RESUMO
Cell polarity - the asymmetric distribution of molecules and cell structures within the cell - is a feature that almost all cells possess. Even though the cytoskeleton and other intracellular organelles can have a direction and guide protein distribution, the plasma membrane is, in many cases, essential for the asymmetric localization of proteins because it helps to concentrate proteins and restrict their localization. Indeed, many proteins that exhibit asymmetric or polarized localization are either embedded in the PM or located close to it in the cellular cortex. Such proteins, which we refer to here as 'polar proteins', use various mechanisms of membrane targeting, including vesicle trafficking, direct phospholipid binding, or membrane anchoring mediated by post-translational modifications or binding to other proteins. These mechanisms are often shared with non-polar proteins, yet the unique combinations of several mechanisms or protein-specific factors assure the asymmetric distribution of polar proteins. Although there is a relatively detailed understanding of polar protein membrane targeting mechanisms in animal and yeast models, knowledge in plants is more fragmented and focused on a limited number of known polar proteins in different contexts. In this Review, we combine the current knowledge of membrane targeting mechanisms and factors for known plant transmembrane and cortical proteins and compare these with the mechanisms elucidated in non-plant systems. We classify the known factors as general or polarity specific, and we highlight areas where more knowledge is needed to construct an understanding of general polar targeting mechanisms in plants or to resolve controversies.
Assuntos
Membrana Celular , Polaridade Celular , Transporte Proteico , Membrana Celular/metabolismo , Animais , Proteínas de Plantas/metabolismo , Humanos , Proteínas de Membrana/metabolismoRESUMO
The rapid growth of bacterial resistance has created obstacles for the effective treatment with conventional antibiotics, simultaneously posing a major threat to public health. In this study, a class of novel amphipathic pyranochromene derivatives were designed and synthesized by mimicking the amphiphilic characteristics of AMPs. Bioactivity screening identified a lead compound 5a with broad-spectrum antibacterial activity against Gram-positive stains (MICs = 1-4 µg/mL) and low hemolytic toxicity (HC50 = 111.6 µg/mL). Additionally, compound 5a displayed rapid bactericidal action, and was unlikely to induce bacterial resistance. Mechanistic investigation further demonstrated that compound 5a was able to disrupt the transmembrane potential and increased membrane permeability of S. aureus, which in turn causes leakage of cell contents such as DNA and proteins, ultimately leading to bacterial death. These findings indicated that compound 5a is a promising lead to combat bacterial infection caused by Gram-positive bacteria.
Assuntos
Antibacterianos , Benzopiranos , Desenho de Fármacos , Bactérias Gram-Positivas , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Benzopiranos/farmacologia , Benzopiranos/química , Benzopiranos/síntese química , Bactérias Gram-Positivas/efeitos dos fármacos , Relação Estrutura-Atividade , Estrutura Molecular , Humanos , Staphylococcus aureus/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hemólise/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacosRESUMO
Extra-large stimulatory Gα (XLαs) is a large variant of G protein αs subunit (Gαs) that uses an alternative promoter and thus differs from Gαs at the first exon. XLαs activation by G protein-coupled receptors mediates cAMP generation, similarly to Gαs; however, Gαs and XLαs have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLαs can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLαs directly and specifically stimulates a specific isoform of phospholipase Cß (PLCß), PLCß4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLαs to activate cAMP generation nor the canonical G protein switch II regions are required for PLCß stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gßγ, suggesting a mechanism of activation that relies on Gßγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLαs relative to Gαs confers the ability to activate PLCß4. We also show that PLCß4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of Gs-coupled receptors that may have a critical role in endocrine physiology.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP , Fosfatos de Inositol , Fosfolipase C beta , Membrana Celular/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Fosfatos de Inositol/metabolismo , Isoenzimas/metabolismo , Isoproterenol/farmacologia , Fosfolipase C beta/metabolismoRESUMO
Protein phosphatase-1 (PP1) is a ubiquitous enzyme involved in multiple processes inside cells. PP1-disrupting peptides (PDPs) are chemical tools that selectively bind to PP1 and release its activity. To restrict the activity of PDPs to a cellular compartment, we developed PDP-Mem, a cell membrane-targeting PDP. The membrane localization was achieved through the introduction of a palmitoylated lysine. PDP-Mem was shown to activate PP1α in vitro and to localize to the membrane of HeLa Kyoto and U2OS cells. However, in cells, the combination of the polybasic sequence for cell penetration and the membrane targeting palmitoylated lysine activates the MAPK signaling pathway and induces cytoplasmic calcium release independently of PP1 activation. Therefore, when targeting peptides to cellular membranes, undesired effects induced by the targeting sequence and lipid modification need to be considered.
