Biological Calorimetry: Old Friend, New Insights

Calorimetry is an old experimental technique (first instrument developed in S. XVIII), but it is broadly used and still provides key information for understanding biological processes at the molecular level, particularly, cooperative phenomena in protein interactions. Here, we review and highlight some key aspects of biological calorimetry. Several biological systems will be described in which calorimetry was instrumental for modeling the behavior of the protein and obtaining further biological insight

Conformational Stability of Hepatitis C Virus NS3 Protease

AbstractThe hepatitis C virus NS3 protease is responsible for the processing of the nonstructural region of viral precursor polyprotein in infected hepatic cells. NS3 has been considered a target for drug discovery for a long time. NS3 is a zinc-dependent serine protease. However, the zinc ion is not involved in the catalytic mechanism, because it is bound far away from the active site. Thus, zinc is essential for the structural integrity of the protein and it is considered to have a structural role. The first thermodynamic study on the conformational equilibrium and stability of NS3 and the effect of zinc on such equilibrium is presented here. In agreement with a previous calorimetric study on the binding of zinc to NS3, the global unfolding heat capacity is dominated by the zinc dissociation step, suggesting that the binding of zinc induces a significant structural rearrangement of the protein. In addition, contrary to other homologous zinc-dependent proteases, the zinc-free NS3 protease is not completely unstructured. It is apparent that the conformational landscape of hepatitis C virus NS3 protease is fairly complex due to its intrinsic plasticity, and to the interactions with its different effectors (zinc and the accessory viral protein NS4A) and their modulation of the population of the different conformational states

Experimental validation of in silico target predictions on synergistic protein targets

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Molecular Context-Dependent Effects Induced by Rett Syndrome-Associated Mutations in MeCP2

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-binding protein involved in neuronal development and maturation. Loss-of-function mutations in MeCP2 result in Rett syndrome (RTT), a neurodevelopmental disorder that is the main cause of mental retardation in females. MeCP2 is an intrinsically disordered protein (IDP) constituted by six domains. Two domains are the main responsible elements for DNA binding (methyl-CpG binding domain, MBD) and recruitment of gene transcription/silencing machinery (transcription repressor domain, TRD). These two domains concentrate most of the RTT-associated mutations. R106W and R133C are associated with severe and mild RTT phenotype, respectively. We have performed a comprehensive characterization of the structural and functional impact of these substitutions at molecular level. Because we have previously shown that the MBD-flanking disordered domains (N-terminal domain, NTD, and intervening domain, ID) exert a considerable influence on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41635), here we report the biophysical study of the influence of the protein scaffold on the structural and functional effect induced by these two RTT-associated mutations. These results represent an example of how a given mutation may show different effects (sometimes opposing effects) depending on the molecular context

Influence of the disordered domain structure of MeCP2 on its structural stability and dsDNA interaction

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-associated structural protein. MeCP2 deregulation results in two neurodevelopmental disorders: MeCP2 dysfunction is associated with Rett syndrome, while excess of activity is associated with MeCP2 duplication syndrome. MeCP2 is an intrinsically disordered protein (IDP) constituted by six structural domains with variable, small percentage of well-defined secondary structure. Two domains, methyl-CpG binding domain (MBD) and transcription repressor domain (TRD), are the elements responsible for dsDNA binding ability and recruitment of the gene transcription/silencing machinery, respectively. Previously we studied the influence of the completely disordered, MBD-flanking domains (N-terminal domain, NTD, and intervening domain, ID) on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41,635). Here we report the biophysical study of the influence of the remaining domains (transcriptional repressor domain, TRD, and C-terminal domains, CTDα and CTDβ) on the structural stability of MBD and the dsDNA binding capabilities of MBD and ID. The influence of distant disordered domains on MBD properties makes it necessary to consider the NTD-MBD-ID variant as the minimal protein construct for studying dsDNA/chromatin binding properties, while the full-length protein should be considered for transcriptional regulation studies

The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site

Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities

Biopsia Líquida Térmica (TLB): Calorimetría diferencial de barrido aplicada a diagnóstico y seguimiento de melanoma y cáncer de pulmón

El càncer supone una patología en aumento en los últimos años, cobrando gran importancia el desarrollo de técnicas que mejoren el diagnóstico precoz y el seguimiento de estos pacientes. La Calorimetría Diferencia de Barrido (DSC) supone una técnica novedosa que tras aplicar calor al plasma sanguíneo se obtiene un termograma específico que ha demostrado ser diferente en pacientes con càncer con respecto a individuos sanos. En este estudio aplicamos esta técnica a pacientes diagnosticados de Cáncer de Pulmón y Melanoma y se obtienen muestras de ambos al diagnóstico y en el seguimiento (en el caso del melanoma).Tras la obtención de la muestra se analizan los termogramas de DSC y se obtienen unos parámetros (en total 14) que se analizan mediante estudios estadísticos multiparamétricos obteniendo un PS score que nos permite clasificar las muestras entre pacientes con cáncer o individuos sanos (grupo caso o control) con una fiabilidad superior al 90%. Este estudio permite avanzar en el desarrollo de técnicas de diagnóstico precoz en estas patologías y por tanto en sistemas de cribaje futuros mediante una extracción sanguínea, siendo esta menos invasiva y de menor coste que las técnicas actuales. De este estudio se han obtenido 2 publicaciones en revistas indexadas de alto factor de impacto (Q1): Adrián Velazquez-Campoy, Sonia Vega, Oscar Sánchez-Gracia, Angel Lanas, Alberto Rodrigo, et al. “Thermal liquid biopsy for monitoring melanoma patients under surveillance during treatment: A pilot study”. Biochim Biophys Acta. 2018 Aug;1862(8):1701-1710Alberto Rodrigo, Jorge L Ojeda, Sonia Vega, Oscar Sánchez-Gracia et al. “Thermal liquid biopsy (TLB): a predictive score derived from serum thermograms as a clinical tool for screening lung cancer patients”. Cancers (Basel). 2019 Jul; 11(7): 1012.<br /

