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Many archetypal and emerging classes of small-molecule therapeutics form covalent protein adducts. In vivo, both the resulting conjugates and their off-target side-conjugates have the potential to elicit antibodies, with implications for allergy and drug sequestration. Although ß-lactam antibiotics are a drug class long associated with these immunological phenomena, the molecular underpinnings of off-target drug-protein conjugation and consequent drug-specific immune responses remain incomplete. Here, using the classical ß-lactam penicillin G (PenG), we probe the B and T cell determinants of drug-specific IgG responses to such conjugates in mice. Deep B cell clonotyping reveals a dominant murine clonal antibody class encompassing phylogenetically-related IGHV1, IGHV5 and IGHV10 subgroup gene segments. Protein NMR and x-ray structural analyses reveal that these drive structurally convergent binding modes in adduct-specific antibody clones. Their common primary recognition mechanisms of the penicillin side-chain moiety (phenylacetamide in PenG)-regardless of CDRH3 length-limits cross-reactivity against other ß-lactam antibiotics. This immunogenetics-guided discovery of the limited binding solutions available to antibodies against side products of an archetypal covalent inhibitor now suggests future potential strategies for the 'germline-guided reverse engineering' of such drugs away from unwanted immune responses.
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Antibacterianos , Animales , Ratones , Antibacterianos/farmacología , Antibacterianos/inmunología , Inmunoglobulina G/inmunología , Penicilina G/inmunología , Penicilina G/química , Linfocitos B/inmunología , Penicilinas/inmunología , Penicilinas/química , Femenino , Reacciones Cruzadas/inmunología , Cristalografía por Rayos XRESUMEN
Mass photometry (MP) is a rapidly growing optical technique for label-free mass measurement of single biomolecules in solution. The underlying measurement principle provides numerous advantages over ensemble-based methods but has been limited to low analyte concentrations due to the need to uniquely and accurately quantify the binding of individual molecules to the measurement surface, which results in diffraction-limited spots. Here, we combine nanoparticle lithography with surface PEGylation to substantially lower surface binding, resulting in a 2 orders of magnitude improvement in the upper concentration limit associated with mass photometry. We demonstrate the facile tunability of degree of passivation, enabling measurements at increased analyte concentrations. These advances provide access to protein-protein interactions in the high nanomolar to low micromolar range, substantially expanding the application space of mass photometry.
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The dense O-glycosylation of mucins plays an important role in the defensive properties of the mucus hydrogel. Aberrant glycosylation is often correlated with inflammation and pathology such as COPD, cancer, and Crohn's disease. The inherent complexity of glycans and the diversity in the O-core structure constitute fundamental challenges for the analysis of mucin-type O-glycans. Due to coexistence of multiple isomers, multidimensional workflows such as LC-MS are required. To separate the highly polar carbohydrates, porous graphitized carbon is often used as a stationary phase. However, LC-MS workflows are time-consuming and lack reproducibility. Here we present a rapid alternative for separating and identifying O-glycans released from mucins based on trapped ion mobility mass spectrometry. Compared to established LC-MS, the acquisition time is reduced from an hour to two minutes. To test the validity, the developed workflow was applied to sputum samples from cystic fibrosis patients to map O-glycosylation features associated with disease.
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Mucinas , Espectrometría de Masas en Tándem , Humanos , Mucinas/metabolismo , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los Resultados , Polisacáridos/química , GlicosilaciónRESUMEN
Sodium bicarbonate (NaHCO3) is commonly utilized as a therapeutic to treat metabolic acidosis in people with chronic kidney disease (CKD). While increased dietary sodium chloride (NaCl) is known to promote volume retention and increase blood pressure, the effects of NaHCO3 loading on blood pressure and volume retention in CKD remain unclear. In the present study, we compared the effects of NaCl and NaHCO3 loading on volume retention, blood pressure, and kidney injury in both 2/3 and 5/6 nephrectomy remnant kidney rats, a well-established rodent model of CKD. We tested the hypothesis that NaCl loading promotes greater volume retention and increases in blood pressure than equimolar NaHCO3. Blood pressure was measured 24 h daily using radio telemetry. NaCl and NaHCO3 were administered in drinking water ad libitum or infused via indwelling catheters. Rats were housed in metabolic cages to determine volume retention. Our data indicate that both NaHCO3 and NaCl promote hypertension and volume retention in remnant kidney rats, with salt-sensitivity increasing with greater renal mass reduction. Importantly, while NaHCO3 intake was less pro-hypertensive than equimolar NaCl intake, NaHCO3 was not benign. NaHCO3 loading significantly elevated blood pressure and promoted volume retention in rats with CKD when compared with control rats receiving tap water. Our findings provide important insight into the effects of sodium loading with NaHCO3 in CKD and indicate that NaHCO3 loading in patients with CKD is unlikely to be benign.