Assuntos
Lisina , Peptídeos , Humanos , Proteína Fosfatase 1/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismo , Células HeLa , Transdução de Sinais , FosforilaçãoRESUMO
Cell-penetrating peptides (CPPs) are prominent scaffolds for drug developments and related research, particularly the endocytic delivery of biomacromolecules. Effective cargo release from endosomes prior to lysosomal degradation is a crucial step, where the rational design and selection of CPPs remains a challenge and calls for deeper mechanistic understandings. Here, we have investigated a strategy of designing CPPs that selectively disrupt endosomal membranes based on bacterial membrane targeting sequences (MTSs). Six synthesized MTS peptides all exhibit cell-penetrating abilities, among which two d-peptides (d-EcMTS and d-TpMTS) are able to escape from endosomes and localize at ER after entering the cell. The utility of this strategy has been demonstrated by the intracellular delivery of green fluorescent protein (GFP). Together, these results suggest that the large pool of bacterial MTSs may be a rich source for the development of novel CPPs.
Assuntos
Peptídeos Penetradores de Células , Peptídeos Penetradores de Células/química , Endossomos/química , Endossomos/metabolismoRESUMO
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 MassasRESUMO
As a central player in the canonical TGF-ß signaling pathway, Smad2 transmits the activation of TGF-ß receptors at the plasma membrane (PM) to transcriptional regulation in the nucleus. Although it has been well established that binding of TGF-ß to its receptors leads to the recruitment and activation of Smad2, the spatiotemporal mechanism by which Smad2 is recruited to the activated TGF-ß receptor complex and activated is not fully understood. Here we show that Smad2 selectively and tightly binds phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the PM. The PI(4,5)P2-binding site is located in the MH2 domain that is involved in interaction with the TGF-ß receptor I that transduces TGF-ß-receptor binding to downstream signaling proteins. Quantitative optical imaging analyses show that PM recruitment of Smad2 is triggered by its interaction with PI(4,5)P2 that is locally enriched near the activated TGF-ß receptor complex, leading to its binding to the TGF-ß receptor I. The PI(4,5)P2-binding activity of Smad2 is essential for the TGF-ß-stimulated phosphorylation, nuclear transport, and transcriptional activity of Smad2. Structural comparison of all Smad MH2 domains suggests that membrane lipids may also interact with other Smad proteins and regulate their function in diverse TGF-ß-mediated biological processes.
Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Transdução de Sinais , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transporte Ativo do Núcleo Celular , Células HeLa , Humanos , Fosfatidilinositol 4,5-Difosfato/genética , Ligação Proteica , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Proteína Smad2/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
MAIN CONCLUSION: The N-myristoylation is required for BSK1 proper plasma membrane targeting and protein turnover. Brassinosteroid (BR) signaling kinase 1 (BSK1), with a myristoylation site at its N-terminus to anchor at plasma membrane (PM), is involved in BR-regulated plant growth and flg22-triggered immunity responses. However, little is known about the role of N-myristoylation in BSK1 protein homeostasis. Here, we revealed that N-myristoylation is critical to the PM targeting and protein stability of BSK1. The N-myristoylation-deficient mutant BSK1G2A mainly distributed in the cytoplasm and retained in the endoplasmic reticulum. We further found that the BSK1G2A proteins were unstable and degraded through ATG8e-labled autophagic pathway. This study provides a new insight into the regulation of plant protein homeostasis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Homeostase , Transdução de Sinais/fisiologiaRESUMO
Infections associated with Gram-positive bacteria like S. aureus pose a major threat as these bacteria can develop resistance and thereby limit the applications of antibiotics. Therefore, there is a need for new antibacterials to mitigate these infections. Bacterial membranes present an attractive therapeutic target as these membranes are anionic in nature and have a low chance of developing modifications in their physicochemical features. Antimicrobial peptides (AMPs) can disrupt the microbial membranes via electrostatic interactions, but the poor stability of AMPs halts their clinical translation. Here, we present the synthesis of eight N-methyl benzimidazole substituted cholic acid amphiphiles as antibacterial agents. We screened these novel heterocyclic cholic acid amphiphiles against different pathogens. Among the series, CABI-6 outperformed the other amphiphiles in terms of bactericidal activity against S. aureus. The membrane disruptive property of CABI-6 using a fluorescence-based assay has also been investigated, and it was inferred that CABI-6 can enhance the production of reactive oxygen species. We further demonstrated that CABI-6 can clear the pre-formed biofilms and can mitigate wound infection in murine models.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecção dos Ferimentos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias , Benzimidazóis/farmacologia , Biofilmes , Ácido Cólico/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Staphylococcus aureusRESUMO
