Gonçalves, AM, Sousa Â, Pedro AQ, Romão MJ, Queiroz JA, Gallardo E, Passarinha LA.
2022.
Advances in Membrane-Bound Catechol-O-Methyltransferase Stability Achieved Using a New Ionic Liquid-Based Storage Formulation. International Journal of Molecular Sciences. 23, Number 13
AbstractMembrane-bound catechol-O-methyltransferase (MBCOMT), present in the brain and involved in the main pathway of the catechol neurotransmitter deactivation, is linked to several types of human dementia, which are relevant pharmacological targets for new potent and nontoxic inhibitors that have been developed, particularly for Parkinson’s disease treatment. However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs’ design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1- dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at −80 °C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.
Santos, MFA, Sciortino G, Correia I, Fernandes ACP, Santos-Silva T, Pisanu F, Garribba E, Pessoa JC.
2022.
Binding of VIVO2+, VIVOL, VIVOL2 and VVO2L Moieties to Proteins: X-ray/Theoretical Characterization and Biological Implications, 2022. Chemistry – A European JournalChemistry – A European Journal. 28(40):e202200105.: John Wiley & Sons, Ltd
AbstractAbstract Vanadium compounds have frequently been proposed as therapeutics, but their application has been hampered by the lack of information on the different V-containing species that may form and how these interact with blood and cell proteins, and with enzymes. Herein, we report several resolved crystal structures of lysozyme with bound VIVO2+ and VIVOL2+, where L=2,2?-bipyridine or 1,10-phenanthroline (phen), and of trypsin with VIVO(picolinato)2 and VVO2(phen)+ moieties. Computational studies complete the refinement and shed light on the relevant role of hydrophobic interactions, hydrogen bonds, and microsolvation in stabilizating the structure. Noteworthy is that the trypsin?VVO2(phen) and trypsin?VIVO(OH)(phen) adducts correspond to similar energies, thus suggesting a possible interconversion under physiological/biological conditions. The obtained data support the relevance of hydrolysis of VIV and VV complexes in the several types of binding established with proteins and the formation of different adducts that might contribute to their pharmacological action, and significantly widen our knowledge of vanadium?protein interactions.
Carvalho, AL, Dias FMV, Prates JAM, Nagy T, Gilbert HJ, Davies GJ, Ferreira LMA, Romao MJ, Fontes C.
2003.
Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex. Proceedings of the National Academy of Sciences of the United States of America. 100:13809-13814., Number 24
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Seixas, JD, Mukhopadhyay A, Santos-Silva T, Otterbein LE, Gallo DJ, Rodrigues SS, Guerreiro BH, Goncalves AML, Penacho N, Marques AR, Coelho AC, Reis PM, Romao MJ, Romao CC.
2013.
Characterization of a versatile organometallic pro-drug (CORM) for experimental CO based therapeutics. Dalton Transactions. 42:5985-5998., Number 17
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Luís, MP, Pereira IS, Bugalhão JN, Simões CN, Mota C, Romão MJ, Mota LJ.
2023.
The Chlamydia trachomatis IncM Protein Interferes with Host Cell Cytokinesis, Centrosome Positioning, and Golgi Distribution and Contributes to the Stability of the Pathogen-Containing Vacuole. Infection and Immunity. 91:e00405-22., Number 4
AbstractChlamydia trachomatis is an obligate intracellular bacterial pathogen that causes ocular and urogenital infections in humans. The ability of C. trachomatis to grow intracellularly in a pathogen-containing vacuole (known as an inclusion) depends on chlamydial effector proteins transported into the host cell by a type III secretion system. Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes ocular and urogenital infections in humans. The ability of C. trachomatis to grow intracellularly in a pathogen-containing vacuole (known as an inclusion) depends on chlamydial effector proteins transported into the host cell by a type III secretion system. Among these effectors, several inclusion membrane proteins (Incs) insert in the vacuolar membrane. Here, we show that human cell lines infected by a C. trachomatis strain deficient for Inc CT288/CTL0540 (renamed IncM) displayed less multinucleation than when infected by IncM-producing strains (wild type or complemented). This indicated that IncM is involved in the ability of Chlamydia to inhibit host cell cytokinesis. The capacity of IncM to induce multinucleation in infected cells was shown to be conserved among its chlamydial homologues and appeared to require its two larger regions predicted to be exposed to the host cell cytosol. C. trachomatis-infected cells also displayed IncM-dependent defects in centrosome positioning, Golgi distribution around the inclusion, and morphology and stability of the inclusion. The altered morphology of inclusions containing IncM-deficient C. trachomatis was further affected by depolymerization of host cell microtubules. This was not observed after depolymerization of microfilaments, and inclusions containing wild-type C. trachomatis did not alter their morphology upon depolymerization of microtubules. Overall, these findings suggest that IncM may exert its effector function by acting directly or indirectly on host cell microtubules.
