RESUMEN
Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.
Asunto(s)
Adipocitos , Proteínas de Unión al Calcio , Metabolismo de los Lípidos , Proteínas de la Membrana , Animales , Femenino , Humanos , Ratones , Adipocitos/citología , Adipocitos/metabolismo , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Placenta , Triglicéridos/metabolismo , Retículo Endoplásmico/metabolismo , Gotas Lipídicas/metabolismo , Ácidos Grasos/metabolismo , Hipotermia/metabolismo , TermogénesisRESUMEN
Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is an established tool for exploring protein structure and dynamics. Although nitroxide side chains attached to a single cysteine via a disulfide linkage are commonly employed in SDSL-EPR, their internal flexibility complicates applications to monitor slow internal motions in proteins and to structure determination by distance mapping. Moreover, the labile disulfide linkage prohibits the use of reducing agents often needed for protein stability. To enable the application of SDSL-EPR to the measurement of slow internal dynamics, new spin labels with hindered internal motion are desired. Here, we introduce a highly ordered nitroxide side chain, designated R9, attached at a single cysteine residue via a non-reducible thioether linkage. The reaction to introduce R9 is highly selective for solvent-exposed cysteine residues. Structures of R9 at two helical sites in T4 Lysozyme were determined by X-ray crystallography and the mobility in helical sequences was characterized by EPR spectral lineshape analysis, Saturation Transfer EPR, and Saturation Recovery EPR. In addition, interspin distance measurements between pairs of R9 residues are reported. Collectively, all data indicate that R9 will be useful for monitoring slow internal structural fluctuations, and applications to distance mapping via dipolar spectroscopy and relaxation enhancement methods are anticipated. Supplementary Information: The online version contains supplementary material available at 10.1007/s00723-023-01618-8.
RESUMEN
Human cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. These processes are known to be regulated by extracellular signal-regulated kinase 2 (ERK2) in other species, but the role of ERK2 in mitosis in mammals remains unclear. Here, we have identified the dual-specificity phosphatase 7 (DUSP7), known to display selectivity for ERK2, as important in regulating chromosome alignment. During mitosis, DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytically inactive mutants, led to a decrease in the levels of phospho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, knockdown or chemical inhibition of ERK2 or chemical inhibition of the MEK kinase that phosphorylates ERK2 led to chromosome alignment defects. Our results support a model wherein MEK-mediated phosphorylation and DUSP7-mediated dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.
Asunto(s)
Cromosomas Humanos/metabolismo , Fosfatasas de Especificidad Dual/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Mitosis , Cromosomas Humanos/genética , Fosfatasas de Especificidad Dual/genética , Células HCT116 , Células HeLa , Humanos , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Mutación , Fosforilación/genéticaRESUMEN
One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.
Asunto(s)
Benchmarking , Espectrometría de Masas/métodos , Proteínas/química , Desnaturalización Proteica , Procesamiento Proteico-Postraduccional , ProteómicaRESUMEN
The assembly of large, multi-cofactor membrane protein complexes like photosystem II (PSII) requires a high level of coordination. The process is facilitated by a large network of auxiliary proteins that bind transiently to unassembled subunits, preassembled modules or intermediate states of PSII, which are comprised of a subset of subunits. However, analysis of these immature, partially assembled PSII complexes is hampered by their low abundance and intrinsic instability. In this study, PSII was purified from the thermophilic cyanobacterium Thermosynechococcus elongatus via Twin-Strep-tagged CP43 and further separated by ion exchange chromatography into mature and immature complexes. Mass spectrometry analysis of the immature Psb27-PSII intermediate revealed six different Psb27 proteoforms with distinct lipid modifications. The maturation and functional role of thylakoid localized lipoproteins are discussed.
Asunto(s)
Cianobacterias , Complejo de Proteína del Fotosistema II , Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Lípidos , Espectrometría de Masas , Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismoRESUMEN
Insulin therapy in the setting of type 1 and advanced type 2 diabetes is complicated by increased risk of hypoglycemia. This potentially fatal complication could be mitigated by a glucose-responsive insulin analog. We report an insulin-facilitated glucose transporter (Glut) inhibitor conjugate, in which the insulin molecule is rendered glucose-responsive via conjugation to an inhibitor of Glut. The binding affinity of this insulin analog to endogenous Glut is modulated by plasma and tissue glucose levels. In hyperglycemic conditions (e.g., uncontrolled diabetes or the postprandial state), the in situ-generated insulin analog-Glut complex is driven to dissociate, freeing the insulin analog and glucose-accessible Glut to restore normoglycemia. Upon overdose, enhanced binding of insulin analog to Glut suppresses the glucose transport activity of Glut to attenuate further uptake of glucose. We demonstrate the ability of this insulin conjugate to regulate blood glucose levels within a normal range while mitigating the risk of hypoglycemia in a type 1 diabetic mouse model.