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Hipertensión , Insuficiencia Renal Crónica , Humanos , Ratas , Animales , Bicarbonato de Sodio/farmacología , Bicarbonato de Sodio/uso terapéutico , Cloruro de Sodio/metabolismo , Cloruro de Sodio/farmacología , Presión Arterial , Riñón/metabolismo , Insuficiencia Renal Crónica/metabolismo , Presión Sanguínea , Cloruro de Sodio Dietético/farmacologíaRESUMEN
Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.
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Inmunoglobulina G , Microfluídica , Humanos , Recién Nacido , Fotometría/métodosRESUMEN
Protein-protein interactions underlie most biological processes, and determining the affinity and abundance of binding partners for each interaction is often a challenging task because these interactions often involve multiple ligands and binding sites. Standard methods for determining the affinity of protein interactions often require a large amount of starting material in addition to potentially disruptive labeling or immobilization of the binding partners. Mass photometry is a bioanalytical technique that measures the mass of single biomolecules in solution, quickly and with minimal sample requirements. This article describes how mass photometry can be used to determine the mass distribution of binding partners, the complexes they form, the relative abundance of each species, and, accordingly, the dissociation constant (KD ) of their interactions. © 2024 Refeyn Ltd. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Using mass photometry to measure protein-protein binding and quantify the KD of this interaction.
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Fotometría , Unión Proteica , Sitios de UniónRESUMEN
BACKGROUND: Obesity is associated with increased risk of cardiovascular disease, but underlying mechanisms remain elusive. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor, but how glucose impacts vascular function is unclear. GAL3 (galectin-3) is a sugar-binding lectin upregulated by hyperglycemia, but its role as a causative mechanism of cardiovascular disease remains poorly understood. Therefore, the objective of this study was to determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity. METHODS: GAL3 was measured and found to be markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate causative mechanisms in cardiovascular disease, mice deficient in GAL3 were bred with obese db/db mice to generate lean, lean GAL3 knockout, obese, and obese GAL3 knockout genotypes. Endothelial cell-specific GAL3 knockout mice with novel AAV-induced obesity recapitulated whole-body knockout studies to confirm cell specificity. RESULTS: Deletion of GAL3 did not alter body mass, adiposity, or plasma indices of glycemia and lipidemia, but levels of plasma reactive oxygen species as assessed by plasma thiobarbituric acid reactive substances were normalized in obese GAL3 knockout mice. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells from obese mice had increased expression of NOX1 (nicotinamide adenine dinucleotide phosphate oxidase 1), which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, which was normalized in microvascular endothelium from mice lacking GAL3. Cell-specific deletion confirmed that endothelial GAL3 regulates obesity-induced NOX1 overexpression and subsequent microvascular function. Furthermore, improvement of metabolic syndrome by increasing muscle mass, improving insulin signaling, or treating with metformin decreased microvascular GAL3, and thereby NOX1, expression levels. CONCLUSIONS: Deletion of GAL3 normalizes microvascular endothelial function in obese db/db mice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3, and in turn NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.
