RESUMEN
Gram-negative bacterial plant pathogens inject effectors into their hosts to hijack and manipulate metabolism, eluding surveillance at the battle frontier on the cell surface. The effector AvrRpm1Pma from Pseudomonas syringae pv. maculicola functions as an ADP-ribosyl transferase that modifies RESISTANCE TO P. SYRINGAE PV MACULICOLA1 (RPM1)-INTERACTING PROTEIN4 (RIN4), leading to the activation of Arabidopsis thaliana (Arabidopsis) resistance protein RPM1. Here we confirmed the ADP-ribosyl transferase activity of another bacterial effector, AvrRpm2Psa from P. syringae pv. actinidiae, via sequential inoculation of Pseudomonas strain Pto DC3000 harboring avrRpm2Psa following Agrobacterium-mediated transient expression of RIN4 in Nicotiana benthamiana. We conducted mutational analysis in combination with mass spectrometry to locate the target site in RIN4. A conserved glutamate residue (Glu156) is the most likely target for AvrRpm2Psa, as only Glu156 could be ADP-ribosylated to activate RPM1 among candidate target residues identified from the MS/MS fragmentation spectra. Soybean (Glycine max) and snap bean (Phaseolus vulgaris) RIN4 homologs without glutamate at the positions corresponding to Glu156 of Arabidopsis RIN4 are not ADP-ribosylated by bacterial AvrRpm2Psa. In contrast to the effector AvrB, AvrRpm2Psa does not require the phosphorylation of Thr166 in RIN4 to activate RPM1. Therefore, separate biochemical reactions by different pathogen effectors may trigger the activation of the same resistance protein via distinct modifications of RIN4.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Ácido Glutámico , Espectrometría de Masas en Tándem , Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pseudomonas syringae/metabolismo , Glycine max/metabolismo , Transferasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Enfermedades de las Plantas/microbiologíaRESUMEN
Endometrial cancer, the most common gynaecological cancer worldwide, is closely linked to obesity and metabolic diseases, particularly in younger women. New circulating biomarkers have the potential to improve diagnosis and treatment selections, which could significantly improve outcomes. Our approach focuses on extracellular vesicle (EV) biomarker discovery by directly profiling the proteome of EVs enriched from frozen biobanked endometrial tumours. We analysed nine tissue samples to compare three clinical subgroups-low BMI (Body Mass Index) Endometrioid, high BMI Endometrioid, and Serous (any BMI)-identifying proteins related to histological subtype, BMI, and shared secreted proteins. Using collagenase digestion and size exclusion chromatography, we successfully enriched generous quantities of EVs (range 204.8-1291.0 µg protein: 1.38 × 1011-1.10 × 1012 particles), characterised by their size (â¼150 nm), expression of EV markers (CD63/81), and proposed endometrial cancer markers (L1CAM, ANXA2). Mass spectrometry-based proteomic profiling identified 2075 proteins present in at least one of the 18 samples. Compared to cell lysates, EVs were successfully depleted for mitochondrial and blood proteins and enriched for common EV markers and large secreted proteins. Further analysis highlighted significant differences in EV protein profiles between the high BMI subgroup and others, underlining the impact of comorbidities on the EV secretome. Interestingly, proteins differentially abundant in tissue subgroups were largely not also differential in matched EVs. This research identified secreted proteins known to be involved in endometrial cancer pathophysiology and proposed novel diagnostic biomarkers (EIF6, MUC16, PROM1, SLC26A2).