Asunto(s)
Glucemia/efectos de los fármacos , Proteínas Facilitadoras del Transporte de la Glucosa/antagonistas & inhibidores , Hipoglucemia/prevención & control , Hipoglucemiantes , Insulina , Animales , Glucemia/análisis , Diabetes Mellitus Experimental , Sistemas de Liberación de Medicamentos/métodos , Hiperglucemia/tratamiento farmacológico , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Insulina/análogos & derivados , Insulina/química , Insulina/farmacología , RatonesRESUMEN
The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule-kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors before proceeding into anaphase. Dysregulation of the SAC leads to chromosome segregation errors that have been linked to human diseases like cancer. Although much has been learned about the composition of the SAC and the factors that regulate its activity, the proximity associations of core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2-proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 in mitotic-enriched populations of cells where the SAC is active. These five protein association maps were integrated to generate a SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein-protein interactions, and proximity associations. Our analysis validated many known SAC complexes and protein-protein interactions. Additionally, it uncovered new protein associations, including the ELYS-MAD1L1 interaction that we have validated, which lend insight into the functioning of core SAC proteins and highlight future areas of investigation to better understand the SAC.
Asunto(s)
Puntos de Control de la Fase M del Ciclo Celular , Huso Acromático , Proteínas de Ciclo Celular/genética , Humanos , Cinetocoros , Proteínas Serina-Treonina Quinasas/genética , ProteómicaRESUMEN
Each of the 30 human amyloid diseases is associated with the aggregation of a particular precursor protein into amyloid fibrils. In transthyretin amyloidosis (ATTR), mutant or wild-type forms of the serum carrier protein transthyretin (TTR), synthesized and secreted by the liver, convert to amyloid fibrils deposited in the heart and other organs. The current standard of care for hereditary ATTR is liver transplantation, which replaces the mutant TTR gene with the wild-type gene. However, the procedure is often followed by cardiac deposition of wild-type TTR secreted by the new liver. Here we find that amyloid fibrils extracted from autopsied and explanted hearts of ATTR patients robustly seed wild-type TTR into amyloid fibrils in vitro. Cardiac-derived ATTR seeds can accelerate fibril formation of wild-type and monomeric TTR at acidic pH and under physiological conditions, respectively. We show that this seeding is inhibited by peptides designed to complement structures of TTR fibrils. These inhibitors cap fibril growth, suggesting an approach for halting progression of ATTR.
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Amiloide/química , Miocardio/química , Prealbúmina/química , Amiloide/metabolismo , Neuropatías Amiloides Familiares/metabolismo , Neuropatías Amiloides Familiares/patología , Femenino , Humanos , Concentración de Iones de Hidrógeno , Masculino , Miocardio/metabolismo , Miocardio/patología , Prealbúmina/metabolismoRESUMEN
Photochemical energy conversion during oxygenic photosynthesis is performed by membrane-embedded chlorophyll-binding protein complexes. The biogenesis and maintenance of these complexes requires auxiliary protein factors that optimize the assembly process and protect nascent complexes from photodamage. In cyanobacteria, several lipoproteins contribute to the biogenesis and function of the photosystem II (PSII) complex. They include CyanoP, CyanoQ, and Psb27, which are all attached to the lumenal side of PSII complexes. Here, we show that the lumenal Ycf48 assembly factor found in the cyanobacterium Synechocystis sp. PCC 6803 is also a lipoprotein. Detailed mass spectrometric analysis of the isolated protein supported by site-directed mutagenesis experiments indicates lipidation of the N-terminal C29 residue of Ycf48 and removal of three amino acids from the C-terminus. The lipobox sequence in Ycf48 contains a cysteine residue at the -3 position compared to Leu/Val/Ile residues found in the canonical lipobox sequence. The atypical Ycf48 lipobox sequence is present in most cyanobacteria but is absent in eukaryotes. A possible role for lipoproteins in the coordinated assembly of cyanobacterial PSII is discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Metabolismo de los Lípidos , Complejo de Proteína del Fotosistema II/metabolismo , Synechocystis/metabolismoRESUMEN
