RESUMEN
Cyclic peptides are increasingly important structures in drugs but their development can be impeded by difficulties associated with their synthesis. Here, we introduce the 3-aminoazetidine (3-AAz) subunit as a new turn-inducing element for the efficient synthesis of small head-to-tail cyclic peptides. Greatly improved cyclizations of tetra-, penta- and hexapeptides (28 examples) under standard reaction conditions are achieved by introduction of this element within the linear peptide precursor. Post-cyclization deprotection of the amino acid side chains with strong acid is realized without degradation of the strained four-membered azetidine. A special feature of this chemistry is that further late-stage modification of the resultant macrocyclic peptides can be achieved via the 3-AAz unit. This is done by: (i) chemoselective deprotection and substitution at the azetidine nitrogen, or by (ii) a click-based approach employing a 2-propynyl carbamate on the azetidine nitrogen. In this way, a range of dye and biotin tagged macrocycles are readily produced. Structural insights gained by XRD analysis of a cyclic tetrapeptide indicate that the azetidine ring encourages access to the less stable, all-trans conformation. Moreover, introduction of a 3-AAz into a representative cyclohexapeptide improves stability towards proteases compared to the homodetic macrocycle.
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Azetidinas , Péptidos Cíclicos , Azetidinas/química , Azetidinas/síntesis química , Ciclización , Péptidos Cíclicos/química , Péptidos Cíclicos/síntesis química , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/síntesis química , Química ClicRESUMEN
Over 500 million people worldwide are affected by diabetes mellitus, a chronic disease that leads to high blood glucose levels and causes severe side effects. The predominant biological marker for diagnosis of diabetes is glycated haemoglobin (GHb). In human blood the predominant reducing sugar, glucose, irreversibly conjugates onto accessible amine groups within Hb. Most methods for diagnosis and monitoring of diabetes selectively detect N-terminal glycation at Val-1 on the ß-globin chain, but not glycation at other sites. Detection of other glycated epitopes of GHb has the potential to provide new information on the extent, duration and timing of elevated glucose, facilitating personalised diagnosis and intelligent diabetic control. In this work, a new anti-GHb Fab antibody (Fab-1) specific for haemoglobin A1c (HbA1c) with nanomolar affinity was discovered via epitope-directed immunisation and phage display. A single chain variable fragment (scFv) antibody derived from Fab-1 retained affinity and specificity for HbA1c, and affinity was enhanced tenfold upon addition of an enhanced green fluorescent protein tag. Both the scFv and Fab-1 recognised an epitope within HbA1c that was distinct from ß-Val-1, and our data suggest that this epitope may include glycation at Lys-66 in the ß-globin chain. To our knowledge, this is the first report of an scFv/Fab anti-glycated epitope antibody that recognises a non-A1c epitope in GHb, and confirms that fructosamine attached to different, discrete glycation sites within the same protein can be resolved from one another by immunoassay.
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Diabetes Mellitus , Anticuerpos de Cadena Única , Oxibato de Sodio , Humanos , Hemoglobina Glucada , Epítopos , Glucosa , Globinas betaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-stranded RNA virus that sits at the centre of the recent global pandemic. As a member of the coronaviridae family of viruses, it shares features such as a very large genome (>30 kb) that is replicated in a purpose-built replication organelle. Biogenesis of the replication organelle requires significant and concerted rearrangement of the endoplasmic reticulum membrane, a job that is carried out by a group of integral membrane non-structural proteins (NSP3, 4 and 6) expressed by the virus along with a host of viral replication enzymes and other factors that support transcription and replication. The primary sites for RNA replication within the replication organelle are double membrane vesicles (DMVs). The small size of DMVs requires generation of high membrane curvature, as well as stabilization of a double-membrane arrangement, but the mechanisms that underlie DMV formation remain elusive. In this review, we discuss recent breakthroughs in our understanding of the molecular basis for membrane rearrangements by coronaviruses. We incorporate established models of NSP3-4 protein-protein interactions to drive double membrane formation, and recent data highlighting the roles of lipid composition and host factor proteins (e.g. reticulons) that influence membrane curvature, to propose a revised model for DMV formation in SARS-CoV-2.
