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1.
Protein Sci ; 33(6): e5026, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38757384

RESUMO

Many biomedical applications, such as classification of binding specificities or bioengineering, depend on the accurate definition of protein binding interfaces. Depending on the choice of method used, substantially different sets of residues can be classified as belonging to the interface of a protein. A typical approach used to verify these definitions is to mutate residues and measure the impact of these changes on binding. Besides the lack of exhaustive data, this approach also suffers from the fundamental problem that a mutation introduces an unknown amount of alteration into an interface, which potentially alters the binding characteristics of the interface. In this study we explore the impact of alternative binding site definitions on the ability of a protein to recognize its cognate ligand using a pharmacophore approach, which does not affect the interface. The study also shows that methods for protein binding interface predictions should perform above approximately F-score = 0.7 accuracy level to capture the biological function of a protein.


Assuntos
Ligação Proteica , Proteínas , Sítios de Ligação , Proteínas/química , Proteínas/metabolismo , Proteínas/genética , Ligantes , Modelos Moleculares
2.
iScience ; 27(4): 109458, 2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38571760

RESUMO

Histone chaperones-structurally diverse, non-catalytic proteins enriched with acidic intrinsically disordered regions (IDRs)-protect histones from spurious nucleic acid interactions and guide their deposition into and out of nucleosomes. Despite their conservation and ubiquity, the function of the chaperone acidic IDRs remains unclear. Here, we show that the Xenopus laevis Npm2 and Nap1 acidic IDRs are substrates for TTLL4 (Tubulin Tyrosine Ligase Like 4)-catalyzed post-translational glutamate-glutamylation. We demonstrate that to bind, stabilize, and deposit histones into nucleosomes, chaperone acidic IDRs function as DNA mimetics. Our biochemical, computational, and biophysical studies reveal that glutamylation of these chaperone polyelectrolyte acidic stretches functions to enhance DNA electrostatic mimicry, promoting the binding and stabilization of H2A/H2B heterodimers and facilitating nucleosome assembly. This discovery provides insights into both the previously unclear function of the acidic IDRs and the regulatory role of post-translational modifications in chromatin dynamics.

3.
bioRxiv ; 2023 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-37790377

RESUMO

Histone chaperones-structurally diverse, non-catalytic proteins enriched with acidic intrinsically disordered regions (IDRs)-protect histones from spurious nucleic acid interactions and guide their deposition into and out of nucleosomes. Despite their conservation and ubiquity, the function of the chaperone acidic IDRs remains unclear. Here, we show that the Xenopus laevis Npm2 and Nap1 acidic IDRs are substrates for TTLL4 (Tubulin Tyrosine Ligase Like 4)-catalyzed post-translational glutamate-glutamylation. We demonstrate that, to bind, stabilize, and deposit histones into nucleosomes, chaperone acidic IDRs function as DNA mimetics. Our biochemical, computational, and biophysical studies reveal that glutamylation of these chaperone polyelectrolyte acidic stretches functions to enhance DNA electrostatic mimicry, promoting the binding and stabilization of H2A/H2B heterodimers and facilitating nucleosome assembly. This discovery provides insights into both the previously unclear function of the acidic IDRs and the regulatory role of post-translational modifications in chromatin dynamics.