Asunto(s)
Biomarcadores de Tumor , Neoplasias Endometriales , Vesículas Extracelulares , Obesidad , Proteoma , Humanos , Femenino , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Vesículas Extracelulares/metabolismo , Proteoma/metabolismo , Proteoma/análisis , Obesidad/metabolismo , Obesidad/patología , Biomarcadores de Tumor/metabolismo , Proteómica/métodos , Índice de Masa Corporal , Persona de Mediana EdadRESUMEN
Growth hormone (GH) has been implicated in cancer progression andis a potential target for anticancer therapy. Currently, pegvisomant is the only GH receptor (GHR) antagonist approved for clinical use. Pegvisomant is a mutated GH molecule (B2036) which is PEGylated on amine groups to extend serum half-life. However, PEGylation significantly reduces the bioactivity of the antagonist in mice. To improve bioactivity, we generated a series of B2036 conjugates with the site-specific attachment of 20, 30, or 40 kDa methoxyPEG maleimide (mPEG maleimide) by introduction of a cysteine residue at amino acid 144 (S144C). Recombinant B2036-S144C was expressed in Escherichia coli, purified, and then PEGylated using cysteine-specific conjugation chemistry. To avoid issues with dimerization due to the introduced cysteine, B2036-S144C was PEGylated while immobilized on an Ni-nitrilotriacetic (Ni-NTA) acid column, which effectively reduced disulfide-mediated dimer formation and allowed efficient conjugation to mPEG maleimide. Following PEGylation, the IC50 values for the 20, 30, and 40 kDa mPEG maleimide B2036-S144C conjugates were 66.2 ± 3.8, 106.1 ± 7.1, and 127.4 ± 3.6 nM, respectively. The circulating half-life of the 40 kDa mPEG conjugate was 58.3 h in mice. Subcutaneous administration of the 40 kDa mPEG conjugate (10 mg/kg/day) reduced serum insulin-like growth factor I (IGF-I) concentrations by 50.6%. This in vivo reduction in serum IGF-I was at a considerably lower dose compared to the higher doses required to observe comparable activity in studies with pegvisomant. In conclusion, we have generated a novel PEGylated GHR antagonist by the solid-phase site-specific attachment of mPEG maleimide at an introduced cysteine residue, which effectively reduces serum IGF-I in vivo.
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Cisteína , Hormona del Crecimiento , Animales , Dimerización , Escherichia coli , Humanos , Ratones , Proteínas RecombinantesRESUMEN
In Escherichia coli, the expression of heterologous genes for the production of recombinant proteins can be challenging due to the codon bias of different organisms. The rare codons AGG and AGA are among the rarest in E. coli. In this work, by using the human gene RioK2 as case study, we found that the presence of consecutive AGG-AGA led to a premature stop, which may be caused by an event of -1 frameshift. We found that translational problems caused by consecutive AGG-AGA are sequence dependent, in particular, in sequences that contain multiple rare AGG or AGA codons elsewhere. Translational problems can be alleviated by different strategies, including codon harmonization, codon optimization, or by substituting the consecutive AGG-AGA codons by more frequent arginine codons. Overall, our results furthered our understanding about the relationship between consecutive rare codons and translational problems. Such information will aid the design of DNA sequence for the production of recombinant proteins.
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Codón , Escherichia coli/metabolismo , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , ARN de Transferencia de Arginina/genética , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Humanos , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , Proteínas Recombinantes/genética , Ribosomas/metabolismoRESUMEN
The cystine-glutamate exchanger (system xc-) is responsible for the exchange of extracellular cystine for intracellular glutamate. In this study, we mapped the expression of xCT, the light chain subunit of system xc- in the different tissues of 3-6-week-old mouse (C57BL/6J) eye and have used an xCT knockout mouse to verify labelling specificity. Moreover, using the xCT knockout mouse, we investigated whether xCT was involved in maintaining extracellular redox balance in the eye. xCT transcript and protein were present in the cornea, lens and retina of wild-type mice, but not knockout mice. xCT was localised to the corneal epithelium, and the lens epithelium and cortical fibre cells but was absent in the iris. xCT localisation could not be determined in the ciliary body or retina, since xCT labelling was also detected in the knockout indicating a lack of specificity of the xCT antibody in tissues of a neural origin. Intracellular cysteine and cystine concentrations were similar in the wild-type and xCT knockout mouse for the cornea, lens, and retina. While extracellular cysteine levels were similar between the plasma, aqueous humour, and vitreous humour of the wild-type and xCT knockout mouse, extracellular cystine levels in the plasma and aqueous were significantly elevated in the xCT knockout mouse relative to the wild type. This suggests that loss of xCT results in an increased oxidative environment, particularly within the anterior chamber of the eye in which the aqueous humour resides. How this oxidative shift impacts ocular tissues that interface with the aqueous humour over time will be the focus of future work.