Seixas, JD, Santos MFA, Mukhopadhyay A, Coelho AC, Reis PM, Veiros LF, Marques AR, Penacho N, Goncalves AML, Romao MJ, Bernardes GJL, Santos-Silva T, Romao CC.
2015.
A contribution to the rational design of Ru(CO)(3)Cl2L complexes for in vivo delivery of CO. Dalton Transactions. 44:5058-5075., Number 11
AbstractA few ruthenium based metal carbonyl complexes, e.g. CORM-2 and CORM-3, have therapeutic activity attributed to their ability to deliver CO to biological targets. In this work, a series of related complexes with the formula [Ru(CO)(3)Cl2L] (L = DMSO (3), L-H3CSO(CH2)(2)CH(NH2)CO2H) (6a); D,L-H3CSO(CH2)(2)CH-(NH2)CO2H (6b); 3-NC5H4(CH2)(2)SO3.Na (7); 4-NC5H4(CH2)(2)SO3Na (8); PTA (9); DAPTA (10); H3CS-(CH2)(2)CH(OH) CO2H (11); CNCMe2CO2Me (12); CNCMeEtCO2Me (13); CN(c-C3H4)CO2Et) (14)) were designed, synthesized and studied. The effects of L on their stability, CO release profile, cytotoxicity and anti-inflammatory properties are described. The stability in aqueous solution depends on the nature of L as shown using HPLC and LC-MS studies. The isocyanide derivatives are the least stable complexes, and the S-bound methionine oxide derivative is the more stable one. The complexes do not release CO gas to the headspace, but release CO2 instead. X-ray diffraction of crystals of the model protein Hen Egg White Lysozyme soaked with 6b (4UWN) and 8 (4UWV) shows the addition of Ru-II(CO)(H2O)(4) at the His15 binding site. Soakings with 7 (4UWU) produced the metallacarboxylate [Ru(COOH)(CO)(H2O)(3)](+) bound to the His15 site. The aqueous chemistry of these complexes is governed by the water-gas shift reaction initiated with the nucleophilic attack of HO- on coordinated CO. DFT calculations show this addition to be essentially barrierless. The complexes have low cytotoxicity and low hemolytic indices. Following i.v. administration of CORM-3, the in vivo bio-distribution of CO differs from that obtained with CO inhalation or with heme oxygenase stimulation. A mechanism for CO transport and delivery from these complexes is proposed.
Palma, AS, Liu Y, Zhang Y, Zhang H, Luis AS, Carvalho AL, Gilbert HJ, Boraston A, Fontes CMGA, Chai W, Ten F.
2012.
Designer-oligosaccharide microarrays to decipher ligands in mammalian and prokaryotic glucan-recognition systems. Glycobiology. 22:1612-1613., Number 11
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Brás, JLA, Pinheiro BA, Cameron K, Cuskin F, Viegas A, Najmudin S, Bule P, Pires VMR, Romão MJ, Bayer EA, Spencer HL, Smith S, Gilbert HJ, Alves VD, Carvalho AL, Fontes CMGA.
2016.
Diverse specificity of cellulosome attachment to the bacterial cell surface, dec. Scientific Reports. 6:38292.: The Author(s)
AbstractDuring the course of evolution, the cellulosome, one of Nature's most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.
Lopes, R, Raya-Barón Á, Robalo PM, Vinagreiro C, Barroso S, Romão MJ, Fernández I, Pereira MM, Royo B.
2021.
Donor Functionalized Iron(II) N-Heterocyclic Carbene Complexes in Transfer Hydrogenation Reactions. European Journal of Inorganic Chemistry. 2021:22-29., Number 1
AbstractTwo piano-stool iron(II) complexes bearing N-heterocyclic carbene ligands outfitted with acetamide- and amine-pendant arms [Cp*Fe(NHCR)(CO)I] {Cp* = η5-tetramethylcyclopentadienyl; R = CH2CONEt2 (3), (CH2)2NEt2 (4)}, have been prepared and fully characterized. Their catalytic activity in transfer hydrogenation (TH) of ketones using iPrOH as a hydrogen source and catalytic amounts of base (LiOtBu) has been explored, along with that of previously reported [CpFe(NHCR)(CO)I] {R = nBu (5), (CH2)2OH (6), Et (7), and (CH2)3OH (8)} complexes containing hydroxyl and nonfunctionalized alkyl arms. Complex 3 displayed the highest catalytic activity of the whole series 3–8, reaching a TOF50 value of 533 h–1. NMR monitoring of the stoichiometric reaction of 3 with LiOtBu, allowed the identification of a new species 3' containing a deprotonated amidate moiety, which has been fully characterized by 1H, 13C, and 15N NMR. Finally, a green protocol for the reduction of ketones through TH using glycerol as a hydrogen source, under microwave irradiation in the presence of catalytic amounts of 3 and base has been developed.
Duarte, M, Viegas A, Alves VD, Prates JAM, Ferreira LMA, Najmudin S, Cabrita EJ, Carvalho AL, Fontes CMGA, Bule P.
