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1.
Proteins ; 89(12): 1618-1632, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34350630

RESUMO

An evolutionary-based definition and classification of target evaluation units (EUs) is presented for the 14th round of the critical assessment of structure prediction (CASP14). CASP14 targets included 84 experimental models submitted by various structural groups (designated T1024-T1101). Targets were split into EUs based on the domain organization of available templates and performance of server groups. Several targets required splitting (19 out of 25 multidomain targets) due in part to observed conformation changes. All in all, 96 CASP14 EUs were defined and assigned to tertiary structure assessment categories (Topology-based FM or High Accuracy-based TBM-easy and TBM-hard) considering their evolutionary relationship to existing ECOD fold space: 24 family level, 50 distant homologs (H-group), 12 analogs (X-group), and 10 new folds. Principal component analysis and heatmap visualization of sequence and structure similarity to known templates as well as performance of servers highlighted trends in CASP14 target difficulty. The assigned evolutionary levels (i.e., H-groups) and assessment classes (i.e., FM) displayed overlapping clusters of EUs. Many viral targets diverged considerably from their template homologs and thus were more difficult for prediction than other homology-related targets. On the other hand, some targets did not have sequence-identifiable templates, but were predicted better than expected due to relatively simple arrangements of secondary structural elements. An apparent improvement in overall server performance in CASP14 further complicated traditional classification, which ultimately assigned EUs into high-accuracy modeling (27 TBM-easy and 31 TBM-hard), topology (23 FM), or both (15 FM/TBM).

2.
Proteins ; 89(12): 1673-1686, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34240477

RESUMO

This report describes the tertiary structure prediction assessment of difficult modeling targets in the 14th round of the Critical Assessment of Structure Prediction (CASP14). We implemented an official ranking scheme that used the same scores as the previous CASP topology-based assessment, but combined these scores with one that emphasized physically realistic models. The top performing AlphaFold2 group outperformed the rest of the prediction community on all but two of the difficult targets considered in this assessment. They provided high quality models for most of the targets (86% over GDT_TS 70), including larger targets above 150 residues, and they correctly predicted the topology of almost all the rest. AlphaFold2 performance was followed by two manual Baker methods, a Feig method that refined Zhang-server models, two notable automated Zhang server methods (QUARK and Zhang-server), and a Zhang manual group. Despite the remarkable progress in protein structure prediction of difficult targets, both the prediction community and AlphaFold2, to a lesser extent, faced challenges with flexible regions and obligate oligomeric assemblies. The official ranking of top-performing methods was supported by performance generated PCA and heatmap clusters that gave insight into target difficulties and the most successful state-of-the-art structure prediction methodologies.

3.
Science ; 373(6557): 871-876, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34282049

RESUMO

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. We explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo-electron microscopy structure modeling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. We make the method available to the scientific community to speed biological research.


Assuntos
Aprendizado Profundo , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas ADAM/química , Sequência de Aminoácidos , Simulação por Computador , Microscopia Crioeletrônica , Cristalografia por Raios X , Bases de Dados de Proteínas , Proteínas de Membrana/química , Modelos Moleculares , Complexos Multiproteicos/química , Redes Neurais de Computação , Subunidades Proteicas/química , Proteínas/fisiologia , Receptores Acoplados a Proteínas G/química , Esfingosina N-Aciltransferase/química
4.
ACS Omega ; 6(24): 15698-15707, 2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34179613

RESUMO

Domain classifications are a useful resource for computational analysis of the protein structure, but elements of their composition are often opaque to potential users. We perform a comparative analysis of our classification ECOD against the SCOPe, SCOP2, and CATH domain classifications with respect to their constituent domain boundaries and hierarchal organization. The coverage of these domain classifications with respect to ECOD and to the PDB was assessed by structure and by sequence. We also conducted domain pair analysis to determine broad differences in hierarchy between domains shared by ECOD and other classifications. Finally, we present domains from the major facilitator superfamily (MFS) of transporter proteins and provide evidence that supports their split into domains and for multiple conformations within these families. We find that the ECOD and CATH provide the most extensive structural coverage of the PDB. ECOD and SCOPe have the most consistent domain boundary conditions, whereas CATH and SCOP2 both differ significantly.