KEY POINTS: We report a novel method for the transient expression of SARS-CoV-2 envelope (E) protein in intracellular organelles and the plasma membrane of mammalian cells and Xenopus oocytes. Intracellular expression of SARS-CoV-2 E protein increases intra-Golgi pH. By targeting the SARS-CoV-2 E protein to the plasma membrane, we show that it forms a cation channel, viroporin, that is modulated by changes of pH. This method for studying the activity of viroporins may facilitate screening for new antiviral drugs to identify novel treatments for COVID-19. ABSTRACT: The envelope (E) protein of coronaviruses such as SARS-CoV-1 is proposed to form an ion channel or viroporin that participates in viral propagation and pathogenesis. Here we developed a technique to study the E protein of SARS-CoV-2 in mammalian cells by directed targeting using a carboxyl-terminal fluorescent protein tag, mKate2. The wild-type SARS-CoV-2 E protein can be trafficked to intracellular organelles, notably the endoplasmic reticulum-Golgi intermediate complex, where its expression increases pH inside the organelle. We also succeeded in targeting SARS-CoV-2 E to the plasma membrane, which enabled biophysical analysis using whole-cell patch clamp recording in a mammalian cell line, HEK 293 cells, and two-electrode voltage clamp electrophysiology in Xenopus oocytes. The results suggest that the E protein forms an ion channel that is permeable to monovalent cations such as Na+ , Cs+ and K+ . The E current is nearly time- and voltage-independent when E protein is expressed in mammalian cells, and is modulated by changes of pH. At pH 6.0 and 7.4, the E protein current is activated, whereas at pH 8.0 and 9.0, the amplitude of E protein current is reduced, and in oocytes the inward E current fades at pH 9 in a time- and voltage-dependent manner. Using this directed targeting method and electrophysiological recordings, potential inhibitors of the E protein can be screened and subsequently investigated for antiviral activity against SARS-CoV-2 in vitro and possible efficacy in treating COVID-19.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Cátions , Células HEK293 , Humanos , Concentração de Íons de HidrogênioRESUMO
The transmembrane recognition complex (TRC) pathway targets tail-anchored (TA) proteins to the membrane of the endoplasmic reticulum (ER). While many TA proteins are known to be able to use this pathway, it is essential for the targeting of only a few. Here, we uncover a large number of TA proteins that engage with TRC40 when other targeting machineries are fully operational. We use a dominant-negative ATPase-impaired mutant of TRC40 in which aspartate 74 was replaced by a glutamate residue to trap TA proteins in the cytoplasm. Manipulation of the hydrophobic TA-binding groove in TRC40 (also known as ASNA1) reduces interaction with most, but not all, substrates suggesting that co-purification may also reflect interactions unrelated to precursor protein targeting. We confirm known TRC40 substrates and identify many additional TA proteins interacting with TRC40. By using the trap approach in combination with quantitative mass spectrometry, we show that Golgi-resident TA proteins such as the golgins golgin-84, CASP and giantin as well as the vesicle-associated membrane-protein-associated proteins VAPA and VAPB interact with TRC40. Thus, our results provide new avenues to assess the essential role of TRC40 in metazoan organisms.This article has an associated First Person interview with the first author of the paper.
Assuntos
ATPases Transportadoras de Arsenito/genética , Mutação/genética , ATPases Transportadoras de Arsenito/metabolismo , Citoplasma/metabolismo , Inativação Gênica , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Ligação Proteica , Frações Subcelulares/metabolismo , Especificidade por SubstratoRESUMO
COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, has resulted in global social and economic disruption, putting the world economy to the largest global recession since the Great Depression. To control the spread of COVID-19, cutting off the transmission route is a critical step. In this work, the efficient inactivation of human coronavirus with photodynamic therapy (PDT) by employing photosensitizers with aggregation-induced emission characteristics (DTTPB) is reported. DTTPB is designed to bear a hydrophilic head and two hydrophobic tails, mimicking the structure of phospholipids on biological membranes. DTTPB demonstrates a broad absorption band covering the whole visible light range and high molar absorptivity, as well as excellent reactive oxygen species sensitizing ability, making it an excellent candidate for PDT. Besides, DTTPB can target membrane structure, and bind to the envelope of human coronaviruses. Upon light irradiation, DTTPB demonstrates highly effective antiviral behavior: human coronavirus treated with DTTPB and white-light irradiation can be efficiently inactivated with complete loss of infectivity, as revealed by the significant decrease of virus RNA and proteins in host cells. Thus, DTTPB sensitized PDT can efficiently prevent the infection and the spread of human coronavirus, which provides a new avenue for photodynamic combating of COVID-19.