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Retículo Endoplásmico , Proteínas no Estructurales Virales , Línea Celular , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Replicación ViralRESUMEN
Management of low-risk ductal carcinoma in situ (DCIS) is controversial, with clinical trials currently assessing the safety of active monitoring amidst concern about overtreatment. Little is known about general community views regarding DCIS and its management. We aimed to explore women's understanding and views about low-risk DCIS and current and potential future management options. This mixed-method study involved qualitative focus groups and brief quantitative questionnaires. Participants were screening-aged (50-74 years) women, with diverse socioeconomic backgrounds and no personal history of breast cancer/DCIS, recruited from across metropolitan Sydney, Australia. Sessions incorporated an informative presentation interspersed with group discussions which were audio-recorded, transcribed and analysed thematically. Fifty-six women took part in six age-stratified focus groups. Prior awareness of DCIS was limited, however women developed reasonable understanding of DCIS and the relevant issues. Overall, women expressed substantial support for active monitoring being offered as a management approach for low-risk DCIS, and many were interested in participating in a hypothetical clinical trial. Although some women expressed concern that current management may sometimes represent overtreatment, there were mixed views about personally accepting monitoring. Women noted a number of important questions and considerations that would factor into their decision making. Our findings about women's perceptions of active monitoring for DCIS are timely while results of ongoing clinical trials of monitoring are awaited, and may inform clinicians and investigators designing future, similar trials. Exploration of offering well-informed patients the choice of non-surgical management of low-risk DCIS, even outside a clinical trial setting, may be warranted.
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Neoplasias de la Mama , Carcinoma Intraductal no Infiltrante , Femenino , Humanos , Australia , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/terapia , Carcinoma Intraductal no Infiltrante/terapia , Carcinoma Intraductal no Infiltrante/patología , Grupos Focales , Persona de Mediana Edad , Anciano , Ensayos Clínicos como AsuntoRESUMEN
MHC class II molecules function to present exogenous antigen-derived peptides to CD4 T cells to both drive T cell activation and to provide signals back into the class II antigen-presenting cell. Previous work established the presence of multiple GxxxG dimerization motifs within the transmembrane domains of MHC class II α and ß chains across a wide range of species and revealed a role for differential GxxxG motif pairing in the formation of two discrete mouse class II conformers with distinct functional properties (i.e., M1-and M2-paired I-Ak class II). Biochemical and mutagenesis studies detailed herein extend this model to human class II by identifying an anti-HLA-DR mAb (Tü36) that selectively binds M1-paired HLA-DR molecules. Analysis of the HLA-DR allele reactivity of the Tü36 mAb helped define other HLA-DR residues involved in mAb binding. In silico modeling of both TM domain interactions and whole protein structure is consistent with the outcome of biochemical/mutagenesis studies and provides insight into the possible structural differences between the two HLA-DR conformers. Cholesterol depletion studies indicate a role for cholesterol-rich membrane domains in the formation/maintenance of Tü36 mAb reactive DR molecules. Finally, phylogenetic analysis of the amino acid sequences of Tü36-reactive HLA-DR ß chains reveals a unique pattern of both Tü36 mAb reactivity and key amino acid polymorphisms. In total, these studies bring the paradigm M1/M2-paired MHC class II molecules to the human HLA-DR molecule and suggest that the functional differences between these conformers defined in mouse class II extend to the human immune system.
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Secuencias de Aminoácidos , Antígenos HLA-DR , Antígenos de Histocompatibilidad Clase II , Animales , Humanos , Ratones , Linfocitos T CD4-Positivos/metabolismo , Dimerización , Antígenos de Histocompatibilidad Clase II/metabolismo , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Filogenia , Secuencias de Aminoácidos/fisiologíaRESUMEN
PURPOSE: Up to 70% of survivors report cognitive symptoms after chemotherapy. We compared two cognitive rehabilitation programs to a control group in cancer survivors. METHODS: Study population were adult cancer survivors with cognitive symptoms 6-60 months after adjuvant chemotherapy. Participants randomised to: Attention Process Training (APT), Compensatory Strategy Training (CST), or control group. Active interventions comprised 6-week, 2-h/week small group sessions. ASSESSMENTS: pre- and post-intervention, 6- and 12-months later. Primary outcome was change in cognitive symptoms (FACT-COG-PCI subscale) between baseline and post-intervention. Secondary endpoints included objective neuropsychological performance, Functional Impact Assessment (FIA), patient-reported outcome measures, and associations. Analyses were on an intention-to-treat basis. Analysis of covariance mixed models were used for continuous outcomes. RESULTS: Sixty-five participants were randomised (APT n = 21; CST n = 24; controls n = 20): 94% breast cancer, median age 54. Median time since chemotherapy 20.7 months. FACT-COG-PCI, clinical neuropsychological T-scores, and FIA improved in all groups over time, but no significant differences between arms. On mean neuropsychological T-scores 19/65 (29%) were impaired at baseline; post-intervention impairment controls 31.3%, CST 16.7%, APT 20.0%. On FIA at baseline, nine were impaired; this decreased to three post-intervention (one/group). FACT-COG-PCI was weakly associated with neuropsychological tests (rho = 0.24, p = 0.051) at baseline, and had no association with FIA. Neuropsychological total mean T-score was moderately positively associated with FIA (rho = 0.37, p = 0.003). CONCLUSION: There were no significant differences between intervention groups and controls using linear mixed models adjusted for baseline scores. IMPLICATIONS FOR CANCER SURVIVORS: Cognitive symptoms and neuropsychological test scores improve over time.