4.
Bioinformatics ; 39(9)2023 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-37603727

RESUMO

MOTIVATION: Molecular-level classification of protein-protein interfaces can greatly assist in functional characterization and rational drug design. The most accurate protein interface predictions rely on finding homologous proteins with known interfaces since most interfaces are conserved within the same protein family. The accuracy of these template-based prediction approaches depends on the correct choice of suitable templates. Choosing the right templates in the immunoglobulin superfamily (IgSF) is challenging because its members share low sequence identity and display a wide range of alternative binding sites despite structural homology. RESULTS: We present a new approach to predict protein interfaces. First, template-specific, informative evolutionary profiles are established using a mutual information-based approach. Next, based on the similarity of residue level conservation scores derived from the evolutionary profiles, a query protein is hierarchically clustered with all available template proteins in its superfamily with known interface definitions. Once clustered, a subset of the most closely related templates is selected, and an interface prediction is made. These initial interface predictions are subsequently refined by extensive docking. This method was benchmarked on 51 IgSF proteins and can predict nontrivial interfaces of IgSF proteins with an average and median F-score of 0.64 and 0.78, respectively. We also provide a way to assess the confidence of the results. The average and median F-scores increase to 0.8 and 0.81, respectively, if 27% of low confidence cases and 17% of medium confidence cases are removed. Lastly, we provide residue level interface predictions, protein complexes, and confidence measurements for singletons in the IgSF. AVAILABILITY AND IMPLEMENTATION: Source code is freely available at: https://gitlab.com/fiserlab.org/interdct_with_refinement.


Assuntos
Benchmarking , Evolução Biológica , Sítios de Ligação , Desenho de Fármacos , Software
5.
Aging Cell ; 22(9): e13908, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37345431

RESUMO

Glaucoma is a leading cause of irreversible blindness, with advanced age being the single most significant risk factor. However, the mechanisms underlying the relationship between aging and glaucoma remain unclear. Genome-wide association studies (GWAS) have successfully identified genetic variants strongly associated with increased glaucoma risk. Understanding how these variants function in pathogenesis is crucial for translating genetic associations into molecular mechanisms and, ultimately, clinical applications. The chromosome 9p21.3 locus is among the most replicated glaucoma risk loci discovered by GWAS. Nonetheless, the absence of protein-coding genes in the locus makes interpreting the disease association challenging, leaving the causal variant and molecular mechanism elusive. In this study, we report the identification of a functional glaucoma risk variant, rs6475604. By employing computational and experimental methods, we demonstrated that rs6475604 resides in a repressive regulatory element. Risk allele of rs6475604 disrupts the binding of YY1, a transcription factor known to repress the expression of a neighboring gene in 9p21.3, p16INK4A, which plays a crucial role in cellular senescence and aging. These findings suggest that the glaucoma disease variant contributes to accelerated senescence, providing a molecular link between glaucoma risk and an essential cellular mechanism for human aging.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina , Glaucoma , Humanos , Inibidor p16 de Quinase Dependente de Ciclina/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Glaucoma/genética , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Fatores de Transcrição/genética
6.
bioRxiv ; 2023 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-37292862

RESUMO

Glaucoma is a leading cause of irreversible blindness, with advanced age being the single most significant risk factor. However, the mechanisms underlying the relationship between aging and glaucoma remain unclear. Genome-wide association studies (GWAS) have successfully identified genetic variants strongly associated with increased glaucoma risk. Understanding how these variants function in pathogenesis is crucial for translating genetic associations into molecular mechanisms and, ultimately, clinical applications. The chromosome 9p21.3 locus is among the most replicated glaucoma risk loci discovered by GWAS. Nonetheless, the absence of protein-coding genes in the locus makes interpreting the disease association challenging, leaving the causal variant and molecular mechanism elusive. In this study, we report the identification of a functional glaucoma risk variant, rs6475604. By employing computational and experimental methods, we demonstrated that rs6475604 resides in a repressive regulatory element. Risk allele of rs6475604 disrupts the binding of YY1, a transcription factor known to repress the expression of a neighboring gene in 9p21.3, p16INK4A, which plays a crucial role in cellular senescence and aging. These findings suggest that the glaucoma disease variant contributes to accelerated senescence, providing a molecular link between glaucoma risk and an essential cellular mechanism for human aging.