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Enfermedades Cardiovasculares , Hiperglucemia , Hipertensión , Animales , Humanos , Ratones , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Hiperglucemia/metabolismo , Ratones Noqueados , Ratones Obesos , NADPH Oxidasa 1/metabolismo , NADPH Oxidasas/metabolismo , Obesidad/complicaciones , Obesidad/genética , Obesidad/metabolismo , Estrés OxidativoRESUMEN
Rationale: Obesity increases the risk of cardiovascular disease (CVD) through mechanisms that remain incompletely defined. Metabolic dysfunction, especially hyperglycemia, is thought to be a major contributor but how glucose impacts vascular function is unclear. Galectin-3 (GAL3) is a sugar binding lectin upregulated by hyperglycemia but its role as a causative mechanism of CVD remains poorly understood. Objective: To determine the role of GAL3 in regulating microvascular endothelial vasodilation in obesity. Methods and Results: GAL3 was markedly increased in the plasma of overweight and obese patients, as well as in the microvascular endothelium of diabetic patients. To investigate a role for GAL3 in CVD, mice deficient in GAL3 were bred with obese db/db mice to generate lean, lean GAL3 knockout (KO), obese, and obese GAL3 KO genotypes. GAL3 KO did not alter body mass, adiposity, glycemia or lipidemia, but normalized elevated markers of reactive oxygen species (TBARS) in plasma. Obese mice exhibited profound endothelial dysfunction and hypertension, both of which were rescued by GAL3 deletion. Isolated microvascular endothelial cells (EC) from obese mice had increased NOX1 expression, which we have previously shown to contribute to increased oxidative stress and endothelial dysfunction, and NOX1 levels were normalized in EC from obese mice lacking GAL3. EC-specific GAL3 knockout mice made obese using a novel AAV-approach recapitulated whole-body knockout studies, confirming that endothelial GAL3 drives obesity-induced NOX1 overexpression and endothelial dysfunction. Improved metabolism through increased muscle mass, enhanced insulin signaling, or metformin treatment, decreased microvascular GAL3 and NOX1. GAL3 increased NOX1 promoter activity and this was dependent on GAL3 oligomerization. Conclusions: Deletion of GAL3 normalizes microvascular endothelial function in obese db/db mice, likely through a NOX1-mediated mechanism. Pathological levels of GAL3 and in turn, NOX1, are amenable to improvements in metabolic status, presenting a potential therapeutic target to ameliorate pathological cardiovascular consequences of obesity.
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BACKGROUND: Emergency Department Observation Unit (EDOU) patients with chest pain have a high prevalence of smoking, a key cardiovascular disease risk factor. While in the EDOU, there is an opportunity to initiate smoking cessation therapy (SCT), but this is not standard practice. This study aims to describe the missed opportunity for EDOU-initiated SCT by determining the proportion of smokers who receive SCT in the EDOU and within 1-year of EDOU discharge and to evaluate if SCT rates vary by race or sex. METHODS: We performed an observational cohort study of patients ≥18 years old being evaluated for chest pain in a tertiary care center EDOU from 3/1/2019-2/28/2020. Demographics, smoking history, and SCT were determined by electronic health record review. Emergency, family medicine, internal medicine, and cardiology records were reviewed to determine if SCT occurred within 1-year of their initial visit. SCT was defined as behavioral interventions or pharmacotherapy. Rates of SCT in the EDOU, 1-year follow-up period, and the EDOU through 1-year of follow-up were calculated. SCT rates from the EDOU through 1-year were compared between white vs. non-white and male vs. female patients using a multivariable logistic regression model including age, sex, and race. RESULTS: Among 649 EDOU patients, 24.0% (156/649) were smokers. These patients were 51.3% (80/156) female and 46.8% (73/156) white, with a mean age of 54.4 ± 10.5 years. From the EDOU encounter through 1-year of follow-up, only 33.3% (52/156) received SCT. In the EDOU, 16.0% (25/156) received SCT. During the 1-year follow-up period, 22.4% (35/156) had outpatient SCT. After adjusting for potential confounders, SCT rates from the EDOU through 1-year were similar among whites vs. non-whites (aOR 1.19, 95% CI 0.61-2.32) and males vs. females (aOR 0.79, 95% CI 0.40-1.56). CONCLUSIONS: SCT was rarely initiated in the EDOU among chest pain patients who smoke and most patients who did not receive SCT in the EDOU never received SCT at 1-year of follow-up. Rates of SCT were similarly low among race and sex subgroups. These data suggest an opportunity exists to improve health by initiating SCT in the EDOU.