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Neoplasias de la Mama , Supervivientes de Cáncer , Disfunción Cognitiva , Intervención Coronaria Percutánea , Adulto , Humanos , Persona de Mediana Edad , Femenino , Supervivientes de Cáncer/psicología , Entrenamiento Cognitivo , Disfunción Cognitiva/etiología , Neoplasias de la Mama/psicología , Pruebas NeuropsicológicasRESUMEN
BACKGROUND: The capacity to handle money and personal finances is crucial for living independently. Cognitive decline can lead to financial mismanagement (e.g., forgetting to pay bills, overspending) and exploitation (e.g., falling for financial scams). While greater susceptibility to financial mismanagement and exploitation is commonly reported by carers of people with dementia, objective neuropsychological measures of these financial skills are scarce. It is also unclear whether profiles of change in financial skills vary across different stages and subtypes of dementia. METHOD: A normative sample of 112 healthy controls and individuals with mild cognitive impairment (MCI; n = 16), Alzheimer's disease (AD; n = 7) or behavioural-variant frontotemporal dementia (bvFTD; n = 18) completed a novel neuropsychological Test of Financial Skills (TOFS), which assessed performance across three key areas: everyday financial tasks, susceptibility to financial scams and financial goal planning. For all patients and controls, measures of dementia severity (Clinical Dementia Rating Scale) and general cognition (Addenbrooke's Cognitive Examination, 3rd Ed.) were also collected. RESULT: Relative to controls, AD and bvFTD patients showed lower overall performance on the TOFS, whereas MCI patients did not differ significantly from controls. While AD and bvFTD patients showed similar levels of impairment on everyday financial tasks and financial goal planning, bvFTD patients performed significantly worse on the financial scams subtest. Across all participants, lower TOFS performance was associated with greater dementia severity and cognitive impairment. CONCLUSION: Our results are consistent with previous studies of carer-reported changes in financial mismanagement and exploitation in people with dementia. Notably, we found important differences in the areas of financial skills impacted by different stages and subtypes of dementia. These findings provide novel insights into the different factors that contribute to deficits in financial skills in people with dementia, highlight the importance of objective neuropsychological assessments in this area, and open avenues for targeted financial support strategies.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Demencia Frontotemporal , Pruebas Neuropsicológicas , Humanos , Masculino , Femenino , Anciano , Pruebas Neuropsicológicas/estadística & datos numéricos , Persona de Mediana EdadRESUMEN
The B-cell receptor (BCR), a complex comprised of a membrane-associated immunoglobulin and the Igα/ß heterodimer, is one of the most important immune receptors in humans and controls B-cell development, activity, selection, and death. BCR signaling plays key roles in autoimmune diseases and lymphoproliferative disorders, yet, despite the clinical significance of this protein complex, key regions (i.e., the transmembrane domains) have yet to be structurally characterized. The mechanism for BCR signaling also remains unclear and has been variously described by the mutually exclusive cross-linking and dissociation activation models. Common to these models is the significance of local plasma membrane composition, which implies that interactions between BCR transmembrane domains (TMDs) play a role in receptor functionality. Here we used an in vivo assay of TMD oligomerization called GALLEX alongside spectroscopic and computational methods to characterize the structures and interactions of human Igα and Igß TMDs in detergent micelles and natural membranes. We observed weak self-association of the Igß TMD and strong self-association of the Igα TMD, which scanning mutagenesis revealed was entirely stabilized by an E-X10-P motif. We also demonstrated strong heterotypic interactions between the Igα and Igß TMDs both in vitro and in vivo, which scanning mutagenesis and computational models suggest is multiconfigurational but can accommodate distinct interaction sites for self-interactions and heterotypic interactions of the Igα TMD. Taken together, these results demonstrate that the TMDs of the human BCR are sites of strong protein-protein interactions that may direct BCR assembly, endoplasmic reticulum retention, and immune signaling.