Somatic mutations that perturb Parkin ubiquitin ligase activity and the misregulation of iron homeostasis have both been linked to Parkinson's disease. Lactotransferrin (LTF) is a member of the family of transferrin iron binding proteins that regulate iron homeostasis, and increased levels of LTF and its receptor have been observed in neurodegenerative disorders like Parkinson's disease. Here, we report that Parkin binds to LTF and ubiquitylates LTF to influence iron homeostasis. Parkin-dependent ubiquitylation of LTF occurred most often on lysines (K) 182 and 649. Substitution of K182 or K649 with alanine (K182A or K649A, respectively) led to a decrease in the level of LTF ubiquitylation, and substitution at both sites led to a major decrease in the level of LTF ubiquitylation. Importantly, Parkin-mediated ubiquitylation of LTF was critical for regulating intracellular iron levels as overexpression of LTF ubiquitylation site point mutants (K649A or K182A/K649A) led to an increase in intracellular iron levels measured by ICP-MS/MS. Consistently, RNAi-mediated depletion of Parkin led to an increase in intracellular iron levels in contrast to overexpression of Parkin that led to a decrease in intracellular iron levels. Together, these results indicate that Parkin binds to and ubiquitylates LTF to regulate intracellular iron levels. These results expand our understanding of the cellular processes that are perturbed when Parkin activity is disrupted and more broadly the mechanisms that contribute to Parkinson's disease.
Asunto(s)
Homeostasis , Hierro/metabolismo , Lactoferrina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Sitios de Unión , Células HEK293 , Humanos , Lactoferrina/química , Modelos Moleculares , Conformación ProteicaRESUMEN
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 , UbiquitinaciónRESUMEN
Multidrug transporters are ubiquitous efflux pumps that provide cells with defense against various toxic compounds. In bacteria, which typically harbor numerous multidrug transporter genes, the majority function as secondary multidrug/proton antiporters. Proton-coupled secondary transport is a fundamental process that is not fully understood, largely owing to the obscure nature of proton-transporter interactions. Here we analyzed the substrate/proton coupling mechanism in MdfA, a model multidrug/proton antiporter. By measuring the effect of protons on substrate binding and by directly measuring proton binding and release, we show that substrates and protons compete for binding to MdfA. Our studies strongly suggest that competition is an integral feature of secondary multidrug transport. We identified the proton-binding acidic residue and show that, surprisingly, the substrate binds at a different site. Together, the results suggest an interesting mode of indirect competition as a mechanism of multidrug/proton antiport.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Diciclohexilcarbodiimida/farmacología , Escherichia coli/citología , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Concentración de Iones de Hidrógeno , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Compuestos Onio/antagonistas & inhibidores , Compuestos Onio/química , Compuestos Onio/farmacología , Compuestos Organofosforados/antagonistas & inhibidores , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacología , Pironina/farmacologíaRESUMEN
Oligomeric forms of α-synuclein are believed to cause mitochondrial injury, which may contribute to neurotoxicity in Parkinson's disease (PD). Here oligomers of α-synuclein were prepared using the dopamine metabolite, DOPAL (3,4-dihydroxyphenyl-acetaldehyde), in the presence of guanidinium hydrochloride. Electron microscopy, mass spectrometry, and Western blotting studies revealed enhanced and stable oligomerization with DOPAL compared with dopamine or CuCl2 /H2 O2 . Using isolated mouse brain mitochondria, DOPAL-oligomerized α-synuclein (DOS) significantly inhibited oxygen consumption rates compared with untreated, control-fibrillated, and dopamine-fibrillated synuclein, or with monomeric α-synuclein. Inhibition was greater in the presence of malate plus pyruvate than with succinate, suggesting the involvement of mitochondrial complex I. Mitochondrial membrane potential studies using fluorescent probes, JC-1, and Safranin O also detected enhanced inhibition by DOS compared with the other aggregated forms of α-synuclein. Testing a small customized chemical library, four compounds were identified that rescued membrane potential from DOS injury. While diverse in chemical structure and mechanism, each compound has been reported to interact with mitochondrial complex I. Western blotting studies revealed that none of the four compounds disrupted the oligomeric banding pattern of DOS, suggesting their protection involved direct mitochondrial interaction. The remaining set of chemicals also did not disrupt oligomeric banding, attesting to the high structural stability of this α-synuclein proteoform. DOPAL and α-synuclein are both found in dopaminergic neurons, where their levels are elevated in PD and in animal models exposed to chemical toxicants, including agricultural pesticides. The current study provides further evidence of α-synuclein-induced mitochondrial injury and a likely role in PD neuropathology.