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Unidades de Observación Clínica , Cese del Hábito de Fumar , Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Adolescente , Estudios Prospectivos , Dolor en el Pecho/epidemiología , Dolor en el Pecho/etiología , Dolor en el Pecho/terapia , Estudios de Cohortes , Servicio de Urgencia en HospitalRESUMEN
Interactions between the SARS-CoV-2 Spike protein and ACE2 are one of the most scrutinized reactions of our time. Yet, questions remain as to the impact of glycans on mediating ACE2 dimerization and downstream interactions with Spike. Here, we address these unanswered questions by combining a glycoengineering strategy with high-resolution native mass spectrometry (MS) to investigate the impact of N-glycan occupancy on the assembly of multiple Spike-ACE2 complexes. We confirmed that intact Spike trimers have all 66 N-linked sites occupied. For monomeric ACE2, all seven N-linked glycan sites are occupied to various degrees; six sites have >90% occupancy, while the seventh site (Asn690) is only partially occupied (â¼30%). By resolving the glycoforms on ACE2, we deciphered the influence of each N-glycan on ACE2 dimerization. Unexpectedly, we found that Asn432 plays a role in mediating dimerization, a result confirmed by site-directed mutagenesis. We also found that glycosylated dimeric ACE2 and Spike trimers form complexes with multiple stoichiometries (Spike-ACE2 and Spike2-ACE2) with dissociation constants (Kds) of â¼500 and <100 nM, respectively. Comparing these values indicates that positive cooperativity may drive ACE2 dimers to complex with multiple Spike trimers. Overall, our results show that occupancy has a key regulatory role in mediating interactions between ACE2 dimers and Spike trimers. More generally, since soluble ACE2 (sACE2) retains an intact SARS-CoV-2 interaction site, the importance of glycosylation in ACE2 dimerization and the propensity for Spike and ACE2 to assemble into higher oligomers are molecular details important for developing strategies for neutralizing the virus.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Unión Proteica , Espectrometría de Masas , PolisacáridosRESUMEN
Fast Blue (FB) and Cholera Toxin-B (CTB) are two retrograde tracers extensively used to label alpha-motoneurons (α-MNs). The overall goals of the present study were to (1) assess the effectiveness of different FB and CTB protocols in labeling α-MNs, (2) compare the labeling quality of these tracers at standard concentrations reported in the literature (FB 2% and CTB 0.1%) versus lower concentrations to overcome tracer leakage, and (3) determine an optimal protocol for labeling α-MNs in young B6SJL and aged C57Bl/J mice (when axonal transport is disrupted by aging). Hindlimb muscles of young B6SJL and aged C57Bl/J mice were intramuscularly injected with different FB or CTB concentrations and then euthanized at either 3 or 5 days after injection. Measurements were performed to assess labeling quality via seven different parameters. Our results show that tracer protocols of lower concentration and shorter labeling durations were generally better in labeling young α-MNs, whereas tracer protocols of higher tracer concentration and longer labeling durations were generally better in labeling aged α-MNs. A 0.2%, 3-day FB protocol provided optimal labeling of young α-MNs without tracer leakage, whereas a 2%, 5-day FB protocol or 0.1% CTB protocol provided optimal labeling of aged α-MNs. These results inform future studies on the selection of optimal FB and CTB protocols for α-MNs labeling in normal, aging, and neurodegenerative disease conditions.