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Receptores de Antígenos de Linfocitos B , Membrana Celular/genética , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Dominios Proteicos , Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de SeñalRESUMEN
Cellular damage is a key issue in the context of cryopreservation. Much of this damage is believed to be caused by extracellular ice formation at temperatures well above the homogeneous freezing point of pure water. Hence the question: what initiates ice nucleation during cryopreservation? In this paper, we assess whether cellular membranes could be responsible for facilitating the ice nucleation process, and what characteristics would make them good or bad ice nucleating agents. By means of molecular dynamics simulations, we investigate a number of phospholipids and lipopolysaccharide bilayers at the interface with supercooled liquid water. While these systems certainly appear to act as ice nucleating agents, it is likely that other impurities might also play a role in initiating extracellular ice nucleation. Furthermore, we elucidate the factors which affect a bilayer's ability to act as an ice nucleating agent; these are complex, with specific reference to both chemical and structural factors. These findings represent a first attempt to pinpoint the origin of extracellular ice nucleation, with important implications for the cryopreservation process.
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Criopreservación , Hielo , Membrana Dobles de Lípidos , Congelación , Simulación de Dinámica Molecular , Agua/químicaRESUMEN
Antibiotic resistance is a significant threat to human health, with natural products remaining the best source for new antimicrobial compounds. Antimicrobial peptides (AMPs) are natural products with great potential for clinical use as they are small, amenable to customization, and show broad-spectrum activities. Lynronne-1 is a promising AMP identified in the rumen microbiome that shows broad-spectrum activity against pathogens such as methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii. Here we investigated the structure of Lynronne-1 using solution NMR spectroscopy and identified a 13-residue amphipathic helix containing all six cationic residues. We used biophysical approaches to observe folding, membrane partitioning and membrane lysis selective to the presence of anionic lipids. We translated our understanding of Lynronne-1 structure to design peptides which varied in the size of their hydrophobic helical face. These peptides displayed the predicted continuum of membrane-lysis activities inâ vitro and inâ vivo, and yielded a new AMP with 4-fold improved activity against A. baumannii and 32-fold improved activity against S. aureus.
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Péptidos AntimicrobianosRESUMEN
The reticulon family of integral membrane proteins are conserved across all eukaryotes and typically localize to the endoplasmic reticulum (ER), where they are involved in generating highly-curved tubules. We recently demonstrated that Reticulon-like protein B13 (RTNLB13) from Arabidopsis thaliana contains a curvature-responsive amphipathic helix (APH) important for the proteins' ability to induce curvature in the ER membrane, but incapable of generating curvature by itself. We suggested it acts as a feedback element, only folding/binding once a sufficient degree of curvature has been achieved, and stabilizes curvature without disrupting the bilayer. However, it remains unclear whether this is unique to RTNLB13 or is conserved across all reticulons-to date, experimental evidence has only been reported for two reticulons. Here we used biophysical methods to characterize a minimal library of putative APH peptides from across the 21 A. thaliana isoforms. We found that reticulons with the closest evolutionary relationship to RTNLB13 contain curvature-sensing APHs in the same location with sequence conservation. Our data reveal that a more distantly-related branch of reticulons developed a ~ 20-residue linker between the transmembrane domain and APH. This may facilitate functional flexibility as previous studies have linked these isoforms not only to ER remodeling but other cellular activities.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Retículo Endoplásmico/genética , Proteínas de la Membrana/genética , Arabidopsis/crecimiento & desarrollo , Fenómenos Biofísicos/genética , Secuencia Conservada/genética , Membranas Intracelulares/metabolismo , Dominios Proteicos/genética , Isoformas de Proteínas/genética , Nicotiana/genéticaRESUMEN
Peptide-based drugs combine advantages of larger biological therapeutics with those of small molecule drugs, but they generally display poor permeability and metabolic stability. Recently, we introduced a new type of peptide bond isostere, in which the backbone carbonyl is replaced with a 3-amino oxetane heterocycle, into short linear peptides with the aim of improving their therapeutic potential. In this study, we have explored the impact of oxetane modification on α-helical peptides to establish whether or not this modification is tolerated in this biologically important structural motif. The oxetane modification was introduced at two positions in a well-characterised helical peptide sequence, and circular dichroism and NMR spectroscopy were used to measure the resulting secondary structure content under different experimental conditions. Our data demonstrated that introduction of an oxetane into the peptide backbone results in a significant loss of helicity, regardless of where in the sequence the modification is placed. The molecular determinants of this destabilisation were then explored using steered molecular dynamics simulations, a computational method analogous to single molecule spectroscopy. Our simulations indicated that oxetane modification introduces a kink in the helical axis, alters the dihedral angles of residues up to three positions away from the modification, and disrupts the (i, i + 4) hydrogen bonding pattern characteristic of α-helices in favour of new, short-range hydrogen bonds. The detailed structural understanding provided in this work can direct future design of chemically modified peptides.
