus, from local infections to systemic dissemination, is mediated by a battery of virulence factors that are regulated by intricate mechanisms, which include regulatory proteins and small RNAs (sRNAs) as key regulatory molecules. We have investigated the involvement of sRNA RsaF, in the regulation of pathogenicity genes hyaluronate lyase (hysA) and serine proteaselike protein D (splD), by employing S. aureus strains with disruption and overexpression of rsaF. Staphylococcus aureus strain with disruption of rsaF exhibited marked down-regulation of hysA transcripts by 0.2 to 0.0002 fold, and hyaluronate lyase activity by 0.2-0.1 fold, as well as increased biofilm formation, during growth from log phase to stationery phase. These mutants also displayed down-regulation of splD transcripts by 0.8 to 0.005 fold, and reduced activity of multiple proteases by zymography. Conversely, overexpression of rsaF resulted in a 2- to 4- fold increase in hysA mRNA levels and hyaluronidase activity. Both hysA and splD mRNAs demonstrated an increased stability in RsaF+ strains. In silico RNA-RNA interaction indicated a direct base pairing of RsaF with hysA and splD mRNAs, which was established in electrophoretic mobility shift assays. The findings demonstrate a positive regulatory role for small RNA RsaF in the expression of the virulence factors, HysA and SplD.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Polissacarídeo-Liases/metabolismo , Serina Proteases/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Humanos , Polissacarídeo-Liases/genética , Sistemas de Translocação de Proteínas , Serina Proteases/genética , Staphylococcus aureus/genética , Fatores de Virulência/genética
11.
PLoS One ; 16(9): e0258005, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34582499

RESUMO

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is a potential host strain for industrial protein production. Heterologous proteins are often retained intracellularly in yeast resulting in endoplasmic reticulum (ER) stress and poor secretion, and despite efforts to engineer protein secretory pathways, heterologous protein production is often lower than expected. We hypothesized that activation of genes involved in the secretory pathway could mitigate ER stress. In this study, we created mutants defective in protein secretory-related functions using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) tools. Secretion of the model protein xylanase was significantly decreased in loss of function mutants for oxidative stress (sod1Δ) and vacuolar and protein sorting (vps1Δ and ypt7Δ) genes. However, xylanase secretion was unaffected in an autophagy related atg12Δ mutant. Then, we developed a system for sequence-specific activation of target gene expression (CRISPRa) in O. thermomethanolica and used it to activate SOD1, VPS1 and YPT7 genes. Production of both non-glycosylated xylanase and glycosylated phytase was enhanced in the gene activated mutants, demonstrating that CRISPR-Cas9 systems can be used as tools for understanding O. thermomethanolica genes involved in protein secretion, which could be applied for increasing heterologous protein secretion in this yeast.


Assuntos
Proteínas Fúngicas/metabolismo , Saccharomycetales/genética , Autofagia , Western Blotting , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Eletroforese em Gel de Poliacrilamida , Estresse do Retículo Endoplasmático , Edição de Genes , Genes Fúngicos/genética , Estresse Oxidativo , Sistemas de Translocação de Proteínas/genética , Transporte Proteico/genética , Reação em Cadeia da Polimerase em Tempo Real , Saccharomycetales/metabolismo , Termotolerância
12.
J Biol Chem ; 297(3): 101055, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34411563

RESUMO

Fibrillin-1 (FBN1) is the major component of extracellular matrix microfibrils, which are required for proper development of elastic tissues, including the heart and lungs. Through protein-protein interactions with latent transforming growth factor (TGF) ß-binding protein 1 (LTBP1), microfibrils regulate TGF-ß signaling. Mutations within the 47 epidermal growth factor-like (EGF) repeats of FBN1 cause autosomal dominant disorders including Marfan Syndrome, which is characterized by disrupted TGF-ß signaling. We recently identified two novel protein O-glucosyltransferases, Protein O-glucosyltransferase 2 (POGLUT2) and 3 (POGLUT3), that modify a small fraction of EGF repeats on Notch. Here, using mass spectral analysis, we show that POGLUT2 and POGLUT3 also modify over half of the EGF repeats on FBN1, fibrillin-2 (FBN2), and LTBP1. While most sites are modified by both enzymes, some sites show a preference for either POGLUT2 or POGLUT3. POGLUT2 and POGLUT3 are homologs of POGLUT1, which stabilizes Notch proteins by addition of O-glucose to Notch EGF repeats. Like POGLUT1, POGLUT2 and 3 can discern a folded versus unfolded EGF repeat, suggesting POGLUT2 and 3 are involved in a protein folding pathway. In vitro secretion assays using the N-terminal portion of recombinant FBN1 revealed reduced FBN1 secretion in POGLUT2 knockout, POGLUT3 knockout, and POGLUT2 and 3 double-knockout HEK293T cells compared with wild type. These results illustrate that POGLUT2 and 3 function together to O-glucosylate protein substrates and that these modifications play a role in the secretion of substrate proteins. It will be interesting to see how disease variants in these proteins affect their O-glucosylation.