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RNA molecules have emerged as increasingly attractive biomaterials with important applications such as RNA interference (RNAi) for cancer treatment and mRNA vaccines against infectious diseases. However, it remains challenging to engineer RNA biomaterials with sophisticated functions such as non-covalent light-switching ability. Herein, light-responsive RNA-protein nanowires are engineered to have such functions. It first demonstrates that the high affinity of RNA aptamer enables the formation of long RNA-protein nanowires through designing a dimeric RNA aptamer and an engineered green fluorescence protein (GFP) that contains two TAT-derived peptides at N- and C- termini. GFP is then replaced with an optogenetic protein pair system, LOV2 (light-oxygen-voltage) protein and its binding partner ZDK (Z subunit of protein A), to confer blue light-controlled photo-switching ability. The light-responsive nanowires are long (>500 nm) in the dark, but small (20-30 nm) when exposed to light. Importantly, the co-assembly of this RNA-protein hybrid biomaterial does not rely on the photochemistry commonly used for light-responsive biomaterials, such as bond formation, cleavage, and isomerization, and is thus reversible. These RNA-protein structures can serve as a new class of light-controlled biocompatible frameworks for incorporating versatile elements such as RNA, DNA, and enzymes.
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Aptámeros de Nucleótidos , Nanocables , ARN/química , Aptámeros de Nucleótidos/química , Interferencia de ARN , Péptidos , Proteínas Fluorescentes VerdesRESUMEN
Here we show using mass photometry how proline substitutions, commonly used for SARS-CoV-2 spike stabilisation in vaccine design, directly affects ACE2 receptor interactions via dynamics of open and closed states. Conformational changes and ACE2 binding were influenced by spike variant and temperature, but independent of site-specific N-glycosylation.
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COVID-19 , SARS-CoV-2 , Humanos , Enzima Convertidora de Angiotensina 2 , Glicoproteína de la Espiga del Coronavirus/química , Peptidil-Dipeptidasa A/metabolismo , Receptores Virales/química , Receptores Virales/metabolismo , Unión Proteica , Fotometría , Simulación de Dinámica Molecular , Sitios de UniónRESUMEN
The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand's ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling-dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation.
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Dominios C2 , Liposomas , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Glicósido Hidrolasas/metabolismo , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Ligandos , Lípidos , Manosa , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Polisacáridos/genética , Receptores Notch/genética , Receptores Notch/metabolismoRESUMEN
Protein assembly is a main route to generating complexity in living systems. Revealing the relevant molecular details is challenging because of the intrinsic heterogeneity of species ranging from few to hundreds of molecules. Here, we use mass photometry to quantify and monitor the full range of actin oligomers during polymerization with single-molecule sensitivity. We find that traditional nucleation-based models cannot account for the observed distributions of actin oligomers. Instead, the key step of filament formation is a slow transition between distinct states of an actin filament mediated by cation exchange or ATP hydrolysis. The resulting model reproduces important aspects of actin polymerization, such as the critical concentration for filament formation and bulk growth behavior. Our results revise the mechanism of actin nucleation, shed light on the role and function of actin-associated proteins, and introduce a general and quantitative means to studying protein assembly at the molecular level.
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Glycosaminoglycans (GAGs) are heterogeneous acidic polysaccharides involved in a range of biological functions. They have a significant influence on the regulation of cellular processes and the development of various diseases and infections. To fully understand the functional roles that GAGs play in mammalian systems, including disease processes, it is essential to understand their structural features. Despite having a linear structure and a repetitive disaccharide backbone, their structural analysis is challenging and requires elaborate preparative and analytical techniques. In particular, the extent to which GAGs are sulfated, as well as variation in sulfate position across the entire oligosaccharide or on individual monosaccharides, represents a major obstacle. Here, we summarize the current state-of-the-art methodologies used for GAG sample preparation and analysis, discussing in detail liquid chromatograpy and mass spectrometry-based approaches, including advanced ion activation methods, ion mobility separations and infrared action spectroscopy of mass-selected species.