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Éteres Cíclicos/química , Éteres Cíclicos/metabolismo , Péptidos/química , Modelos Moleculares , Conformación Proteica en Hélice alfa , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: Ductal carcinoma in situ (DCIS) is an in-situ (pre-cancerous) breast malignancy whereby malignant cells are contained within the basement membrane of the breast ducts. Increasing awareness that some low-risk forms of DCIS might remain indolent for many years has led to concern about overtreatment, with at least 3 clinical trials underway internationally assessing the safety of active monitoring for low-risk DCIS. This study aimed to understand healthcare professionals' (HCPs) views on the management options for patients with DCIS. METHODS: Qualitative study using semi-structured interviews with HCPs involved in the diagnosis and management of DCIS in Australia and New Zealand. Interviews were audio-recorded, transcribed and analysed thematically using Framework Analysis method. RESULTS: Twenty-six HCPs including 10 breast surgeons, 3 breast physicians, 6 radiation oncologists, and 7 breast care nurses participated. There was a strong overall consensus that DCIS requires active treatment. HCPs generally felt uncomfortable recommending active monitoring as a management option for low-risk DCIS as they viewed this as outside current standard care. Overall, HCPs felt that active monitoring was an unproven strategy in need of an evidence base; however, many acknowledged that active monitoring for low-risk DCIS could be appropriate for patients with significant co-morbidities or limited life expectancy. They believed that most patients would opt for surgery wherever possible. CONCLUSIONS: This study highlights the important need for robust randomised controlled trial data about active monitoring for women with low-risk DCIS, to provide HCPs with confidence in their management recommendations and decision-making.
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Actitud del Personal de Salud , Neoplasias de la Mama/terapia , Carcinoma Intraductal no Infiltrante/terapia , Personal de Salud/psicología , Espera Vigilante , Adulto , Australia , Neoplasias de la Mama/psicología , Carcinoma Intraductal no Infiltrante/psicología , Femenino , Humanos , Masculino , Uso Excesivo de los Servicios de Salud , Persona de Mediana Edad , Nueva Zelanda , Enfermeras y Enfermeros/psicología , Médicos/psicología , Rol Profesional/psicología , Relaciones Profesional-Paciente , Investigación Cualitativa , Oncólogos de Radiación/psicología , Cirujanos/psicologíaRESUMEN
The glycopeptide antibiotic vancomycin has been widely used to treat infections of Gram-positive bacteria including Clostridium difficile and methicillin-resistant Staphylococcus aureus. However, since its introduction, high level vancomycin resistance has emerged. The genes responsible require the action of the two-component regulatory system VanSR to induce expression of resistance genes. The mechanism of detection of vancomycin by this two-component system has yet to be elucidated. Diverging evidence in the literature supports activation models in which the VanS protein binds either vancomycin, or Lipid II, to induce resistance. Here we investigated the interaction between vancomycin and VanS from Streptomyces coelicolor (VanSSC), a model Actinomycete. We demonstrate a direct interaction between vancomycin and purified VanSSC, and traced these interactions to the extracellular region of the protein, which we reveal adopts a predominantly α-helical conformation. The VanSSC-binding epitope within vancomycin was mapped to the N-terminus of the peptide chain, distinct from the binding site for Lipid II. In targeting a separate site on vancomycin, the effective VanS ligand concentration includes both free and lipid-bound molecules, facilitating VanS activation. This is the first molecular description of the VanS binding site within vancomycin, and could direct engineering of future therapeutics.