5.
Sci Rep ; 11(1): 7996, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846496

RESUMO

Bumble bees exhibit exceptional diversity in their segmental body coloration largely as a result of mimicry. In this study we sought to discover genes involved in this variation through studying a lab-generated mutant in bumble bee Bombus terrestris, in which the typical black coloration of the pleuron, scutellum, and first metasomal tergite is replaced by yellow, a color variant also found in sister lineages to B. terrestris. Utilizing a combination of RAD-Seq and whole-genome re-sequencing, we localized the color-generating variant to a single SNP in the protein-coding sequence of transcription factor cut. This mutation generates an amino acid change that modifies the conformation of a coiled-coil structure outside DNA-binding domains. We found that all sequenced Hymenoptera, including sister lineages, possess the non-mutant allele, indicating different mechanisms are involved in the same color transition in nature. Cut is important for multiple facets of development, yet this mutation generated no noticeable external phenotypic effects outside of setal characteristics. Reproductive capacity was reduced, however, as queens were less likely to mate and produce female offspring, exhibiting behavior similar to that of workers. Our research implicates a novel developmental player in pigmentation, and potentially caste, thus contributing to a better understanding of the evolution of diversity in both of these processes.


Assuntos
Abelhas/genética , Genoma de Inseto , Sequenciamento de Nucleotídeos em Larga Escala , Mutação/genética , Pigmentação/genética , Sequenciamento Completo do Genoma , Sequência de Aminoácidos , Animais , Sequência Conservada/genética , Genes de Insetos , Estudo de Associação Genômica Ampla , Proteínas de Insetos/química , Proteínas de Insetos/genética , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Domínios Proteicos
6.
mSystems ; 6(1)2021 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563785

RESUMO

Diverse bacterial pathogens employ effector delivery systems to disrupt vital cellular processes in the host (N. M. Alto and K. Orth, Cold Spring Harbor Perspect Biol 4:a006114, 2012, https://doi.org/10.1101/cshperspect.a006114). The type III secretion system 1 of the marine pathogen Vibrio parahaemolyticus utilizes the sequential action of four effectors to induce a rapid, proinflammatory cell death uniquely characterized by a prosurvival host transcriptional response (D. L. Burdette, M. L. Yarbrough, A Orvedahl, C. J. Gilpin, and K. Orth, Proc Natl Acad Sci USA 105:12497-12502, 2008, https://doi.org/10.1073/pnas.0802773105; N. J. De Nisco, M. Kanchwala, P. Li, J. Fernandez, C. Xing, and K. Orth, Sci Signal 10:eaa14501, 2017, https://doi.org/10.1126/scisignal.aal4501). Herein, we show that this prosurvival response is caused by the action of the channel-forming effector VopQ that targets the host V-ATPase, resulting in lysosomal deacidification and inhibition of lysosome-autophagosome fusion. Recent structural studies have shown how VopQ interacts with the V-ATPase and, while in the ER, a V-ATPase assembly intermediate can interact with VopQ, causing a disruption in membrane integrity. Additionally, we observed that VopQ-mediated disruption of the V-ATPase activates the IRE1 branch of the unfolded protein response (UPR), resulting in an IRE1-dependent activation of ERK1/2 MAPK signaling. We also find that this early VopQ-dependent induction of ERK1/2 phosphorylation is terminated by the VopS-mediated inhibitory AMPylation of Rho GTPase signaling. Since VopS dampens VopQ-induced IRE1-dependent ERK1/2 activation, we propose that IRE1 activates ERK1/2 phosphorylation at or above the level of Rho GTPases. This study illustrates how temporally induced effectors can work as in tandem as agonist/antagonist to manipulate host signaling and reveals new connections between V-ATPase function, UPR, and MAPK signaling.IMPORTANCE Vibrio parahaemolyticus is a seafood-borne pathogen that encodes two type 3 secretion systems (T3SS). The first system, T3SS1, is thought to be maintained in all strains of V. parahaemolyticus to maintain survival in the environment, whereas the second system, T3SS2, is linked to clinical isolates and disease in humans. Here, we found that first system targets evolutionarily conserved signaling systems to manipulate host cells, eventually causing a rapid, orchestrated cells death within 3 h. We have found that the T3SS1 injects virulence factors that temporally manipulate host signaling. Within the first hour of infection, the effector VopQ acts first by activating host survival signals while diminishing the host cell apoptotic machinery. Less than an hour later, another effector, VopS, reverses activation and inhibition of these signaling systems, ultimately leading to death of the host cell. This work provides example of how pathogens have evolved to manipulate the interplay between T3SS effectors to regulate host signaling pathways.