Asunto(s)
Dopamina/metabolismo , Mitocondrias/metabolismo , alfa-Sinucleína/metabolismo , Animales , Dopamina/química , Dopamina/farmacología , Femenino , Masculino , Potencial de la Membrana Mitocondrial , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Consumo de Oxígeno , Enfermedad de Parkinson , Agregado de Proteínas/efectos de los fármacos , alfa-Sinucleína/química , alfa-Sinucleína/farmacología , alfa-Sinucleína/ultraestructuraRESUMEN
BACKGROUND: Loss of intestinal barrier integrity plays a fundamental role in the pathogenesis of various gastrointestinal diseases and is implicated in the onset of sepsis and multiple organ failure. An array of methods to assess different aspects of intestinal barrier function suffers from lack of sensitivity, prolonged periods of specimen collection, or high expense. We have developed a technique to measure the concentration of the food dye FD&C Blue #1 from blood and sought to assess its utility in measuring intestinal barrier function in humans. MATERIALS AND METHODS: Four healthy volunteers and 10 critically ill subjects in the intensive care unit were recruited in accordance with an institutional review board approved protocol. Subjects were given 0.5 mg/kg Blue #1 enterally as an aqueous solution of diluted food coloring. Five blood specimens were drawn per subject: 0 h (before dose), 1, 2, 4, and 8 h. After plasma isolation, organic extracts were analyzed by high-performance liquid chromatography/mass spectrometry detecting the presence of unmodified dye. RESULTS: We found no baseline detectable absorption in healthy volunteers. After including the subjects in the intensive care unit, we compared dye absorption in the six subjects who met criteria for septic shock with the eight who did not. Septic patients demonstrated significantly greater absorption of Blue #1 after 2 h. CONCLUSIONS: We have developed a novel, easy-to-use method to measure intestinal barrier integrity using a food grade dye detectable by mass spectrometry analysis of patient blood following oral administration.
Asunto(s)
Colorantes de Alimentos/farmacocinética , Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Choque Séptico/diagnóstico , Administración Oral , Adulto , Bencenosulfonatos/administración & dosificación , Bencenosulfonatos/sangre , Bencenosulfonatos/farmacocinética , Enfermedad Crítica , Estudios de Factibilidad , Femenino , Colorantes de Alimentos/administración & dosificación , Colorantes de Alimentos/análisis , Voluntarios Sanos , Humanos , Unidades de Cuidados Intensivos , Masculino , Permeabilidad , Estudios Prospectivos , Choque Séptico/sangre , Choque Séptico/fisiopatologíaRESUMEN
Voltage-dependent anion channel-1 (VDAC1) is a highly regulated ß-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr83 and Glu73, respectively. When Glu73 was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr62 within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu73 residue.
Asunto(s)
Colesterol/química , Canal Aniónico 1 Dependiente del Voltaje/química , Marcadores de Afinidad , Animales , Sitios de Unión , Ratones , Resonancia Magnética Nuclear Biomolecular , Canal Aniónico 1 Dependiente del Voltaje/genéticaRESUMEN
Multiple secretion pathways are known for export of protein(s) forming the S-layer in bacteria. The unicellular model cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter S. 6803) also possesses a well-defined S-layer composed of Sll1951 protein. However, the mechanism of its secretion is not completely understood. In the present study, the putative T1SS (Type I secretion system) components, Sll1180 and Sll1181 [inner membrane ABC transporter and membrane fusion protein (MFP), respectively] were characterized for their role in Sll1951 secretion. The corresponding ORFs i.e. sll1180 and sll1181 were inactivated by insertion of a spectinomycin resistance gene. The viability of the homozygous mutants of both the genes indicated dispensability of the corresponding proteins under the experimental conditions. Interestingly, the culture supernatants of the mutants i.e. Δsll1180 and Δsll1181, lacked Sll1951 as observed on SDS-PAGE and confirmed by mass spectrometry. Immunofluorescence delineated a distinct outer ring of Sll1951 in S. 6803 cells only that was further iterated by transmission and scanning electron microscopy. The loss of S-layer imparted an aggregative phenotype to both the mutants. Surprisingly, Δsll1181 cells showed increased sensitivity to different antibiotics indicating a role in multidrug efflux. This is the first report establishing Sl1180 and Sll1181 proteins as partners of the previously characterized Slr1270, for Sll1951 secretion and thus S-layer biogenesis in S. 6803. Sll1181 (in conjunction with Slr1270) also acts as MFP in multidrug efflux along with a yet uncharacterized inner membrane protein.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Proteínas Bacterianas/fisiología , Proteínas de la Fusión de la Membrana/fisiología , Synechocystis/fisiología , Antibacterianos/farmacología , Microscopía Electrónica , Transporte de Proteínas , Synechocystis/efectos de los fármacosRESUMEN
Background and Aims: Cultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described. Methods: Time-course experiments were carried out to analyse gene expression, and protein and metabolite levels. Key Results: Both MeJA treatment and the overexpression of COI1 modify growth, by altering cell proliferation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as ß-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby providing a connection between JA-inhibited growth and stress responses. Conclusions: This study contributes to the understanding of the regulation of growth and the production of metabolic resources by JAs and COI1. This will have important implications in dissecting the complex relationships between hormonal and cell wall signalling in plants. The work also provides tools to uncover novel mechanisms co-ordinating cell division and post-mitotic cell expansion in the absence of organ developmental control.