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Proteínas Bacterianas/metabolismo , Streptomyces coelicolor/metabolismo , Factores de Transcripción/metabolismo , Resistencia a la Vancomicina/genética , Vancomicina/farmacología , Proteínas Bacterianas/genética , Sitios de Unión , Regulación Bacteriana de la Expresión Génica , Streptomyces coelicolor/efectos de los fármacos , Streptomyces coelicolor/genética , Factores de Transcripción/genéticaRESUMEN
Membrane curvature sensing via helical protein domains, such as those identified in Amphiphysin and ArfGAP1, have been linked to a diverse range of cellular processes. However, these regions can vary significantly between different protein families and thus remain challenging to identify from sequence alone. Greater insight into the protein-lipid interactions that drive this behavior could lead to production of therapeutics that specifically target highly curved membranes. Here we demonstrate the curvature-dependence of membrane binding for an amphipathic helix (APH) in a plant reticulon, namely RTNLB13 from A. thaliana. We utilize solution-state nuclear magnetic resonance spectroscopy to establish the exact location of the APH and map the residues involved in protein-membrane interactions at atomic resolution. We find that the hydrophobic residues making up the membrane binding site are conserved throughout all A. thaliana reticulons. Our results also provide mechanistic insight that leads us to propose that membrane binding by this APH may act as a feedback element, only forming when ER tubules reach a critical size and adding stabilization to these structures without disrupting the bilayer. A shallow hydrophobic binding interface appears to be a feature shared more broadly across helical curvature sensors and would automatically restrict the penetration depth of these structures into the membrane. We also suggest this APH is highly tuned to the composition of the membrane in which it resides, and that this property may be universal in curvature sensors thus rationalizing the variety of mechanisms reported for these functional elements.
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Retículo Endoplásmico/metabolismo , Lípidos/fisiología , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fenómenos Biofísicos , Membrana Celular/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos/análisis , Liposomas/química , Dominios Proteicos , Pliegue de ProteínaRESUMEN
The activation mechanism of the ErbB family of receptors is of considerable medical interest as they are linked to a number of human cancers, including an aggressive form of breast cancer. In the rat analogue of the human ErbB2 receptor, referred to as Neu, a point mutation in the transmembrane domain (V664E) has been shown to trigger oncogenic transformation. While the structural impact of this mutation has been widely studied in the past to yield models for the active state of the Neu receptor, little is known about the impact of cholesterol on its structure. Given previous reports of the influence of cholesterol on other receptor tyrosine kinases (RTKs), as well as the modulation of lipid composition in cancer cells, we wished to investigate how cholesterol content impacts the structure of the Neu transmembrane domain. We utilized high-resolution magic angle spinning solid-state NMR to measure 13C-13C coupling of selectively labelled probe residues in the Neu transmembrane domain in lipid bilayers containing cholesterol. We observe inter-helical coupling between residues that support helix-helix interactions on both dimerization motifs reported in the literature (A661-XXX-G665 and I659-XXX-V663). We further explore how changes in cholesterol concentration alter transmembrane domain interactions and the properties and mechanics of the bilayer. We interpret our results in light of previous studies relating RTK activity to cholesterol enrichment and/or depletion, and propose a novel model to explain our data that includes the recognition and binding of cholesterol by the Neu transmembrane domain through a putative cholesterol-recognition/interaction amino acid consensus sequence.
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Colesterol/química , Membrana Dobles de Lípidos/química , Fluidez de la Membrana , Receptor ErbB-2/química , Colesterol/metabolismo , Humanos , Membrana Dobles de Lípidos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Receptor ErbB-2/metabolismoRESUMEN
Nicotinamide (vitamin B3) has photoprotective effects and reduces skin cancer incidence in high risk patients. Nicotinamide also improves cognition in animal models. As part of the ONTRAC (Oral Nicotinamide To Reduce Actinic Cancer) phase III placebo-controlled, randomized trial to assess nicotinamide's efficacy in skin cancer prevention, we included clinical neurocognitive function and patient-reported quality of life assessments at baseline and after 12 months of intervention in individuals with previous skin cancer in order to assess any effect of oral nicotinamide (500 mg po twice daily) on cognitive function and quality of life. In our sample of 310 participants who completed neurocognitive function testing at baseline and at 12 months, we were not able to detect any significant effect of oral nicotinamide on cognitive function nor on quality of life. Further studies of nicotinamide's effects on cognition in humans might include individuals with pre-existing mild cognitive impairment, and it may be that higher doses of nicotinamide are required to significantly influence cognitive function compared to doses required to reduce skin cancer.