Assuntos
Fibrilina-1/metabolismo , Fibrilina-2/metabolismo , Proteínas de Ligação a TGF-beta Latente/metabolismo , Síndrome de Marfan/metabolismo , Motivos de Aminoácidos , Fibrilina-1/química , Fibrilina-1/genética , Fibrilina-2/química , Fibrilina-2/genética , Glicosilação , Humanos , Proteínas de Ligação a TGF-beta Latente/química , Proteínas de Ligação a TGF-beta Latente/genética , Síndrome de Marfan/enzimologia , Síndrome de Marfan/genética , Sistemas de Translocação de Proteínas , Transdução de Sinais
13.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202771

RESUMO

In this article, we review the biological and clinical implication of the Recruitment-Secretory Block ("R-SB") phenomenon. The phenomenon refers to the reaction of the liver with regard to protein secretion in conditions of clinical stimulation. Our basic knowledge of the process is due to the experimental work in animal models. Under basal conditions, the protein synthesis is mainly carried out by periportal (zone 1) hepatocytes that are considered the "professional" synthesizing protein cells. Under stimulation, midlobular and centrolobular (zones 2 and 3) hepatocytes, are progressively recruited according to lobular gradients and contribute to the increase of synthesis and secretion. The block of secretion, operated by exogenous agents, causes intracellular retention of all secretory proteins. The Pi MZ phenotype of Alpha-1-antitrypsin deficiency (AATD) has turned out to be the key for in vivo studies of the reaction of the liver, as synthesis and block of secretion are concomitant. Indeed, the M fraction of AAT is stimulated for synthesis and regularly exported while the Z fraction is mostly retained within the cell. For that reason, the phenomenon has been designated "Recruitment-Secretory Block" ("R-SB"). The "R-SB" phenomenon explains why: (a) the MZ individuals can correct the serum deficiency; (b) the resulting immonohistochemical and electron microscopic (EM) patterns are very peculiar and specific for the diagnosis of the Z mutation in tissue sections in the absence of genotyping; (c) the term carrier is no longer applicable for the heterozygous condition as all Pi MZ individuals undergo storage and the storage predisposes to liver damage. The storage represents the true elementary lesion and consequently reflects the phenotype-genotype correlation; (d) the site and function of the extrahepatic AAT and the relationship between intra and extracellular AAT; (e) last but not least, the concept of Endoplasmic Reticulum Storage Disease (ERSD) and of a new disease, hereditary hypofibrinogenemia with hepatic storage (HHHS). In the light of the emerging phenomenon, described in vitro, namely that M and Z AAT can form heteropolymers within hepatocytes as well as in circulation, we have reviewed the whole clinical and experimental material collected during forty years, in order to evaluate to what extent the polymerization phenomenon occurs in vivo. The paper summarizes similarities and differences between AAT and Fibrinogen as well as between the related diseases, AATD and HHHS. Indeed, fibrinogen gamma chain mutations undergo an aggregation process within the RER of hepatocytes similar to AATD. In addition, this work has clarified the intriguing phenomenon underlying a new syndrome, hereditary hypofibrinogenemia and hypo-APO-B-lipoproteinemia with hepatic storage of fibrinogen and APO-B lipoproteins. It is hoped that these studies could contribute to future research and select strategies aimed to simultaneously correct the hepatocytic storage, thus preventing the liver damage and the plasma deficiency of the two proteins.


Assuntos
Suscetibilidade a Doenças , Retículo Endoplasmático/metabolismo , Sistemas de Translocação de Proteínas/metabolismo , Animais , Animais Geneticamente Modificados , Biomarcadores , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Fígado/metabolismo , Fígado/patologia , Fígado/ultraestrutura , Redes e Vias Metabólicas , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Transporte Proteico , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismo
14.
Mol Biochem Parasitol ; 244: 111393, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34197864

RESUMO

Mitochondrial protein import depends on heterooligomeric translocases in the outer and inner membranes. Using import substrates consisting of various lengths of the N-terminal part of mitochondrial dihydrolipoamide dehydrogenase (LDH) fused to dihydrofolate reductase we present an in vivo analysis showing that in Trypanosoma brucei at least 96 aa of mature LDH are required to efficiently produce an import intermediate that spans both translocases. This is different to yeast, where around 50 aa are sufficient to achieve the same task and likely reflects the different arrangement and architecture of the trypanosomal mitochondrial translocases. Furthermore, we show that formation of the stuck import intermediate leads to a strong growth inhibition suggesting that, depending on the length of the LDH, the import channels in the translocases are quantitatively blocked.