7.
J Mol Biol ; 433(4): 166788, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33387532

RESUMO

The Rossmann-like fold is the most prevalent and diversified doubly-wound superfold of ancient evolutionary origin. Rossmann-like domains are present in a variety of metabolic enzymes and are capable of binding diverse ligands. Discerning evolutionary relationships among these domains is challenging because of their diverse functions and ancient origin. We defined a minimal Rossmann-like structural motif (RLM), identified RLM-containing domains among known 3D structures (20%) and classified them according to their homologous relationships. New classifications were incorporated into our Evolutionary Classification of protein Domains (ECOD) database. We defined 156 homology groups (H-groups), which were further clustered into 123 possible homology groups (X-groups). Our analysis revealed that RLM-containing proteins constitute approximately 15% of the human proteome. We found that disease-causing mutations are more frequent within RLM domains than within non-RLM domains of these proteins, highlighting the importance of RLM-containing proteins for human health.


Assuntos
Motivos de Aminoácidos , Modelos Moleculares , Conformação Proteica , Proteínas/química , Sítios de Ligação , Evolução Biológica , Bases de Dados de Proteínas , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas/genética , Proteínas/metabolismo
8.
Mol Biol Evol ; 38(5): 2166-2176, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33502509

RESUMO

Centuries of zoological studies have amassed billions of specimens in collections worldwide. Genomics of these specimens promises to reinvigorate biodiversity research. However, because DNA degrades with age in historical specimens, it is a challenge to obtain genomic data for them and analyze degraded genomes. We developed experimental and computational protocols to overcome these challenges and applied our methods to resolve a series of long-standing controversies involving a group of butterflies. We deduced the geographical origins of several historical specimens of uncertain provenance that are at the heart of these debates. Here, genomics tackles one of the greatest problems in zoology: countless old specimens that serve as irreplaceable embodiments of species concepts cannot be confidently assigned to extant species or population due to the lack of diagnostic morphological features and clear documentation of the collection locality. The ability to determine where they were collected will resolve many on-going disputes. More broadly, we show the utility of applying genomics to historical museum specimens to delineate the boundaries of species and populations, and to hypothesize about genotypic determinants of phenotypic traits.


Assuntos
Borboletas/genética , DNA Antigo/análise , Genômica/métodos , Adaptação Biológica/genética , Altitude , Animais , Pigmentação/genética
9.
Nat Struct Mol Biol ; 27(12): 1194-1201, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33106659

RESUMO

De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.


Assuntos
Autofagossomos/química , Proteínas Relacionadas à Autofagia/química , Proteínas de Membrana/química , Fosfolipídeos/química , Proteolipídeos/química , Proteínas de Transporte Vesicular/química , Animais , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Sítios de Ligação , Transporte Biológico , Linhagem Celular , Microscopia Crioeletrônica , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Expressão Gênica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Fosfolipídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteolipídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
10.
Proteins ; 88(11): 1513-1527, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32543729

RESUMO

Protein domains exist by themselves or in combination with other domains to form complex multidomain proteins. Defining domain boundaries in proteins is essential for understanding their evolution and function but is not trivial. More specifically, partitioning domains that interact by forming a single ß-sheet is known to be particularly troublesome for automatic structure-based domain decomposition pipelines. Here, we study edge-to-edge ß-strand interactions between domains in a protein chain, to help define the boundaries for some more difficult cases where a single ß-sheet spanning over two domains gives an appearance of one. We give a number of examples where ß-strands belonging to a single ß-sheet do not belong to a single domain and highlight the difficulties of automatic domain parsers on these examples. This work can be used as a baseline for defining domain boundaries in homologous proteins or proteins with similar domain interactions in the future.