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Cyclic peptides are an important source of new drugs but are challenging to produce synthetically. We show that head-to-tail peptide macrocyclisations are greatly improved, as measured by isolated yields, reaction rates and product distribution, by substitution of one of the backbone amide C[double bond, length as m-dash]O bonds with an oxetane ring. The cyclisation precursors are easily made by standard solution- or solid-phase peptide synthesis techniques. Macrocyclisations across a range of challenging ring sizes (tetra-, penta- and hexapeptides) are enabled by incorporation of this turn-inducing element. Oxetane incorporation is shown to be superior to other established amino acid modifications such as N-methylation. The positional dependence of the modification on cyclisation efficiency is mapped using a cyclic peptide of sequence LAGAY. We provide the first direct experimental evidence that oxetane modification induces a turn in linear peptide backbones, through the observation of d NN (i, i + 2) and d αN (i, i + 2) NOEs, which offers an explanation for these improvements. For cyclic peptide, cLAGAY, a combination of NMR derived distance restraints and molecular dynamics simulations are used to show that this modification alters the backbone conformation in proximity to the oxetane, with the flexibility of the ring reduced and a new intramolecular H-bond established. Finally, we incorporated an oxetane into a cyclic pentapeptide inhibitor of Aminopeptidase N, a transmembrane metalloprotease overexpressed on the surface of cancer cells. The inhibitor, cCNGRC, displayed similar IC50 values in the presence or absence of an oxetane at the glycine residue, indicating that bioactivity is fully retained upon amide C[double bond, length as m-dash]O bond replacement.
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Class II Major Histocompatibility complex (MHC-II) is a polymorphic heterodimer that binds antigen-derived peptides and presents them on the surface of antigen presenting cells. This mechanism of antigen presentation leads to recognition by CD4 T-cells and T-cell activation, making it a critical element of adaptive immune response. For this reason, the structural determinants of MHC-II function have been of great interest for the past 30â¯years, resulting in a robust structural understanding of the extracellular regions of the complex. However, the membrane-localized regions have also been strongly implicated in protein-protein and protein-lipid interactions that facilitate Class II assembly, transport and function, and it is these regions that are the focus of this review. Here we describe studies that reveal the strong and selective interactions between the transmembrane domains of the MHC α, and invariant chains which, when altered, have broad reaching impacts on antigen presentation and Class II function. We also summarize work that clearly demonstrates the link between membrane lipid composition (particularly the presence of cholesterol) and MHC-II conformation, subsequent peptide binding, and downstream T-cell activation. We have integrated these studies into a comprehensive view of Class II transmembrane domain biology.
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Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos/inmunología , Membrana Celular/química , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Modelos Animales de Enfermedad , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Activación de Linfocitos/inmunología , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
The enzyme carboxypeptidaseâ G2 (CPG2) is used in antibody-directed enzyme prodrug therapy (ADEPT) to catalyse the formation of an active drug from an inert prodrug. Free CPG2 in the bloodstream must be inhibited before administration of the prodrug in order to avoid a systemic reaction in the patient. Although a few small-molecule CPG2 inhibitors have been reported, none has been taken forward thus far. This lack of progress is due in part to a lack of structural understanding of the CPG2 active site as well as the absence of small molecules that can block the active site whilst targeting the complex for clearance. The work described here aimed to address both areas. We report the structural/functional impact of extensive point mutation across the putative CPG2 catalytic site and adjacent regions for the first time, revealing that residues outside the catalytic region (K208A, S210A and T357A) are crucial to enzyme activity. We also describe novel molecules that inhibit CPG2 whilst maintaining the accessibility of galactosylated moieties aimed at targeting the enzyme for clearance. This work acts as a platform for the future development of high-affinity CPG2 inhibitors that occupy new chemical space and will advance the safe application of ADEPT in cancer treatment.