Thermal liquid biopsy (Tlb): A predictive score derived from serum thermograms as a clinical tool for screening lung cancer patients

Risk population screening programs are instrumental for advancing cancer management and reducing economic costs of therapeutic interventions and the burden of the disease, as well as increasing the survival rate and improving the quality of life for cancer patients. Lung cancer, with high incidence and mortality rates, is not excluded from this situation. The success of screening programs relies on many factors, with some of them being the appropriate definition of the risk population and the implementation of detection techniques with an optimal discrimination power and strong patient adherence. Liquid biopsy based on serum or plasma detection of circulating tumor cells or DNA/RNA is increasingly employed nowadays, but certain limitations constrain its wide application. In this work, we present a new implementation of thermal liquid biopsy (TLB) for lung cancer patients. TLB provides a prediction score based on the ability to detect plasma/serum proteome alterations through calorimetric thermograms that strongly correlates with the presence of lung cancer disease (91% accuracy rate, 90% sensitivity, 92% specificity, diagnostic odds ratio 104). TLB is a quick, minimally-invasive, low-risk technique that can be applied in clinical practice for evidencing lung cancer, and it can be used in screening and monitoring actions

Repositioning small molecule drugs as allosteric inhibitors of the BFT-3 toxin from enterotoxigenic Bacteroides fragilis

Bacteroides fragilis is an abundant commensal component of the healthy human colon. However, under dysbiotic conditions, enterotoxigenic B. fragilis (ETBF) may arise and elicit diarrhea, anaerobic bacteremia, inflammatory bowel disease, and colorectal cancer. Most worrisome, ETBF is resistant to many disparate antibiotics. ETBF's only recognized specific virulence factor is a zinc-dependent metallopeptidase (MP) called B. fragilis toxin (BFT) or fragilysin, which damages the intestinal mucosa and triggers disease-related signaling mechanisms. Thus, therapeutic targeting of BFT is expected to limit ETBF pathogenicity and improve the prognosis for patients. We focused on one of the naturally occurring BFT isoforms, BFT-3, and managed to repurpose several approved drugs as BFT-3 inhibitors through a combination of biophysical, biochemical, structural, and cellular techniques. In contrast to canonical MP inhibitors, which target the active site of mature enzymes, these effectors bind to a distal allosteric site in the proBFT-3 zymogen structure, which stabilizes a partially unstructured, zinc-free enzyme conformation by shifting a zinc-dependent disorder-to-order equilibrium. This yields proBTF-3 incompetent for autoactivation, thus ablating hydrolytic activity of the mature toxin. Additionally, a similar destabilizing effect is observed for the activated protease according to biophysical and biochemical data. Our strategy paves a novel way for the development of highly specific inhibitors of ETBF-mediated enteropathogenic conditions

Blocking Nupr1 Protein, A Successful Approach for Pancreatic Adenocarcinoma Treatment

Background: Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic malignancy. Nuclear protein 1 (NUPR1) is a recognized protein, over-expressed and involved in PDAC development. NUPR1 belongs to the special class of intrinsically disordered proteins (IDPs) and it is implicated in cell signaling and regulatory functions. The multifunctional nature of NUPR1 renders it as an attractive target for drug design and development. Aim: Identify a small molecule inhibiting protein-protein interactions in NUPR1 and able to interfere with any of NUPR1 key oncogenic activities, thus, constituting a new chemotherapy strategy against PDAC. Methods: Ligand-induced stabilization against thermal denaturation (thermal-shift assay) was employed for identifying potential NUPR1-interacting compounds. An in vitro molecular screening based on thermal denaturation of NUPR1 in the presence of a variety of potential ligands was performed using a collection of 1120 compounds. All compounds are FDA-approved drugs for different therapeutic indications, exhibiting high chemical and pharmacological diversity, as well as good bioavailability and safety parameters in humans. Direct interaction of selected compounds with NUPR1 was assessed experimentally (calorimetry, fluorescence spectroscopy, nuclear magnetic resonance, and proximity ligation assay) and computationally (molecular dynamics simulations). Compound efficacy was determined in PDAC-derived cell-based assays and in vivo assays on xenografted PDACderived cells in mice. Comparisons of treatment outcome were tested for statistical significance by using the t-test, and statistical significance was assumed at a p-value lower than 0.05. Results: Fifteen candidates were selected, and their interactions with NUPR1 were characterized. In vitro experiments with MiaPaCa-2 cells treated using 10 µM of compounds for 6 days showed that two of the compounds (C13 and C15) were very efficient in diminishing cell viability (10 ± 3% and 26 ± 7%, respectively; assays in triplicates (n = ) p= 0.01). These values were similar to those obtained with oxaliplatin (10 ± 2%; p= 0.01). Also, they reduced cell migration (from 10-20% wound-healing ability compared to 50% in control assays; p= 0.05) and colony formation (completely suppressed in the presence of both compounds; p= 0.01). In addition, the most promising compound, C15, interfered with the interaction of NUPR1 with MSL1, one of the NUPR1 binding partners (Figure 1). The administration of a 10 mg/Kg dose of C15 promoted complete arrest of tumor development on xenografted PDAC-derived cells in mice (Figure 2). Conclusion: We report the discovery of a compound specifically active against PDAC and interfering with NUPR1. In addition, we demonstrate that it is possible to identify small molecules able to modulate the function of complex targets such as IDPs