Assuntos
Isomerases de Aminoácido/química , Proteínas de Ligação às Penicilinas/química , Domínios e Motivos de Interação entre Proteínas , Racemases e Epimerases/química , Isomerases de Aminoácido/metabolismo , Sequência de Aminoácidos , Animais , Bactérias/química , Sítios de Ligação , Bases de Dados de Proteínas , Conjuntos de Dados como Assunto , Humanos , Modelos Moleculares , Proteínas de Ligação às Penicilinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Racemases e Epimerases/metabolismo , Termodinâmica
11.
PLoS Comput Biol ; 16(5): e1007775, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32413045

RESUMO

The human genome harbors a variety of genetic variations. Single-nucleotide changes that alter amino acids in protein-coding regions are one of the major causes of human phenotypic variation and diseases. These single-amino acid variations (SAVs) are routinely found in whole genome and exome sequencing. Evaluating the functional impact of such genomic alterations is crucial for diagnosis of genetic disorders. We developed DeepSAV, a deep-learning convolutional neural network to differentiate disease-causing and benign SAVs based on a variety of protein sequence, structural and functional properties. Our method outperforms most stand-alone programs, and the version incorporating population and gene-level information (DeepSAV+PG) has similar predictive power as some of the best available. We transformed DeepSAV scores of rare SAVs in the human population into a quantity termed "mutation severity measure" for each human protein-coding gene. It reflects a gene's tolerance to deleterious missense mutations and serves as a useful tool to study gene-disease associations. Genes implicated in cancer, autism, and viral interaction are found by this measure as intolerant to mutations, while genes associated with a number of other diseases are scored as tolerant. Among known disease-associated genes, those that are mutation-intolerant are likely to function in development and signal transduction pathways, while those that are mutation-tolerant tend to encode metabolic and mitochondrial proteins.


Assuntos
Doença/genética , Previsões/métodos , Genoma Humano/genética , Alelos , Sequência de Aminoácidos/genética , Biologia Computacional/métodos , Aprendizado Profundo , Redes Reguladoras de Genes/genética , Humanos , Mutação/genética , Mutação de Sentido Incorreto/genética , Rede Nervosa , Fases de Leitura Aberta/genética , Análise de Sequência/métodos , Sequenciamento Completo do Exoma/métodos
12.
J Lipid Res ; 61(5): 746-757, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32188638

RESUMO

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K2 subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Binding to UBIAD1 inhibits sterol-accelerated ER-associated degradation (ERAD) of reductase and permits continued synthesis of GGpp in cholesterol-replete cells. GGpp disrupts UBIAD1-reductase binding and thereby allows for maximal ERAD of reductase as well as ER-to-Golgi translocation of UBIAD1. SCD-associated UBIAD1 is refractory to GGpp-mediated dissociation from reductase and remains sequestered in the ER to inhibit ERAD. Here, we report development of a biochemical assay for UBIAD1-mediated synthesis of MK-4 in isolated membranes and intact cells. Using this assay, we compared enzymatic activity of WT UBIAD1 with that of SCD-associated variants. Our studies revealed that SCD-associated UBIAD1 exhibited reduced MK-4 synthetic activity, which may result from its reduced affinity for GGpp. Sequestration in the ER protects SCD-associated UBIAD1 from autophagy and allows intracellular accumulation of the mutant protein, which amplifies the inhibitory effect on reductase ERAD. These findings have important implications not only for the understanding of SCD etiology but also for the efficacy of cholesterol-lowering statin therapy, which becomes limited, in part, because of UBIAD1-mediated inhibition of reductase ERAD.


Assuntos
Autofagia/genética , Distrofias Hereditárias da Córnea/genética , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Variação Genética , Proteólise , Vitamina K 2/análogos & derivados , Linhagem Celular , Humanos , Espaço Intracelular/metabolismo , Transporte Proteico , Vitamina K 2/metabolismo
14.
Nat Chem Biol ; 16(3): 337-344, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932719

RESUMO

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu+ import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1, that is strongly induced during Cu limitation and which encodes a protein related to lytic polysaccharide monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient, and Bim1 function in Cu accumulation depends on Cu2+ coordination and cell-surface association via a glycophosphatidyl inositol anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis.