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Disacáridos , Glicosaminoglicanos , Animales , Glicosaminoglicanos/análisis , Glicosaminoglicanos/química , Mamíferos , Espectrometría de Masas/métodos , Monosacáridos , Oligosacáridos , Polisacáridos , Sulfatos/análisisRESUMEN
BACKGROUND: To describe the Strategic Allocation of Fundamental Epidemic Resources (SAFER) model as a method to inform equitable community distribution of critical resources and testing infrastructure. METHODS: The SAFER model incorporates a four-quadrant design to categorize a given community based on two scales: testing rate and positivity rate. Three models for stratifying testing rates and positivity rates were applied to census tracts in Milwaukee County, Wisconsin: using median values (MVs), cluster-based classification and goal-oriented values (GVs). RESULTS: Each of the three approaches had its strengths. MV stratification divided the categories most evenly across geography, aiding in assessing resource distribution in a fixed resource and testing capacity environment. The cluster-based stratification resulted in a less broad distribution but likely provides a truer distribution of communities. The GVs grouping displayed the least variation across communities, yet best highlighted our areas of need. CONCLUSIONS: The SAFER model allowed the distribution of census tracts into categories to aid in informing resource and testing allocation. The MV stratification was found to be of most utility in our community for near real time resource allocation based on even distribution of census tracts. The GVs approach was found to better demonstrate areas of need.
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Epidemias , Recursos en Salud , Asignación de Recursos , Asignación de Recursos para la Atención de Salud/organización & administración , Equidad en Salud/economía , Equidad en Salud/organización & administración , Recursos en Salud/organización & administración , Humanos , Asignación de Recursos/organización & administraciónRESUMEN
The neurotransmitter acetylcholine (ACh) acts in part through a family of nicotinic ACh receptors (nAChRs), which mediate diverse physiological processes including muscle contraction, neurotransmission, and sensory transduction. Pharmacologically, nAChRs are responsible for tobacco addiction and are targeted by medicines for hypertension and dementia. Nicotinic AChRs were the first ion channels to be isolated. Recent studies have identified molecules that control nAChR biogenesis, trafficking, and function. These nAChR accessories include protein and chemical chaperones as well as auxiliary subunits. Whereas some factors act on many nAChRs, others are receptor specific. Discovery of these regulatory mechanisms is transforming nAChR research in cells and tissues ranging from central neurons to spinal ganglia to cochlear hair cells. Nicotinic AChR-specific accessories also enable drug discovery on high-confidence targets for psychiatric, neurological, and auditory disorders.
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Chaperonas Moleculares/metabolismo , Neuronas/metabolismo , Proteínas/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Membrana Celular/metabolismo , Descubrimiento de Drogas , Retículo Endoplásmico/metabolismo , Humanos , Ligandos , Músculo Esquelético/metabolismo , Neurofarmacología , Nicotina/metabolismo , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/químicaRESUMEN
Structural determination of N-glycans by mass spectrometry is ideally performed by negative ion collision-induced dissociation because the spectra are dominated by cross-ring fragments leading to ions that reveal structural details not available by many other methods. Most glycans form [M - H]- or [M + adduct]- ions but larger ones (above approx. m/z 2000) typically form doubly charged ions. Differences have been reported between the fragmentation of singly and doubly charged ions but a detailed comparison does not appear to have been reported. In addition to [M + adduct]- ions (this paper uses phosphate as the adduct) other doubly, triply, and quadruply charged ions of composition [Mn + (H2PO4)n]n- have been observed in mixtures of N-glycans released from viral and other glycoproteins. This paper explores the formation and fragmentation of these different types of multiply charged ions with particular reference to the presence of diagnostic fragments in the CID spectra and comments on how these ions can be used to characterize these glycans.
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Glicoproteínas/química , Polisacáridos/análisis , Polisacáridos/química , Espectrometría de Movilidad Iónica/métodos , Iones , Espectrometría de Masa por Ionización de Electrospray/métodos , ortoaminobenzoatos/químicaRESUMEN
Protein-protein interactions are involved in the regulation and function of the majority of cellular processes. As a result, much effort has been aimed at the development of methodologies capable of quantifying protein-protein interactions, with label-free methods being of particular interest due to the associated simplified workflows and minimisation of label-induced perturbations. Here, we review recent advances in optical technologies providing label-free in vitro measurements of affinities and kinetics. We provide an overview and comparison of existing techniques and their principles, discussing advantages, limitations, and recent applications.