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Sistema de Transporte de Aminoácidos y+/análisis , Sistema de Transporte de Aminoácidos y+/metabolismo , Ojo/química , Ojo/metabolismo , Sistema de Transporte de Aminoácidos y+/deficiencia , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-ReducciónRESUMEN
Organic micropollutants (OMPs) are pervasive anthropogenic contaminants of receiving waters where they can induce various adverse effects to aquatic life. Their ubiquitous environmental occurrence is primarily attributed to discharge from wastewater treatment plants due to incomplete removal by common biological wastewater treatment processes. Here, we assess a new strategy for promoting the degradation of six representative OMPs (i.e., sulfamethoxazole, carbamazepine, tylosin, atrazine, naproxen, and ibuprofen) by intentionally stimulating the production of microbial oxidoreductases to counter oxidative stress caused by oxygen perturbations. Mixed microbial cultures from a dairy farm wastewater were subjected to cyclic perturbations of dissolved oxygen (DO). A distance-based redundancy analysis was used to show that DO perturbations correlate with the abundance of Pseudomonadaceae and Rhodocyclaceae families, activities of peroxidases and cytochromes, and the degradation of OMPs. DO perturbation of 0.25 and 0.5 cycles/h led to most abundance of Pseudomonadaceae and Rhodocyclaceae families, showed higher activity of peroxidase and cytochrome, and gave largest removal of OMPs (removal of 92 ± 3% for sulfamethoxazole, 84 ± 3% for naproxen, 82 ± 3% for ibuprofen, 66 ± 2% for carbamazepine, 57 ± 15% for tylosin, and 88 ± 1% for atrazine).
Asunto(s)
Aguas Residuales , Contaminantes Químicos del Agua , Estrés Oxidativo , Sulfametoxazol , Eliminación de Residuos LíquidosRESUMEN
Advanced glycation end-products (AGEs) comprise a group of non-enzymatic post-translational modifications of proteins and are elevated in diabetic tissues. AGE-modification impairs the digestibility of collagen in vitro but little is known about its relation to collagen-degrading proteinases in vivo. N(ε)-carboxymethyllysine (CML) is a stable AGE that forms on lysyl side-chains in the presence of glucose, probably via a transition metal-catalysed mechanism. Here, rats with streptozotocin-induced diabetes and non-diabetic controls were treated for 8weeks with placebo or the Cu(II)-selective chelator, triethylenetetramine (TETA), commencing 8weeks after disease induction. Actions of diabetes and drug treatment were measured on collagen and collagen-degrading proteinases in kidney tissue. The digestibility and CML content of collagen, and corresponding levels of mRNAs and collagen, were related to changes in collagen-degrading-proteinases. Collagen-degrading proteinases, cathepsin L (CTSL) and matrix metalloproteinase-2 (MMP-2) were increased in diabetic rats. CTSL-levels correlated strongly and positively with increased collagen-CML levels and inversely with decreased collagen digestibility in diabetes. The collagen-rich mesangium displayed a strong increase of CTSL in diabetes. TETA treatment normalised kidney collagen content and partially normalised levels of CML and CTSL. These data provide evidence for an adaptive proteinase response in diabetic kidneys, affected by excessive collagen-CML formation and decreased collagen digestibility. The normalisation of collagen and partial normalisation of CML- and CTSL-levels by TETA treatment supports the involvement of Cu(II) in CML formation and altered collagen metabolism in diabetic kidneys. Cu(II)-chelation by TETA may represent a treatment option to rectify collagen metabolism in diabetes independent of alterations in blood glucose levels.
Asunto(s)
Quelantes/metabolismo , Colágeno/metabolismo , Cobre/metabolismo , Diabetes Mellitus Experimental/metabolismo , Riñón/metabolismo , Lisina/análogos & derivados , Péptido Hidrolasas/metabolismo , Animales , Quelantes/farmacología , Diabetes Mellitus Experimental/patología , Riñón/efectos de los fármacos , Riñón/patología , Lisina/metabolismo , Masculino , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Ratas Wistar , Estreptozocina , Trientina/farmacologíaRESUMEN
The homeostatic chemokine CCL21 has a pivotal role in lymphocyte homing and compartment localisation within the lymph node, and also affects adhesion between immune cells. The effects of CCL21 are modulated by its mode of presentation, with different cellular responses seen for surface-bound and soluble forms. Here we show that plasmin cleaves surface-bound CCL21 to release the C-terminal peptide responsible for CCL21 binding to glycosaminoglycans on the extracellular matrix and cell surfaces, thereby generating the soluble form. Loss of this anchoring peptide enabled the chemotactic activity of CCL21 and reduced cell tethering. Tissue plasminogen activator did not cleave CCL21 directly but enhanced CCL21 processing through generation of plasmin from plasminogen. The tissue plasminogen activator inhibitor neuroserpin prevented processing of CCL21 and blocked the effects of soluble CCL21 on cell migration. Similarly, the plasmin-specific inhibitor α2-antiplasmin inhibited CCL21-mediated migration of human T cells and dendritic cells and tethering of T cells to APCs. We conclude that the plasmin system proteins plasmin, tissue plasminogen activator and neuroserpin regulate CCL21 function in the immune system by controlling the balance of matrix- and cell-bound CCL21.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Quimiocina CCL21/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Plasminógeno/farmacología , Linfocitos T/citología , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Adhesión Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Quimiocina CCL21/química , Células Dendríticas/efectos de los fármacos , Humanos , Neuropéptidos/farmacología , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Serpinas/farmacología , Linfocitos T/efectos de los fármacos , Activador de Tejido Plasminógeno/farmacología , alfa 2-Antiplasmina/farmacología , NeuroserpinaRESUMEN