Assuntos
Cobre/metabolismo , Cryptococcus neoformans/metabolismo , Oxigenases de Função Mista/metabolismo , Animais , Criptococose/metabolismo , Cryptococcus neoformans/patogenicidade , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Meningite/metabolismo , Meningite/fisiopatologia , Camundongos , Camundongos Endogâmicos A , Polissacarídeos/metabolismo , Virulência
15.
Cancer Cell ; 37(2): 183-199.e5, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-31978326

RESUMO

We developed neratinib-resistant HER2-mutant cancer cells by gradual dose escalation. RNA sequencing identified TORC1 signaling as an actionable mechanism of drug resistance. Primary and acquired neratinib resistance in HER2-mutant breast cancer patient-derived xenografts (PDXs) was also associated with TORC1 hyperactivity. Genetic suppression of RAPTOR or RHEB ablated P-S6 and restored sensitivity to the tyrosine kinase inhibitor. The combination of the TORC1 inhibitor everolimus and neratinib potently arrested the growth of neratinib-resistant xenografts and organoids established from neratinib-resistant PDXs. RNA and whole-exome sequencing revealed RAS-mediated TORC1 activation in a subset of neratinib-resistant models. DNA sequencing of HER2-mutant tumors clinically refractory to neratinib, as well as circulating tumor DNA profiling of patients who progressed on neratinib, showed enrichment of genomic alterations that converge to activate the mTOR pathway.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Quinolinas/farmacologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/efeitos dos fármacos , Receptor ErbB-2/genética , Transdução de Sinais/efeitos dos fármacos
16.
Cell Chem Biol ; 27(1): 105-121.e14, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31883965

RESUMO

RUVBL1 and RUVBL2 (collectively RUVBL1/2) are essential AAA+ ATPases that function as co-chaperones and have been implicated in cancer. Here we investigated the molecular and phenotypic role of RUVBL1/2 ATPase activity in non-small cell lung cancer (NSCLC). We find that RUVBL1/2 are overexpressed in NSCLC patient tumors, with high expression associated with poor survival. Utilizing a specific inhibitor of RUVBL1/2 ATPase activity, we show that RUVBL1/2 ATPase activity is necessary for the maturation or dissociation of the PAQosome, a large RUVBL1/2-dependent multiprotein complex. We also show that RUVBL1/2 have roles in DNA replication, as inhibition of its ATPase activity can cause S-phase arrest, which culminates in cancer cell death via replication catastrophe. While in vivo pharmacological inhibition of RUVBL1/2 results in modest antitumor activity, it synergizes with radiation in NSCLC, but not normal cells, an attractive property for future preclinical development.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Replicação do DNA , Neoplasias Pulmonares/metabolismo , Complexos Multiproteicos/metabolismo , ATPases Associadas a Diversas Atividades Celulares/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/genética , Antineoplásicos/química , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Estrutura Molecular , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Tolerância a Radiação
17.
PLoS Comput Biol ; 15(12): e1007569, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31869345

RESUMO

Rossmann folds are ancient, frequently diverged domains found in many biological reaction pathways where they have adapted for different functions. Consequently, discernment and classification of their homologous relations and function can be complicated. We define a minimal Rossmann-like structure motif (RLM) that corresponds for the common core of known Rossmann domains and use this motif to identify all RLM domains in the Protein Data Bank (PDB), thus finding they constitute about 20% of all known 3D structures. The Evolutionary Classification of protein structure Domains (ECOD) classifies RLM domains in a number of groups that lack evidence for homology (X-groups), which suggests that they could have evolved independently multiple times. Closely related, homologous RLM enzyme families can diverge to bind different ligands using similar binding sites and to catalyze different reactions. Conversely, non-homologous RLM domains can converge to catalyze the same reactions or to bind the same ligand with alternate binding modes. We discuss a special case of such convergent evolution that is relevant to the polypharmacology paradigm, wherein the same drug (methotrexate) binds to multiple non-homologous RLM drug targets with different topologies. Finally, assigning proteins with RLM domain to the Enzyme Commission classification suggest that RLM enzymes function mainly in metabolism (and comprise 38% of reference metabolic pathways) and are overrepresented in extant pathways that represent ancient biosynthetic routes such as nucleotide metabolism, energy metabolism, and metabolism of amino acids. In fact, RLM enzymes take part in five out of eight enzymatic reactions of the Wood-Ljungdahl metabolic pathway thought to be used by the last universal common ancestor (LUCA). The prevalence of RLM domains in this ancient metabolism might explain their wide distribution among enzymes.