7.
Cancer Res ; 83(12): 1984-1999, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37101376

RESUMO

Chitinase 3-like 1 (Chi3l1) is a secreted protein that is highly expressed in glioblastoma. Here, we show that Chi3l1 alters the state of glioma stem cells (GSC) to support tumor growth. Exposure of patient-derived GSCs to Chi3l1 reduced the frequency of CD133+SOX2+ cells and increased the CD44+Chi3l1+ cells. Chi3l1 bound to CD44 and induced phosphorylation and nuclear translocation of ß-catenin, Akt, and STAT3. Single-cell RNA sequencing and RNA velocity following incubation of GSCs with Chi3l1 showed significant changes in GSC state dynamics driving GSCs towards a mesenchymal expression profile and reducing transition probabilities towards terminal cellular states. ATAC-seq revealed that Chi3l1 increases accessibility of promoters containing a Myc-associated zinc finger protein (MAZ) transcription factor footprint. Inhibition of MAZ downregulated a set of genes with high expression in cellular clusters that exhibit significant cell state transitions after treatment with Chi3l1, and MAZ deficiency rescued the Chi3L-induced increase of GSC self-renewal. Finally, targeting Chi3l1 in vivo with a blocking antibody inhibited tumor growth and increased the probability of survival. Overall, this work suggests that Chi3l1 interacts with CD44 on the surface of GSCs to induce Akt/ß-catenin signaling and MAZ transcriptional activity, which in turn upregulates CD44 expression in a pro-mesenchymal feed-forward loop. The role of Chi3l1 in regulating cellular plasticity confers a targetable vulnerability to glioblastoma. SIGNIFICANCE: Chi3l1 is a modulator of glioma stem cell states that can be targeted to promote differentiation and suppress growth of glioblastoma.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Glioblastoma/patologia , beta Catenina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células-Tronco Neoplásicas/patologia , Glioma/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células
8.
ACS Infect Dis ; 9(4): 966-978, 2023 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-36920074

RESUMO

Helicobacter pylori is found in the gut lining of more than half of the world's population, causes gastric ulcers, and contributes to stomach cancers. Menaquinone synthesis in H. pylori relies on the rare futalosine pathway, where H. pylori 5'-methylthioadenosine nucleosidase (MTAN) is proposed to play an essential role. Transition state analogues of MTAN, including BuT-DADMe-ImmA (BTDIA) and MeT-DADMe-ImmA (MTDIA), exhibit bacteriostatic action against numerous diverse clinical isolates of H. pylori with minimum inhibitory concentrations (MIC's) of <2 ng/mL. Three H. pylori BTDIA-resistant clones were selected under increasing BTDIA pressure. Whole genome sequencing showed no mutations in MTAN. Instead, resistant clones had mutations in metK, methionine adenosyltransferase (MAT), feoA, a regulator of the iron transport system, and flhF, a flagellar synthesis regulator. The mutation in metK causes expression of a MAT with increased catalytic activity, leading to elevated cellular S-adenosylmethionine. Metabolite analysis and the mutations associated with resistance suggest multiple inputs associated with BTDIA resistance. Human gut microbiome exposed to MTDIA revealed no growth inhibition under aerobic or anaerobic conditions. Transition state analogues of H. pylori MTAN have potential as agents for treating H. pylori infection without disruption of the human gut microbiome or inducing resistance in the MTAN target.


Assuntos
Helicobacter pylori , Humanos , Helicobacter pylori/genética , Purina-Núcleosídeo Fosforilase , N-Glicosil Hidrolases
9.
bioRxiv ; 2023 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-36747792

RESUMO

Many biomedical applications, such as classification of binding specificities or bioengineering, depend on the accurate definition of protein binding interfaces. Depending on the choice of method used, substantially different sets of residues can be classified as belonging to the interface of a protein. A typical approach used to verify these definitions is to mutate residues and measure the impact of these changes on binding. Besides the lack of exhaustive data this approach generates, it also suffers from the fundamental problem that a mutation introduces an unknown amount of alteration into an interface, which potentially alters the binding characteristics of the interface. In this study we explore the impact of alternative binding site definitions on the ability of a protein to recognize its cognate ligand using a pharmacophore approach, which does not affect the interface. The study also provides guidance on the minimum expected accuracy of interface definition that is required to capture the biological function of a protein.

