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1.
Nat Commun ; 15(1): 5503, 2024 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-38951531

RESUMEN

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.


Asunto(s)
Enzima Convertidora de Angiotensina 2 , Microscopía por Crioelectrón , Prolina , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/genética , Prolina/metabolismo , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , COVID-19/virología , COVID-19/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/química , Modelos Moleculares
2.
J Vis Exp ; (199)2023 09 29.
Artículo en Inglés | MEDLINE | ID: mdl-37843272

RESUMEN

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.


Asunto(s)
Proteínas de Transporte de Membrana , Proteínas Transportadoras de Solutos , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteínas Transportadoras de Solutos/química , Proteínas Transportadoras de Solutos/metabolismo , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento , Proteínas de la Membrana/metabolismo , Preparaciones Farmacéuticas
3.
J Biol Chem ; 295(46): 15511-15526, 2020 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-32878987

RESUMEN

Encapsulated ferritins belong to the universally distributed ferritin superfamily, whose members function as iron detoxification and storage systems. Encapsulated ferritins have a distinct annular structure and must associate with an encapsulin nanocage to form a competent iron store that is capable of holding significantly more iron than classical ferritins. The catalytic mechanism of iron oxidation in the ferritin family is still an open question because of the differences in organization of the ferroxidase catalytic site and neighboring secondary metal-binding sites. We have previously identified a putative metal-binding site on the inner surface of the Rhodospirillum rubrum encapsulated ferritin at the interface between the two-helix subunits and proximal to the ferroxidase center. Here we present a comprehensive structural and functional study to investigate the functional relevance of this putative iron-entry site by means of enzymatic assays, MS, and X-ray crystallography. We show that catalysis occurs in the ferroxidase center and suggest a dual role for the secondary site, which both serves to attract metal ions to the ferroxidase center and acts as a flow-restricting valve to limit the activity of the ferroxidase center. Moreover, confinement of encapsulated ferritins within the encapsulin nanocage, although enhancing the ability of the encapsulated ferritin to undergo catalysis, does not influence the function of the secondary site. Our study demonstrates a novel molecular mechanism by which substrate flux to the ferroxidase center is controlled, potentially to ensure that iron oxidation is productively coupled to mineralization.


Asunto(s)
Proteínas Bacterianas/metabolismo , Ceruloplasmina/metabolismo , Metales/metabolismo , Rhodospirillum rubrum/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dominio Catalítico , Ceruloplasmina/química , Ceruloplasmina/genética , Cristalografía por Rayos X , Hierro/química , Hierro/metabolismo , Metales/química , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Zinc/química , Zinc/metabolismo
4.
Chem Commun (Camb) ; 56(23): 3417-3420, 2020 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-32090213

RESUMEN

Encapsulated ferritins (EncFtn) are a recently characterised member of the ferritin superfamily. EncFtn proteins are sequestered within encapsulin nanocompartments and form a unique biological iron storage system. Here, we use native mass spectrometry and hydrogen-deuterium exchange mass spectrometry to elucidate the metal-mediated assembly pathway of EncFtn.


Asunto(s)
Ceruloplasmina/química , Ferritinas/química , Espectrometría de Masas/métodos , Myxococcales/enzimología , Multimerización de Proteína
5.
Front Microbiol ; 10: 2035, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31551972

RESUMEN

Characterizing and engineering microbial communities for lignocellulosic biofuel production has received widespread attention. Previous research has established that Clostridium thermocellum JN4 and Thermoanaerobacterium thermosaccharolyticum GD17 coculture significantly improves overall cellulosic biofuel production efficiency. Here, we investigated this interaction and revealed the mechanism underlying the improved efficiency observed. In contrast to the previously reported mutualistic relationship, a harmful effect toward C. thermocellum JN4 was observed in these microbial consortia. Although T. thermosaccharolyticum GD17 relieves the carbon catabolite repression of C. thermocellum JN4 regarding obtaining more cellobiose or glucose released from lignocellulose, T. thermosaccharolyticum GD17 significantly hampers the growth of C. thermocellum JN4 in coculture. The increased formation of end products is due to the strong competitive metabolic advantage of T. thermosaccharolyticum GD17 over C. thermocellum JN4 in the conversion of glucose or cellobiose into final products. The possibility of controlling and rebalancing these microbial consortia to modulate cellulose degradation was achieved by adding T. thermosaccharolyticum GD17 stimulants into the system. As cellulolytic bacteria are usually at a metabolic disadvantage, these discoveries may apply to a large proportion of cellulosic biofuel-producing microbial consortia. These findings provide a reference for engineering efficient and modular microbial consortia for modulating cellulosic conversion.