Assuntos
Evolução Molecular , Domínios Proteicos/genética , Sítios de Ligação/genética , Domínio Catalítico/genética , Biologia Computacional , Bases de Dados de Proteínas , Enzimas/química , Enzimas/genética , Enzimas/metabolismo , Humanos , Ligantes , Redes e Vias Metabólicas/genética , Modelos Moleculares , Ligação Proteica/genética , Software , Homologia Estrutural de Proteína
18.
JCI Insight ; 4(21)2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672938

RESUMO

X-linked reticulate pigmentary disorder (XLPDR, Mendelian Inheritance in Man #301220) is a rare syndrome characterized by recurrent infections and sterile multiorgan inflammation. The syndrome is caused by an intronic mutation in POLA1, the gene encoding the catalytic subunit of DNA polymerase-α (Pol-α), which is responsible for Okazaki fragment synthesis during DNA replication. Reduced POLA1 expression in this condition triggers spontaneous type I interferon expression, which can be linked to the autoinflammatory manifestations of the disease. However, the history of recurrent infections in this syndrome is as yet unexplained. Here we report that patients with XLPDR have reduced NK cell cytotoxic activity and decreased numbers of NK cells, particularly differentiated, stage V, cells (CD3-CD56dim). This phenotype is reminiscent of hypomorphic mutations in MCM4, which encodes a component of the minichromosome maintenance (MCM) helicase complex that is functionally linked to Pol-α during the DNA replication process. We find that POLA1 deficiency leads to MCM4 depletion and that both can impair NK cell natural cytotoxicity and show that this is due to a defect in lytic granule polarization. Altogether, our study provides mechanistic connections between Pol-α and the MCM complex and demonstrates their relevance in NK cell function.


Assuntos
Amiloidose Familiar/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Células Matadoras Naturais/imunologia , Transtornos da Pigmentação/imunologia , Dermatopatias Genéticas/imunologia , Amiloidose Familiar/genética , Citotoxicidade Imunológica , Reparo do DNA , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Células K562 , Componente 4 do Complexo de Manutenção de Minicromossomo/genética , Transtornos da Pigmentação/genética , Recombinação Genética , Dermatopatias Genéticas/genética
19.
Proteins ; 87(12): 1021-1036, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31294862

RESUMO

Protein target structures for the Critical Assessment of Structure Prediction round 13 (CASP13) were split into evaluation units (EUs) based on their structural domains, the domain organization of available templates, and the performance of servers on whole targets compared to split target domains. Eighty targets were split into 112 EUs. The EUs were classified into categories suitable for assessment of high accuracy modeling (or template-based modeling [TBM]) and topology (or free modeling [FM]) based on target difficulty. Assignment into assessment categories considered the following criteria: (a) the evolutionary relationship of target domains to existing fold space as defined by the Evolutionary Classification of Protein Domains (ECOD) database; (b) the clustering of target domains using eight objective sequence, structure, and performance measures; and (c) the placement of target domains in a scatter plot of target difficulty against server performance used in the previous CASP. Generally, target domains with good server predictions had close template homologs and were classified as TBM. Alternately, targets with poor server predictions represent a mixture of fast evolving homologs, structure analogs, and new folds, and were classified as FM or FM/TBM overlap.


Assuntos
Sequência de Aminoácidos/genética , Biologia Computacional , Estrutura Terciária de Proteína/genética , Proteínas/ultraestrutura , Bases de Dados de Proteínas , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Proteínas/genética , Alinhamento de Sequência
20.
Hum Mutat ; 40(9): 1463-1473, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31283071

RESUMO

This paper reports the evaluation of predictions for the "CALM1" challenge in the fifth round of the Critical Assessment of Genome Interpretation held in 2018. In the challenge, the participants were asked to predict effects on yeast growth caused by missense variants of human calmodulin, a highly conserved protein in eukaryotic cells sensing calcium concentration. The performance of predictors implementing different algorithms and methods is similar. Most predictors are able to identify the deleterious or tolerated variants with modest accuracy, with a baseline predictor based purely on sequence conservation slightly outperforming the submitted predictions. Nevertheless, we think that the accuracy of predictions remains far from satisfactory, and the field awaits substantial improvements. The most poorly predicted variants in this round surround functional CALM1 sites that bind calcium or peptide, which suggests that better incorporation of structural analysis may help improve predictions.


Assuntos
Calmodulina/química , Calmodulina/genética , Biologia Computacional/métodos , Mutação de Sentido Incorreto , Leveduras/crescimento & desenvolvimento , Algoritmos , Sítios de Ligação , Cálcio/metabolismo , Calmodulina/metabolismo , Evolução Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Aptidão Genética , Humanos , Modelos Genéticos , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas , Leveduras/genética
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