2021.
A dual cohesin–dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome. Journal of Biological Chemistry. 296:100552.
AbstractThe Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin–dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin–dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.
Barroca-Ferreira, J, Cruz-Vicente P, Santos MFA, Rocha SM, Santos-Silva T, Maia CJ, Passarinha LA.
2021.
Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure. International Journal of Molecular Sciences. 22, Number 18
AbstractBackground: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of 55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment.
Carvalho, AL, Dias FMV, Nagy T, Prates JAM, Proctor MR, Smith N, Bayer EA, Davies GJ, Ferreira LMA, Romao MJ, Fontes CMGA, Gilbert HJ.
2007.
Evidence for a dual binding mode of dockerin modules to cohesins. Proceedings of the National Academy of Sciences of the United States of America. 104:3089-3094., Number 9
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Carvalho, AL, Goyal A, Prates JAM, Bolam DN, Gilbert HJ, Pires VMR, Ferreira LMA, Planas A, Romao MJ, Fontes C.
2004.
The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site. Journal of Biological Chemistry. 279:34785-34793., Number 33
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Ribeiro, T, Santos-Silva T, Alves VD, Dias FMV, Luis AS, Prates JAM, Ferreira LMA, Romao MJ, Fontes CMGA.
2010.
Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities. Biochimica Et Biophysica Acta-Proteins and Proteomics. 1804:2054-2062., Number 10
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Dias, AMGC, Moreira IP, Lychko I, Lopes Soares C, Nurrito A, Moura Barbosa AJ, Lutz-Bueno V, Mezzenga R, Carvalho AL, Pina AS, Roque ACA.
2023.
Hierarchical self-assembly of a reflectin-derived peptide. Frontiers in Chemistry. 11
AbstractReflectins are a family of intrinsically disordered proteins involved in cephalopod camouflage, making them an interesting source for bioinspired optical materials. Understanding reflectin assembly into higher-order structures by standard biophysical methods enables the rational design of new materials, but it is difficult due to their low solubility. To address this challenge, we aim to understand the molecular self-assembly mechanism of reflectin’s basic unit—the protopeptide sequence YMDMSGYQ—as a means to understand reflectin’s assembly phenomena. Protopeptide self-assembly was triggered by different environmental cues, yielding supramolecular hydrogels, and characterized by experimental and theoretical methods. Protopeptide films were also prepared to assess optical properties. Our results support the hypothesis for the protopeptide aggregation model at an atomistic level, led by hydrophilic and hydrophobic interactions mediated by tyrosine residues. Protopeptide-derived films were optically active, presenting diffuse reflectance in the visible region of the light spectrum. Hence, these results contribute to a better understanding of the protopeptide structural assembly, crucial for the design of peptide- and reflectin-based functional materials.
Bule, P, Pires VMR, Alves VD, Carvalho AL, Prates JAM, Ferreira LMA, Smith SP, Gilbert HJ, Noach I, Bayer EA, Najmudin S, Fontes CMGA.
2018.
Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction, 2018. Scientific Reports. 8(1):6987.
AbstractCellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.
Otrelo-Cardoso, AR, Nair RR, Correia MAS, Cordeiro RCS, Panjkovich A, Svergun DI, Santos-Silva T, Rivas MG.
2017.
Highly selective tungstate transporter protein TupA from Desulfovibrio alaskensis G20, 2017. Scientific Reports. 7(1):5798.
AbstractMolybdenum and tungsten are taken up by bacteria and archaea as their soluble oxyanions through high affinity transport systems belonging to the ATP-binding cassette (ABC) transporters. The component A (ModA/TupA) of these transporters is the first selection gate from which the cell differentiates between MoO4 2−, WO4 2− and other similar oxyanions. We report the biochemical characterization and the crystal structure of the apo-TupA from Desulfovibrio desulfuricans G20, at 1.4 Å resolution. Small Angle X-ray Scattering data suggests that the protein adopts a closed and more stable conformation upon ion binding. The role of the arginine 118 in the selectivity of the oxyanion was also investigated and three mutants were constructed: R118K, R118E and R118Q. Isothermal titration calorimetry clearly shows the relevance of this residue for metal discrimination and oxyanion binding. In this sense, the three variants lost the ability to coordinate molybdate and the R118K mutant keeps an extremely high affinity for tungstate. These results contribute to an understanding of the metal-protein interaction, making it a suitable candidate for a recognition element of a biosensor for tungsten detection.
Carvalho, AL, Pires VMR, Gloster TM, Turkenburg JP, Prates JAM, Ferreira LMA, Romao MJ, Davies GJ, Fontes C, Gilbert HJ.
2005.
Insights into the structural determinants of cohesin dockerin specificity revealed by the crystal structure of the type II cohesin from Clostridium thermocellum SdbA. Journal of Molecular Biology. 349:909-915., Number 5
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