Assuntos
Di-Hidrolipoamida Desidrogenase/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Sistemas de Translocação de Proteínas/genética , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Di-Hidrolipoamida Desidrogenase/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Sistemas de Translocação de Proteínas/metabolismo , Transporte Proteico , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade da Espécie , Tetra-Hidrofolato Desidrogenase/metabolismo , Trypanosoma brucei brucei/enzimologia
15.
Nat Commun ; 12(1): 3743, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145238

RESUMO

The extracellular Contractile Injection System (eCIS) is a toxin-delivery particle that evolved from a bacteriophage tail. Four eCISs have previously been shown to mediate interactions between bacteria and their invertebrate hosts. Here, we identify eCIS loci in 1,249 bacterial and archaeal genomes and reveal an enrichment of these loci in environmental microbes and their apparent absence from mammalian pathogens. We show that 13 eCIS-associated toxin genes from diverse microbes can inhibit the growth of bacteria and/or yeast. We identify immunity genes that protect bacteria from self-intoxication, further supporting an antibacterial role for some eCISs. We also identify previously undescribed eCIS core genes, including a conserved eCIS transcriptional regulator. Finally, we present our data through an extensive eCIS repository, termed eCIStem. Our findings support eCIS as a toxin-delivery system that is widespread among environmental prokaryotes and likely mediates antagonistic interactions with eukaryotes and other prokaryotes.


Assuntos
Archaea/genética , Bactérias/genética , Proteínas Contráteis/genética , Sistemas de Translocação de Proteínas/genética , Toxinas Biológicas/metabolismo , Animais , Archaea/metabolismo , Bactérias/metabolismo , Bacteriófagos/metabolismo , Fungos , Nematoides , Sistemas de Translocação de Proteínas/metabolismo , Transporte Proteico/fisiologia , Toxinas Biológicas/genética
16.
Plant J ; 107(2): 448-466, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33932060

RESUMO

The xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in cell wall assembly and growth regulation, cleaving and re-joining hemicellulose chains in the xyloglucan-cellulose network. Here, in a homologous system, we compare the secretion patterns of XTH11, XTH33 and XTH29, three members of the Arabidopsis thaliana XTH family, selected for the presence (XTH11 and XTH33) or absence (XTH29) of a signal peptide, and the presence of a transmembrane domain (XTH33). We show that XTH11 and XTH33 reached, respectively, the cell wall and plasma membrane through a conventional protein secretion (CPS) pathway, whereas XTH29 moves towards the apoplast following an unconventional protein secretion (UPS) mediated by exocyst-positive organelles (EXPOs). All XTHs share a common C-terminal functional domain (XET-C) that, for XTH29 and a restricted number of other XTHs (27, 28 and 30), continues with an extraterminal region (ETR) of 45 amino acids. We suggest that this region is necessary for the correct cell wall targeting of XTH29, as the ETR-truncated protein never reaches its final destination and is not recruited by EXPOs. Furthermore, quantitative real-time polymerase chain reaction analyses performed on 4-week-old Arabidopsis seedlings exposed to drought and heat stress suggest a different involvement of the three XTHs in cell wall remodeling under abiotic stress, evidencing stress-, organ- and time-dependent variations in the expression levels. Significantly, XTH29, codifying the only XTH that follows a UPS pathway, is highly upregulated with respect to XTH11 and XTH33, which code for CPS-secreted proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Glicosiltransferases/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/fisiologia , Brefeldina A/farmacologia , Membrana Celular/metabolismo , Desidratação , Glicosiltransferases/fisiologia , Complexo de Golgi/metabolismo , Resposta ao Choque Térmico , Sistemas de Translocação de Proteínas/efeitos dos fármacos
17.
Sci Rep ; 11(1): 163, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420256