Assuntos
COVID-19 , Fotoquimioterapia , Humanos , Pandemias , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , SARS-CoV-2RESUMO
Ras-related C3 botulinum toxin substrate 1 (Rac1) is a member of the Rho family of GTPases that functions as a molecular switch to regulate many important cellular events including actin cytoskeleton remodeling during neurite outgrowth. Engulfment and cell motility 1 (ELMO1)-dedicator of cytokinesis 1 (DOCK180) is a bipartite guanine nucleotide exchange factor (GEF) complex that has been reported to activate Rac1 on the plasma membrane (PM). Emerging evidence suggests that the small GTPase ADP ribosylation factor 6 (ARF6) activates Rac1 via the ELMO1/DOCK180 complex. However, the exact mechanism by which ARF6 triggers ELMO1/DOCK180-mediated Rac1 signaling remains unclear. Here, we report that the neuronal scaffold protein FE65 serves as a functional link between ARF6 and ELMO1, allowing the formation of a multimeric signaling complex. Interfering with formation of this complex by transfecting either FE65-binding-defective mutants or FE65 siRNA attenuates both ARF6-ELMO1-mediated Rac1 activation and neurite elongation. Notably, the PM trafficking of ELMO1 is markedly decreased in cells with suppressed expression of either FE65 or ARF6. Likewise, this process is attenuated in the FE65-binding-defective mutants transfected cells. Moreover, overexpression of FE65 increases the amount of ELMO1 in the recycling endosome, an organelle responsible for returning proteins to the PM, whereas knockout of FE65 shows opposite effect. Together, our data indicates that FE65 potentiates ARF6-Rac1 signaling by orchestrating ARF6 and ELMO1 to promote the PM trafficking of ELMO1 via the endosomal recycling pathway, and thus, promotes Rac1-mediated neurite outgrowth.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Crescimento Neuronal/fisiologia , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Fator 6 de Ribosilação do ADP , Animais , Células CHO , Células COS , Linhagem Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetulus , Endossomos/metabolismo , Células HEK293 , Humanos , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologiaRESUMO
(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, ß1, ß2, ß3, and phospholemman. The α1, α2, ß1, and ß2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.
Assuntos
Aquaporina 1/fisiologia , Plexo Corióideo/metabolismo , Células Epiteliais/metabolismo , Microvilosidades/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/fisiologia , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Mitochondria constantly divide and fuse. Homotypic fusion of the outer mitochondrial membranes requires the mitofusin (MFN) proteins, a family of dynamin-like GTPases. MFNs are anchored in the membrane by transmembrane (TM) segments, exposing both the N-terminal GTPase domain and the C-terminal tail (CT) to the cytosol. This arrangement is very similar to that of the atlastin (ATL) GTPases, which mediate fusion of endoplasmic reticulum (ER) membranes. We engineered various MFN-ATL chimeras to gain mechanistic insight into MFN-mediated fusion. When MFN1 is localized to the ER by TM swapping with ATL1, it functions in the maintenance of ER morphology and fusion. In addition, an amphipathic helix in the CT of MFN1 is exchangeable with that of ATL1 and critical for mitochondrial localization of MFN1. Furthermore, hydrophobic residues N-terminal to the TM segments of MFN1 play a role in membrane targeting but not fusion. Our findings provide important insight into MFN-mediated membrane fusion.
Assuntos
Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Fusão de Membrana/fisiologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Animais , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Microscopia de Fluorescência , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Conformação Proteica , Alinhamento de Sequência , LevedurasRESUMO
Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29-VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes.