6.
Biochem J ; 476(6): 975-989, 2019 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-30837306

RESUMEN

Ferritins are a large family of intracellular proteins that protect the cell from oxidative stress by catalytically converting Fe(II) into less toxic Fe(III) and storing iron minerals within their core. Encapsulated ferritins (EncFtn) are a sub-family of ferritin-like proteins, which are widely distributed in all bacterial and archaeal phyla. The recently characterized Rhodospirillum rubrum EncFtn displays an unusual structure when compared with classical ferritins, with an open decameric structure that is enzymatically active, but unable to store iron. This EncFtn must be associated with an encapsulin nanocage in order to act as an iron store. Given the wide distribution of the EncFtn family in organisms with diverse environmental niches, a question arises as to whether this unusual structure is conserved across the family. Here, we characterize EncFtn proteins from the halophile Haliangium ochraceum and the thermophile Pyrococcus furiosus, which show the conserved annular pentamer of dimers topology. Key structural differences are apparent between the homologues, particularly in the centre of the ring and the secondary metal-binding site, which is not conserved across the homologues. Solution and native mass spectrometry analyses highlight that the stability of the protein quaternary structure differs between EncFtn proteins from different species. The ferroxidase activity of EncFtn proteins was confirmed, and we show that while the quaternary structure around the ferroxidase centre is distinct from classical ferritins, the ferroxidase activity is still inhibited by Zn(II). Our results highlight the common structural organization and activity of EncFtn proteins, despite diverse host environments and contexts within encapsulins.


Asunto(s)
Proteínas Arqueales/química , Proteínas Bacterianas/química , Ferritinas/química , Myxococcales/química , Pyrococcus furiosus/química , Rhodospirillum rubrum/química , Dominios Proteicos , Homología Estructural de Proteína , Relación Estructura-Actividad
7.
Elife ; 52016 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-27529188

RESUMEN

Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins.


Asunto(s)
Ferritinas/química , Ferritinas/metabolismo , Hierro/metabolismo , Rhodospirillum rubrum/enzimología , Rhodospirillum rubrum/metabolismo , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Cristalografía por Rayos X , Microscopía Electrónica de Transmisión , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína
8.
N Biotechnol ; 32(6): 651-7, 2015 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-25573765

RESUMEN

Ferritin family proteins are found in all kingdoms of life and act to store iron within a protein cage and to protect the cell from oxidative damage caused by the Fenton reaction. The structural and biochemical features of the ferritins have been widely exploited in bionanotechnology applications: from the production of metal nanoparticles; as templates for semi-conductor production; and as scaffolds for vaccine design and drug delivery. In this review we first discuss the structural properties of the main ferritin family proteins, and describe how their organisation specifies their functions. Second, we describe materials science applications of ferritins that rely on their ability to sequester metal within their cavities. Finally, we explore the use of ferritin as a container for drug delivery and as a scaffold for the production of vaccines.


Asunto(s)
Medios de Contraste/síntesis química , Ferritinas/química , Imagen por Resonancia Magnética/métodos , Nanopartículas de Magnetita/química , Biotecnología/tendencias , Nanopartículas de Magnetita/ultraestructura , Nanotecnología/tendencias
9.
J Ind Microbiol Biotechnol ; 40(9): 1077-82, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23817671

RESUMEN

Improvement of agitation is a commonly used approach for the optimization of fermentation processes. In this report, the response to improving agitation rate from 150 to 250 rpm on cellulase production from Penicillium decumbens JUA10-1 was investigated. It was shown that the production of all the major components of the cellulase mixture increased following improved agitation. Further investigations showed that at least three factors are involved in this improvement: the improved biomass accumulation, proportion of active/mature cellulases, and cellulase transcription level. The transcription levels of the cellulase repressing transcription factor ace1 and the cellulase activating transcription factor xlnR, however, both declined when a higher agitation was applied. These observations, along with our analysis of the carbon catabolite repressor CreA, lead to the suggestion that the molecular mechanism underlying improved cellulase transcription is the competition of two concurrent effects following improved agitation: CreA-mediated derepression induced by the downregulation of ace1, and CreA-mediated deactivation induced by the downregulation of xlnR.


Asunto(s)
Reactores Biológicos , Celulasa/biosíntesis , Proteínas Fúngicas/metabolismo , Regulación Bacteriana de la Expresión Génica , Penicillium/enzimología , Factores de Transcripción/metabolismo , Adenosina Trifosfato/metabolismo , Biomasa , Celulasa/genética , Regulación hacia Abajo , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Penicillium/genética , Penicillium/crecimiento & desarrollo , Penicillium/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción Genética
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