RESUMO

Secretome derived from human amniotic fluid stem cells (AFSC-S) is rich in soluble bioactive factors (SBF) and offers untapped therapeutic potential for regenerative medicine while avoiding putative cell-related complications. Characterization and optimal generation of AFSC-S remains challenging. We hypothesized that modulation of oxygen conditions during AFSC-S generation enriches SBF and confers enhanced regenerative and cardioprotective effects on cardiovascular cells. We collected secretome at 6-hourly intervals up to 30 h following incubation of AFSC in normoxic (21%O2, nAFSC-S) and hypoxic (1%O2, hAFSC-S) conditions. Proliferation of human adult cardiomyocytes (hCM) and umbilical cord endothelial cells (HUVEC) incubated with nAFSC-S or hAFSC-S were examined following culture in normoxia or hypoxia. Lower AFSC counts and richer protein content in AFSC-S were observed in hypoxia. Characterization of AFSC-S by multiplex immunoassay showed higher concentrations of pro-angiogenic and anti-inflammatory SBF. hCM demonstrated highest proliferation with 30h-hAFSC-S in hypoxic culture. The cardioprotective potential of concentrated 30h-hAFSC-S treatment was demonstrated in a myocardial ischemia-reperfusion injury mouse model by infarct size and cell apoptosis reduction and cell proliferation increase when compared to saline treatment controls. Thus, we project that hypoxic-generated AFSC-S, with higher pro-angiogenic and anti-inflammatory SBF, can be harnessed and refined for tailored regenerative applications in ischemic cardiovascular disease.


Assuntos
Líquido Amniótico/citologia , Hipóxia/metabolismo , Isquemia/fisiopatologia , Miócitos Cardíacos/citologia , Sistemas de Translocação de Proteínas/metabolismo , Células-Tronco/citologia , Líquido Amniótico/metabolismo , Animais , Diferenciação Celular , Hipóxia Celular , Proliferação de Células , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hipóxia/genética , Hipóxia/fisiopatologia , Isquemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Oxigênio/metabolismo , Sistemas de Translocação de Proteínas/genética , Células-Tronco/metabolismo
18.
Mol Microbiol ; 115(3): 478-489, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33410158

RESUMO

Type VII secretion systems (T7SSs) are poorly understood protein export apparatuses found in mycobacteria and many species of Gram-positive bacteria. To date, this pathway has predominantly been studied in Mycobacterium tuberculosis, where it has been shown to play an essential role in virulence; however, much less studied is an evolutionarily divergent subfamily of T7SSs referred to as the T7SSb. The T7SSb is found in the major Gram-positive phylum Firmicutes where it was recently shown to target both eukaryotic and prokaryotic cells, suggesting a dual role for this pathway in host-microbe and microbe-microbe interactions. In this review, we compare the current understanding of the molecular architectures and substrate repertoires of the well-studied mycobacterial T7SSa systems to that of recently characterized T7SSb pathways and highlight how these differences may explain the observed biological functions of this understudied protein export machine.


Assuntos
Bactérias Gram-Positivas/metabolismo , Bactérias Gram-Positivas/patogenicidade , Mycobacterium tuberculosis/fisiologia , Mycobacterium tuberculosis/patogenicidade , Sistemas de Secreção Tipo VII/fisiologia , Virulência , Animais , Proteínas de Bactérias/metabolismo , Bactérias Gram-Positivas/ultraestrutura , Interações entre Hospedeiro e Microrganismos , Humanos , Interações Microbianas , Domínios Proteicos , Sistemas de Translocação de Proteínas/metabolismo , Sistemas de Translocação de Proteínas/ultraestrutura , Tuberculose/microbiologia , Sistemas de Secreção Tipo VII/ultraestrutura
19.
J Hepatol ; 74(1): 156-167, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32763266

RESUMO

BACKGROUND & AIMS: Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is unknown. METHODS: We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia). RESULTS: We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1ß secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B. CONCLUSIONS: These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development. LAY SUMMARY: Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.


Assuntos
Hepatócitos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Cirrose Hepática , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/imunologia , Animais , Caspase 1/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Cirrose Hepática/imunologia , Cirrose Hepática/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Sistemas de Translocação de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
20.
Mol Microbiol ; 115(3): 356-365, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32979868

RESUMO

Lipoproteins are characterized by a fatty acid moiety at their amino-terminus through which they are anchored into membranes. They fulfill a variety of essential functions in bacterial cells, such as cell wall maintenance, virulence, efflux of toxic elements including antibiotics, and uptake of nutrients. The posttranslational modification process of lipoproteins involves the sequential action of integral membrane enzymes and phospholipids as acyl donors. In recent years, the structures of the lipoprotein modification enzymes have been solved by X-ray crystallography leading to a greater insight into their function and the molecular mechanism of the reactions. The catalytic domains of the enzymes are exposed to the periplasm or external milieu and are readily accessible to small molecules. Since the lipoprotein modification pathway is essential in proteobacteria, it is a potential target for the development of novel antibiotics. In this review, we discuss recent literature on the structural characterization of the enzymes, and the in vitro activity assays compatible with high-throughput screening for inhibitors, with perspectives on the development of new antimicrobial agents.


Assuntos
Bactérias/enzimologia , Bactérias/metabolismo , Domínio Catalítico , Enzimas/química , Enzimas/metabolismo , Lipoproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Lipoproteínas/química , Sistemas de Translocação de Proteínas
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