The human pathogen Streptococcus pyogenes produces pili that are essential for adhesion to host surface receptors. Cpa, the adhesin at the pilus tip, was recently shown to have a thioester-containing domain. The thioester bond is believed to be important in adhesion, implying a mechanism of covalent attachment analogous to that used by human complement factors. Here, we have characterized a second active thioester-containing domain on Cpa, the N-terminal domain of Cpa (CpaN). Expression of CpaN in Escherichia coli gave covalently linked dimers. These were shown by x-ray crystallography and mass spectrometry to comprise two CpaN molecules cross-linked by the polyamine spermidine following reaction with the thioester bonds. This cross-linked CpaN dimer provides a model for the covalent attachment of Cpa to target receptors and thus the streptococcal pilus to host cells. Similar thioester domains were identified in cell wall proteins of other Gram-positive pathogens, suggesting that thioester domains are more widely used and provide a mechanism of adhesion by covalent bonding to target molecules on host cells that mimics that used by the human complement system to eliminate pathogens.
Asunto(s)
Adhesinas Bacterianas/química , Fimbrias Bacterianas/química , Modelos Moleculares , Multimerización de Proteína , Streptococcus pyogenes/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Secuencia de Bases , Proteínas del Sistema Complemento/química , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Cristalografía por Rayos X , Escherichia coli , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Humanos , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidadRESUMEN
Lymph nodes (LNs) form the intersection between the vascular and lymphatic systems. Lymphocytes and antigen-presenting cells (APCs) traffic between these systems, but the barriers crossed during this trafficking in human LNs are poorly defined. We identified a population of cells in human LNs that lines the boundary between the parenchyma and lymphatic sinuses, consistent with descriptions of marginal reticular cells (MRCs) in murine LNs. Human MRCs are CD141(high) podoplanin(+), CD90(+), ICAM1(+), and VCAM1(+) but lack endothelial and hematopoietic cell markers, or alpha-smooth muscle actin. We then examined expression of the enzyme sphingosine-1-phosphate (S1P) lyase (SGPL1) relative to the boundary defined by MRCs. SGPL1 expression was almost exclusively restricted to cells on the parenchymal side of MRCs, consistent with a role in maintaining the S1P gradient between the sinuses and the parenchyma. Surprisingly the cells expressing SGPL1 in the parenchyma were CD68(+) APCs. CD68(+) APCs generated from human monocytes were able to internalize and irreversibly degrade S1P, and this activity was inhibited by the S1P analogue FTY720. This work provides a map of the key structures at the boundary where human lymphocytes egress into sinuses, and identifies a novel potential mechanism for the activity of S1P analogues in humans.
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Aldehído-Liasas/biosíntesis , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Ganglios Linfáticos/enzimología , Células del Mesófilo/enzimología , Movimiento Celular/fisiología , Humanos , Ganglios Linfáticos/citología , Ganglios Linfáticos/metabolismo , Sistema Linfático/citología , Sistema Linfático/enzimología , Sistema Linfático/metabolismo , Linfocitos/citología , Linfocitos/enzimología , Linfocitos/metabolismo , Lisofosfolípidos/metabolismo , Células del Mesófilo/citología , Células del Mesófilo/metabolismo , Monocitos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
Kiwellin is a cysteine-rich, cell wall-associated protein with no known structural homologues. It is one of the most abundant proteins in kiwifruit (Actinidia spp.), and has been shown to be recognised by IgE of some patients allergic to kiwifruit. Cleavage of kiwellin into an N-terminal 4 kDa peptide called kissper and a core domain called KiTH is mediated by actinidin in vitro, and isolation of the kissper peptide from green-fleshed kiwifruit extracts suggested it may result from in vivo processing of kiwellin. In solution, kissper is highly flexible and displays pore-forming activity in synthetic lipid-bilayers. We present here the 2.05 Å resolution crystal structure of full-length kiwellin, purified from its native source, Actinidia chinensis (gold-fleshed kiwifruit). The structure confirms the modularity of the protein and the intrinsic flexibility of kissper and reveals that KiTH harbours a double-psi ß-barrel fold hooked to an N-terminal ß hairpin. Comparisons with structurally-related proteins suggest that a deep gorge located at the protein surface forms a binding site for endogenous ligands.