Assuntos
Proteínas de Bactérias/química , Legionella pneumophila/química , Multimerização Proteica , Fatores de Virulência/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidade , Domínios Proteicos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Interkingdom polymicrobial biofilms formed by Gram-positive Staphylococcus aureus and Candida albicans pose serious threats of chronic systemic infections due to the absence of any common therapeutic target for their elimination. Herein, we present the structure-activity relationship (SAR) of membrane-targeting cholic acid-peptide conjugates (CAPs) against Gram-positive bacterial and fungal strains. Structure-activity investigations validated by mechanistic studies revealed that valine-glycine dipeptide-derived CAP 3 was the most effective broad-spectrum antimicrobial against S. aureus and C. albicans CAP 3 was able to degrade the preformed single-species and polymicrobial biofilms formed by S. aureus and C. albicans, and CAP 3-coated materials prevented the formation of biofilms. Murine wound and catheter infection models further confirmed the equally potent bactericidal and fungicidal effect of CAP 3 against bacterial, fungal, and polymicrobial infections. Taken together, these results demonstrate that CAPs, as potential broad-spectrum antimicrobials, can effectively clear the frequently encountered polymicrobial infections and can be fine-tuned further for future applications.
Assuntos
Anti-Infecciosos/farmacologia , Biofilmes/efeitos dos fármacos , Ácido Cólico/farmacologia , Peptídeos/farmacologia , Animais , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana/métodos , Viabilidade Microbiana/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacosRESUMO
Biological membranes display a staggering complexity of lipids and proteins orchestrating cellular functions. Superior analytical tools coupled with numerous functional cellular screens have enabled us to query their role in cellular signalling, trafficking, guiding protein structure and function-all of which rely on the dynamic membrane lipid properties indispensable for proper cellular functions. Alteration of these has led to emergence of various pathological conditions, thus opening an area of lipid-centric therapeutic approaches. This perspective is a short summary of the dynamic properties of membranes essential for proper cellular functions, dictating both protein and lipid functions, and mis-regulated in diseases. Towards the end, we focus on some challenges lying ahead and potential means to tackle the same, mainly underscored by multi-disciplinary approaches.
Assuntos
Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais , Animais , Membrana Celular/patologia , Humanos , Transporte ProteicoRESUMO
A marine, sand-dwelling, golden-brown alga is described from clonal cultures established from a high intertidal pool in southeastern Australia. This tiny, unicellular species, which we call the "golden paradox" (Chrysoparadoxa australica gen. et sp. nov.), is benthic, surrounded by a multilayered cell wall and attached to the substratum by a complex adhesive plug. Each vegetative cell gives rise to a single, naked zoospore with heterokont flagella that settles and may become briefly amoeboid prior to dividing. Daughter cells are initially amoeboid, then either permanently attach and return to the benthic stage or become motile again prior to final settlement. Two deeply lobed chloroplasts occupy opposite ends of the cell and are surrounded by only two membranes. The outer chloroplast membrane is continuous between the two chloroplasts via the outer membrane of the nuclear envelope. Only two membranes occupy the chloroplast-nucleus interface, the inner membrane of the nuclear envelope and the inner chloroplast membrane. A small pyrenoid is found in each chloroplast and closely abuts the nucleus or protrudes into it. It contains an unusual, membrane-bound inclusion that stains with SYBR green but is unlikely to be a nucleomorph. Phylogenies inferred from a 10-gene concatenated alignment show an early-branching position within the PX clade. The unusual morphological features and phylogenetic position indicate C. australica should be classified as a new class, Chrysoparadoxophyceae. Despite an atypical plastid, exploration of the C. australica transcriptome revealed typical heterokont protein targeting to the plastid.
Assuntos
Cloroplastos , Estramenópilas , Austrália , Filogenia , PlastídeosRESUMO
Isoprenylcysteine-O-Carboxyl Methyltransferase (ICMT) catalyzes the final step in the prenylation process of different proteins including members of the Ras superfamily of GTPases. While cysteine methylation is essential in mammalian cells for growth, membrane association, and signalling by Ras and Rho GTPases, its role during signal transduction events in simple eukaryotes like yeasts appears irrelevant. By using a multidisciplinary approach our group has recently shown that, contrary to this initial assumption, in the fission yeast Schizosaccharomyces pombe ICMT activity encoded by the Mam4 gene is not only important to promote selective plasma membrane targeting of Ras and specific Rho GTPases, but also to allow precise downstream signalling to the mitogen-activated protein kinase and target of rapamycin pathways in response to diverse environmental cues. Thus, the dynamic regulation of in vivo methylation as a modulator of GTPase localization and function is an evolutionary conserved mechanism, making fission yeast an appealing model organism to study the regulation of this process.