10.
Prog Retin Eye Res ; 91: 101093, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35817658

RESUMO

The development of the vertebrate eyes is a complex process starting from anterior-posterior and dorso-ventral patterning of the anterior neural tube, resulting in the formation of the eye field. Symmetrical separation of the eye field at the anterior neural plate is followed by two symmetrical evaginations to generate a pair of optic vesicles. Next, reciprocal invagination of the optic vesicles with surface ectoderm-derived lens placodes generates double-layered optic cups. The inner and outer layers of the optic cups develop into the neural retina and retinal pigment epithelium (RPE), respectively. In vitro produced retinal tissues, called retinal organoids, are formed from human pluripotent stem cells, mimicking major steps of retinal differentiation in vivo. This review article summarizes recent progress in our understanding of early eye development, focusing on the formation the eye field, optic vesicles, and early optic cups. Recent single-cell transcriptomic studies are integrated with classical in vivo genetic and functional studies to uncover a range of cellular mechanisms underlying early eye development. The functions of signal transduction pathways and lineage-specific DNA-binding transcription factors are dissected to explain cell-specific regulatory mechanisms underlying cell fate determination during early eye development. The functions of homeodomain (HD) transcription factors Otx2, Pax6, Lhx2, Six3 and Six6, which are required for early eye development, are discussed in detail. Comprehensive understanding of the mechanisms of early eye development provides insight into the molecular and cellular basis of developmental ocular anomalies, such as optic cup coloboma. Lastly, modeling human development and inherited retinal diseases using stem cell-derived retinal organoids generates opportunities to discover novel therapies for retinal diseases.


Assuntos
Doenças Retinianas , Fatores de Transcrição , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Diferenciação Celular/fisiologia , Olho , Retina/metabolismo , Transdução de Sinais , Doenças Retinianas/metabolismo
11.
Stem Cell Reports ; 17(6): 1411-1427, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35523180

RESUMO

The insulin receptor (INSR) is an evolutionarily conserved signaling protein that regulates development and cellular metabolism. INSR signaling promotes neurogenesis in Drosophila; however, a specific role for the INSR in maintaining adult neural stem cells (NSCs) in mammals has not been investigated. We show that conditionally deleting the Insr gene in adult mouse NSCs reduces subventricular zone NSCs by ∼70% accompanied by a corresponding increase in progenitors. Insr deletion also produced hyposmia caused by aberrant olfactory bulb neurogenesis. Interestingly, hippocampal neurogenesis and hippocampal-dependent behaviors were unperturbed. Highly aggressive proneural and mesenchymal glioblastomas had high INSR/insulin-like growth factor (IGF) pathway gene expression, and isolated glioma stem cells had an aberrantly high ratio of INSR:IGF type 1 receptor. Moreover, INSR knockdown inhibited GBM tumorsphere growth. Altogether, these data demonstrate that the INSR is essential for a subset of normal NSCs, as well as for brain tumor stem cell self-renewal.


Assuntos
Células-Tronco Adultas , Ventrículos Laterais/metabolismo , Células-Tronco Neurais , Receptor de Insulina/metabolismo , Somatomedinas , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Ventrículos Laterais/citologia , Camundongos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Somatomedinas/metabolismo
12.
Bioinformatics ; 38(2): 554-555, 2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34499117

RESUMO

SUMMARY: The Interface Contact definition with Adaptable Atom Types (INTERCAAT) was developed to determine the atomic interactions between molecules that form a known three dimensional structure. First, INTERCAAT creates a Voronoi tessellation where each atom acts as a seed. Interactions are defined by atoms that share a hyperplane and whose distance is less than the sum of each atoms' Van der Waals radii plus the diameter of a solvent molecule. Interacting atoms are then classified and interactions are filtered based on compatibility. INTERCAAT implements an adaptive atom classification method; therefore, it can explore interfaces between a variety macromolecules. AVAILABILITY AND IMPLEMENTATION: Source code is freely available at: https://gitlab.com/fiserlab.org/intercaat. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Software , Solventes , Substâncias Macromoleculares
13.
Curr Res Struct Biol ; 3: 337-345, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34917954