Asunto(s)
Actinidia/metabolismo , Antígenos de Plantas/química , Pared Celular/metabolismo , Frutas/metabolismo , Proteínas de Plantas/química , Actinidia/genética , Secuencia de Aminoácidos , Antígenos de Plantas/genética , Antígenos de Plantas/metabolismo , Pared Celular/genética , Quitina/metabolismo , Cristalografía por Rayos X , Frutas/genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polisacáridos/metabolismo , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys-Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys-Asn isopeptide-bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its isopeptide bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.
Asunto(s)
Corynebacterium diphtheriae/química , Proteínas Fimbrias/química , Cristalografía por Rayos X , Disulfuros/química , Proteínas Fimbrias/metabolismo , Lisina/química , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine-rich snakin-1 and -2 antimicrobial peptides by using a combination of solid-phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40-50 % yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin-2 compared to natural snakin-2, we demonstrated that synthetic snakin-2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin-2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.
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Antiinfecciosos/síntesis química , Productos Biológicos/síntesis química , Péptidos/síntesis química , Proteínas de Plantas/síntesis química , Solanum tuberosum/química , Secuencia de Aminoácidos , Antiinfecciosos/química , Productos Biológicos/química , Cisteína/química , Cistina/química , Disulfuros/química , Datos de Secuencia Molecular , Péptidos/química , Proteínas de Plantas/química , Pliegue de Proteína , Técnicas de Síntesis en Fase SólidaRESUMEN
We evaluated statistical approaches to facilitate and improve multi-stage designs for clinical proteomic studies which plan to transit from laboratory discovery to clinical utility. To find the design with the greatest expected number of true discoveries under constraints on cost and false discovery, the operating characteristics of the multi-stage study were optimized as a function of sample sizes and nominal type-I error rates at each stage. A nested simulated annealing algorithm was used to find the best solution in the bounded spaces constructed by multiple design parameters. This approach is demonstrated to be feasible and lead to efficient designs. The use of biological grouping information in the study design was also investigated using synthetic datasets based on a cardiac proteomic study, and an actual dataset from a clinical immunology proteomic study. When different protein patterns presented, performance improved when the grouping was informative, with little loss in performance when the grouping was uninformative.
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Estudios de Asociación Genética/métodos , Estudios de Asociación Genética/normas , Modelos Estadísticos , Proteómica , Proyectos de Investigación , Algoritmos , Biomarcadores , Estudios de Casos y Controles , Inmunodeficiencia Variable Común/genética , Inmunodeficiencia Variable Común/metabolismo , Simulación por Computador , Humanos , Proteómica/métodosRESUMEN
This study evaluates the effects of alkaline and micellisation extraction methods, alongside freeze-drying and spray-drying, on the protein subunits, amino acid profiles, and proteome data of hempseed protein isolate (HPI). Findings revealed that the extraction methods affect protein profiles more than the drying methods. Micellisation-extracted HPI showed higher albumin, oleosin, and sulphur-containing protein levels than alkaline-extracted HPI. The alkali-extracted undried sample (AU) gave more potentially allergenic proteins, including Hsp70 and triosephosphate isomerase, than its micellization-extracted counterpart (MU). Unique potential allergens were identified, including malate dehydrogenase and enolase in AU, and RuBisCo in MU samples. Both drying processes impacted the HPI proteome and reduced RuBisCo in the micellisation-extracted HPI. These insights highlight the crucial role of method selection in HPI processing for optimising production in the food industry.
RESUMEN
Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F420. Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L-glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.