Asunto(s)
Acetatos/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/fisiología , Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Transducción de Señal , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , División Celular/genética , Pared Celular/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Genes cdc/fisiología , Proteínas de Plantas/metabolismoRESUMEN
The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule-severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan-ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule-severing activity in vitro Interestingly, during interphase, KATNBL1 is sequestered in the nucleus through an N-terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule-severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Microtúbulos/metabolismo , Proteómica/métodos , Adenosina Trifosfatasas/química , Núcleo Celular/metabolismo , Células HeLa , Humanos , Katanina , Espectrometría de Masas , Meiosis , Mapas de Interacción de ProteínasRESUMEN
Elongation factor P (EF-P) accelerates diprolyl synthesis and requires a posttranslational modification to maintain proteostasis. Two phylogenetically distinct EF-P modification pathways have been described and are encoded in the majority of Gram-negative bacteria, but neither is present in Gram-positive bacteria. Prior work suggested that the EF-P-encoding gene (efp) primarily supports Bacillus subtilis swarming differentiation, whereas EF-P in Gram-negative bacteria has a more global housekeeping role, prompting our investigation to determine whether EF-P is modified and how it impacts gene expression in motile cells. We identified a 5-aminopentanol moiety attached to Lys(32) of B. subtilis EF-P that is required for swarming motility. A fluorescent in vivo B. subtilis reporter system identified peptide motifs whose efficient synthesis was most dependent on 5-aminopentanol EF-P. Examination of the B. subtilis genome sequence showed that these EF-P-dependent peptide motifs were represented in flagellar genes. Taken together, these data show that, in B. subtilis, a previously uncharacterized posttranslational modification of EF-P can modulate the synthesis of specific diprolyl motifs present in proteins required for swarming motility.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/fisiología , Factores de Elongación de Péptidos/fisiología , Secuencias de Aminoácidos , Bacillus subtilis/citología , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Genes Bacterianos , Lisina/química , Movimiento , Pentanoles/química , Factores de Elongación de Péptidos/química , Factores de Elongación de Péptidos/genética , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Trans-membrane signaling involving a serine/threonine kinase (Stt7 in Chlamydomonas reinhardtii) directs light energy distribution between the two photosystems of oxygenic photosynthesis. Oxidation of plastoquinol mediated by the cytochrome b6f complex on the electrochemically positive side of the thylakoid membrane activates the kinase domain of Stt7 on the trans (negative) side, leading to phosphorylation and redistribution ("state transition") of the light-harvesting chlorophyll proteins between the two photosystems. The molecular description of the Stt7 kinase and its interaction with the cytochrome b6f complex are unknown or unclear. In this study, Stt7 kinase has been cloned, expressed, and purified in a heterologous host. Stt7 kinase is shown to be active in vitro in the presence of reductant and purified as a tetramer, as determined by analytical ultracentrifugation, electron microscopy, and electrospray ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41-kDa monomer. Far-UV circular dichroism spectra show Stt7 to be mostly α-helical and document a physical interaction with the b6f complex through increased thermal stability of Stt7 secondary structure. The activity of wild-type Stt7 and its Cys-Ser mutant at positions 68 and 73 in the presence of a reductant suggest that the enzyme does not require a disulfide bridge for its activity as suggested elsewhere. Kinase activation in vivo could result from direct interaction between Stt7 and the b6f complex or long-range reduction of Stt7 by superoxide, known to be generated in the b6f complex by quinol oxidation.