RESUMO

Molecular interactions mediated by engagement of the Herpes virus entry mediator (HVEM) with members of TNF and Ig superfamily generate distinct signals in T cell activation pathways that modulate inflammatory and inhibitory responses. HVEM interacts with CD160 and B and T lymphocyte attenuator (BTLA), both members of the immunoglobulin (Ig) superfamily, which share a common binding site that is unique from that of LIGHT, a TNF ligand. BTLA or CD160 engagement with HVEM deliver inhibitory or stimulatory signals to the host immune response in a context dependent fashion, whereas HVEM engagement with LIGHT results in pro-inflammatory responses. We identified a mutation in human HVEM, G89F, which directly interferes with the human LIGHT interaction, but interestingly, also differentially modulates the binding of human BTLA and CD160 via an apparent allosteric mechanism involving recognition surfaces remote from the site of the mutation. Specifically, the G89F mutation enhances binding of CD160, while decreasing that of BTLA to HVEM in cell-based assays. Molecular dynamics simulations for wild-type and G89F mutant HVEM, bound to different sets of ligands, were performed to define the molecular basis of this unexpected allosteric effect. These results were leveraged to design additional human HVEM mutants with altered binding specificities.

14.
PLoS Genet ; 17(3): e1009086, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33684100

RESUMO

Within the glioblastoma cellular niche, glioma stem cells (GSCs) can give rise to differentiated glioma cells (DGCs) and, when necessary, DGCs can reciprocally give rise to GSCs to maintain the cellular equilibrium necessary for optimal tumor growth. Here, using ribosome profiling, transcriptome and m6A RNA sequencing, we show that GSCs from patients with different subtypes of glioblastoma share a set of transcripts, which exhibit a pattern of m6A loss and increased protein translation during differentiation. The target sequences of a group of miRNAs overlap the canonical RRACH m6A motifs of these transcripts, many of which confer a survival advantage in glioblastoma. Ectopic expression of the RRACH-binding miR-145 induces loss of m6A, formation of FTO/AGO1/ILF3/miR-145 complexes on a clinically relevant tumor suppressor gene (CLIP3) and significant increase in its nascent translation. Inhibition of miR-145 maintains RRACH m6A levels of CLIP3 and inhibits its nascent translation. This study highlights a critical role of miRNAs in assembling complexes for m6A demethylation and induction of protein translation during GSC state transition.


Assuntos
Adenosina/análogos & derivados , Glioblastoma/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Biossíntese de Proteínas , Regiões 3' não Traduzidas , Adenosina/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Humanos , Metilação , Proteínas Associadas aos Microtúbulos/genética , Interferência de RNA , RNA Mensageiro/genética , Transcriptoma , Células Tumorais Cultivadas
15.
Curr Opin Struct Biol ; 67: 205-211, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33486430

RESUMO

This review focuses on pharmacophore approaches in researching protein interfaces that bind protein ligands. Pharmacophore descriptions of binding interfaces that employ molecular dynamics simulation can account for effects of solvation and conformational flexibility. In addition, these calculations provide an approximation to entropic considerations and as such, a better approximation of the free energy of binding. Residue-based pharmacophore approaches can facilitate a variety of drug discovery tasks such as the identification of receptor-ligand partners, identifying their binding poses, designing protein interfaces for selectivity, or defining a reduced mutational combinatorial exploration for subsequent experimental engineering techniques by orders of magnitudes.


Assuntos
Descoberta de Drogas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Sítios de Ligação , Ligantes , Conformação Molecular , Mapas de Interação de Proteínas
16.
Proteins ; 89(6): 632-638, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33483991