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Ácido Fólico , Ácido Glutámico , Humanos , Péptido Sintasas/metabolismo , Bacterias/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
New Zealand manuka (Leptospermum scoparium) honey is a premium food product. Unfortunately, its high demand has led to "not true to label" marketed manuka honey. Robust methods are therefore required to determine authenticity. We previously identified three unique nectar-derived proteins in manuka honey, detected as twelve tryptic peptide markers, and hypothesized these could be used to determine authenticity. We invoked a targeted proteomic approach based on parallel reaction-monitoring (PRM) to selectively monitor relative abundance of these peptides in sixteen manuka and twenty six non-manuka honey samples of various floral origin. We included six tryptic peptide markers derived from three bee-derived major royal jelly proteins as potential internal standards. The twelve manuka-specific tryptic peptide markers were present in all manuka honeys with minor regional variation. By comparison, they had negligible presence in non-manuka honeys. Bee-derived peptides were detected in all honeys with similar relative abundance but with sufficient variation precluding their utility as internal standards. Manuka honeys displayed an inverse relationship between total protein content and the ratio between nectar- to bee-derived peptide abundance. This trend reveals an association between protein content on possible nectar processing time by bees. Overall, these findings demonstrate the first successful application of peptide profiling as an alternative and potentially more robust approach for manuka honey authentication.
RESUMEN
Growth hormone (GH) actions are mediated through binding to its cell-surface receptor, the GH receptor (GHR), with consequent activation of downstream signalling. However, nuclear GHR localisation has also been observed and is associated with increased cancer cell proliferation. Here we investigated the functional implications of nuclear translocation of the GHR in the human endometrial cancer cell-line, RL95-2, and human mammary epithelial cell-line, MCF-10A. We found that following GH treatment, the GHR rapidly translocates to the nucleus, with maximal localisation at 5-10 min. Combined immunoprecipitation-mass spectrometry analysis of RL95-2 whole cell lysates identified 40 novel GHR binding partners, including the transcriptional regulator, HMGN1. Moreover, microarray analysis demonstrated that the gene targets of HMGN1 were differentially expressed following GH treatment, and co-immunoprecipitation showed that HMGN1 associates with the GHR in the nucleus. Therefore, our results suggest that GHR nuclear translocation might mediate GH actions via interaction with chromatin factors that then drive changes in specific downstream transcriptional programs.
RESUMEN
Cell-surface proteins known as adhesins enable bacteria to colonize particular environments, and in Gram-positive bacteria often contain autocatalytically formed covalent intramolecular cross-links. While investigating the prevalence of such cross-links, a remarkable example was discovered in Mobiluncus mulieris, a pathogen associated with bacterial vaginosis. This organism encodes a putative adhesin of 7651 residues. Crystallography and mass spectrometry of two selected domains, and AlphaFold structure prediction of the remainder of the protein, were used to show that this adhesin belongs to the family of thioester, isopeptide and ester-bond-containing proteins (TIE proteins). It has an N-terminal domain homologous to thioester adhesion domains, followed by 51 immunoglobulin (Ig)-like domains containing ester- or isopeptide-bond cross-links. The energetic cost to the M. mulieris bacterium in retaining such a large adhesin as a single gene or protein construct suggests a critical role in pathogenicity and/or persistence.
Asunto(s)
Adhesinas Bacterianas , Mobiluncus , Femenino , Humanos , Mobiluncus/metabolismo , Adhesinas Bacterianas/química , Ésteres/químicaRESUMEN
Enzymes of the de novo purine biosynthetic pathway have been identified as essential for the growth and survival of Mycobacterium tuberculosis and thus have potential for the development of anti-tuberculosis drugs. The final two steps of this pathway are carried out by the bifunctional enzyme 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC), also known as PurH. This enzyme has already been the target of anti-cancer drug development. We have determined the crystal structures of the M. tuberculosis ATIC (Rv0957) both with and without the substrate 5-aminoimidazole-4-carboxamide ribonucleotide, at resolutions of 2.5 and 2.2 Å, respectively. As for other ATIC enzymes, the protein is folded into two domains, the N-terminal domain (residues 1-212) containing the cyclohydrolase active site and the C-terminal domain (residues 222-523) containing the formyltransferase active site. An adventitiously bound nucleotide was found in the cyclohydrolase active site in both structures and was identified by NMR and mass spectral analysis as a novel 5-formyl derivative of an earlier intermediate in the biosynthetic pathway 4-carboxy-5-aminoimidazole ribonucleotide. This result and other studies suggest that this novel nucleotide is a cyclohydrolase inhibitor. The dimer formed by M. tuberculosis ATIC is different from those seen for human and avian ATICs, but it has a similar â¼50-Å separation of the two active sites of the bifunctional enzyme. Evidence in M. tuberculosis ATIC for reactivity of half-the-sites in the cyclohydrolase domains can be attributed to ligand-induced movements that propagate across the dimer interface and may be a common feature of ATIC enzymes.