RESUMO

Secreted and membrane-bound members of the immunoglobulin superfamily (IgSF) encompass a large, diverse array of proteins that play central roles in immune response and neural development, and are implicated in diseases ranging from cancer to rheumatoid arthritis. Despite the potential biomedical benefits of understanding IgSF:IgSF cognate receptor-ligand interactions, relatively little about them is known at a molecular level, and experimentally probing all possible receptor-ligand pairs is prohibitively costly. The Protein Ligand Interface Design (ProtLID) algorithm is a computational pharmacophore-based approach to identify cognate receptor-ligand pairs that was recently validated in a pilot study on a small set of IgSF complexes. Although ProtLID has shown a success rate of 61% at identifying at least one cognate ligand for a given receptor, it currently lacks any form of confidence measure that can prioritize individual receptor-ligand predictions to pursue experimentally. In this study, we expanded the application of ProtLID to cover all IgSF complexes with available structural data. In addition, we introduced an approach to estimate the confidence of predictions made by ProtLID based on a statistical analysis of how the ProtLID-constructed pharmacophore matches the structures of candidate ligands. The confidence score combines the physicochemical compatibility, spatial consistency, and mathematical skewness of the distribution of matches throughout a set of candidate ligands. Our results suggest that a subset of cases meeting stringent confidence criteria will always have at least one successful receptor-ligand prediction.


Assuntos
Algoritmos , Imunoglobulinas/química , Proteínas de Membrana/química , Família Multigênica , Software , Conjuntos de Dados como Assunto , Humanos , Imunoglobulinas/metabolismo , Ligantes , Proteínas de Membrana/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Projetos de Pesquisa
17.
Structure ; 28(10): 1083-1084, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33027689

RESUMO

Monoclonal antibodies are attractive but, in certain applications, limited therapeutic modalities due to their large size and high specificity. In this issue of Structure, Sevy at al. describe a computationally designed cyclic peptide mimicking the CDRH3 loop of the C05 antibody against influenza showing the potential utility of designer biologics.


Assuntos
Influenza Humana , Anticorpos Monoclonais , Fenômenos Biofísicos , Humanos , Influenza Humana/diagnóstico , Peptídeos Cíclicos
18.
J Biol Chem ; 295(46): 15650-15661, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-32893190

RESUMO

The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss of function with charged (Lys, Asp, and Glu) and polar (Thr, Ser, and Gln) Phe-392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with N-biotinyl aminoethyl methanethiosulfonate labeling), Phe-392 mutations causing loss of function, although preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly-305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations), as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT, was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe-392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V and other inactivating Phe-392 PCFT mutations lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe-392 appear to be features required for full activity.


Assuntos
Transportador de Folato Acoplado a Próton/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Cisteína/química , Cisteína/metabolismo , Deficiência de Ácido Fólico/patologia , Células HeLa , Humanos , Cinética , Síndromes de Malabsorção/patologia , Metotrexato/metabolismo , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Transportador de Folato Acoplado a Próton/química , Transportador de Folato Acoplado a Próton/genética
19.
Structure ; 28(11): 1197-1205.e2, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-32795404

RESUMO

Herpes virus entry mediator (HVEM) regulates positive and negative signals for T cell activation through co-signaling pathways. Dysfunction of the HVEM co-signaling network is associated with multiple pathologies related to autoimmunity, infectious disease, and cancer, making the associated molecules biologically and therapeutically attractive targets. HVEM interacts with three ligands from two different superfamilies using two different binding interfaces. The engagement with ligands CD160 and B- and T-lymphocyte attenuator (BTLA), members of immunoglobulin superfamily, is associated with inhibitory signals, whereas inflammatory responses are regulated through the interaction with LIGHT from the TNF superfamily. We computationally redesigned the HVEM recognition interfaces using a residue-specific pharmacophore approach, ProtLID, to achieve switchable-binding specificity. In subsequent cell-based binding assays the new interfaces, designed with only single or double mutations, exhibited selective binding to only one or two out of the three cognate ligands.


Assuntos
Antígenos CD/química , Receptores Imunológicos/química , Membro 14 de Receptores do Fator de Necrose Tumoral/química , Receptores Virais/química , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/química , Antígenos CD/genética , Antígenos CD/metabolismo , Sítios de Ligação , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Células HEK293 , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Linfócitos T/metabolismo , Linfócitos T/virologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo
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