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1.
Nat Commun ; 12(1): 4848, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381037

RESUMO

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.


Assuntos
Metiltransferases/química , RNA Helicases/química , SARS-CoV-2/química , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Conformação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
2.
Nucleic Acids Res ; 49(16): 9294-9309, 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34387694

RESUMO

The SNM1 nucleases which help maintain genome integrity are members of the metallo-ß-lactamase (MBL) structural superfamily. Their conserved MBL-ß-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5' phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres.

3.
Nucleic Acids Res ; 49(16): 9310-9326, 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34387696

RESUMO

Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-ß-lactamase (MBL) and ß-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its ß-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.

4.
Life Sci Alliance ; 4(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33199508

RESUMO

Loss of WRN, a DNA repair helicase, was identified as a strong vulnerability of microsatellite instable (MSI) cancers, making WRN a promising drug target. We show that ATP binding and hydrolysis are required for genome integrity and viability of MSI cancer cells. We report a 2.2-Å crystal structure of the WRN helicase core (517-1,093), comprising the two helicase subdomains and winged helix domain but not the HRDC domain or nuclease domains. The structure highlights unusual features. First, an atypical mode of nucleotide binding that results in unusual relative positioning of the two helicase subdomains. Second, an additional ß-hairpin in the second helicase subdomain and an unusual helical hairpin in the Zn2+ binding domain. Modelling of the WRN helicase in complex with DNA suggests roles for these features in the binding of alternative DNA structures. NMR analysis shows a weak interaction between the HRDC domain and the helicase core, indicating a possible biological role for this association. Together, this study will facilitate the structure-based development of inhibitors against WRN helicase.


Assuntos
Domínio Catalítico , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Helicase da Síndrome de Werner/química , Helicase da Síndrome de Werner/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/genética , Cristalização , DNA/metabolismo , Dano ao DNA/genética , Inativação Gênica , Células HCT116 , Humanos , Hidrólise , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transfecção , Zinco/metabolismo
5.
Methods Mol Biol ; 2199: 23-43, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33125643

RESUMO

Structural genomics groups have identified the need to generate multiple truncated versions of each target to improve their success in producing a well-expressed, soluble, and stable protein and one that crystallizes and diffracts to a sufficient resolution for structural determination. At the Structural Genomics Consortium, we opted for the ligation-independent cloning (LIC) method which provides the throughput we desire to produce and screen many proteins in a parallel process. Here, we describe our LIC protocol for generating constructs in 96-well format and provide a choice of vectors suitable for expressing proteins in both E. coli and the baculovirus expression vector system (BEVS).


Assuntos
Baculoviridae/genética , Clonagem Molecular , Escherichia coli , Expressão Gênica , Vetores Genéticos/genética , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
6.
Methods Mol Biol ; 2199: 45-66, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33125644

RESUMO

In Chapter 3 , we described the Structural Genomics Consortium (SGC) process for generating multiple constructs of truncated versions of each protein using LIC. In this chapter we provide a step-by-step procedure of our E. coli system for test expressing intracellular (soluble) proteins in a 96-well format that enables us to identify which proteins or truncated versions are expressed in a soluble and stable form suitable for structural studies. In addition, we detail the process for scaling up cultures for large-scale protein purification. This level of production is required to obtain sufficient quantities (i.e., milligram amounts) of protein for further characterization and/or structural studies (e.g., crystallization or cryo-EM experiments). Our standard process is purification by immobilized metal affinity chromatography (IMAC) using nickel resin followed by size exclusion chromatography (SEC), with additional procedures arising from the complexity of the protein itself.


Assuntos
Cromatografia de Afinidade , Cromatografia em Gel , Escherichia coli , Proteínas Recombinantes , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
7.
Methods Mol Biol ; 2199: 67-94, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33125645

RESUMO

This chapter describes the step-by-step methods employed by the Structural Genomics Consortium (SGC) for screening and producing proteins in the baculovirus expression vector system (BEVS). This eukaryotic expression system was selected and a screening process established in 2007 as a measure to tackle the more challenging kinase, RNA-DNA processing, and integral membrane protein families on our target list. Here, we discuss our platform for identifying soluble proteins from 3 mL of insect cell culture and describe the procedures involved in producing protein from liter-scale cultures.


Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Proteínas de Membrana , Animais , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera
8.
Essays Biochem ; 64(5): 819-830, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33095241

RESUMO

Helicases are enzymes that use the energy derived from ATP hydrolysis to catalyze the unwinding of DNA or RNA. The RecQ family of helicases is conserved through evolution from prokaryotes to higher eukaryotes and plays important roles in various DNA repair pathways, contributing to the maintenance of genome integrity. Despite their roles as general tumor suppressors, there is now considerable interest in exploiting RecQ helicases as synthetic lethal targets for the development of new cancer therapeutics. In this review, we summarize the latest developments in the structural and mechanistic study of RecQ helicases and discuss their roles in various DNA repair pathways. Finally, we consider the potential to exploit RecQ helicases as therapeutic targets and review the recent progress towards the development of small molecules targeting RecQ helicases as cancer therapeutics.


Assuntos
Reparo do DNA , Neoplasias/terapia , RecQ Helicases/metabolismo , Instabilidade Genômica , Humanos , Neoplasias/genética
9.
DNA Repair (Amst) ; 95: 102941, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32866775

RESUMO

Unrepaired, or misrepaired, DNA damage can contribute to the pathogenesis of a number of conditions, or disease states; thus, DNA damage repair pathways, and the proteins within them, are required for the safeguarding of the genome. Human SNM1A is a 5'-to-3' exonuclease that plays a role in multiple DNA damage repair processes. To date, most data suggest a role of SNM1A in primarily ICL repair: SNM1A deficient cells exhibit hypersensitivity to ICL-inducing agents (e.g. mitomycin C and cisplatin); and both in vivo and in vitro experiments demonstrate SNM1A and XPF-ERCC1 can function together in the 'unhooking' step of ICL repair. SNM1A further interacts with a number of other proteins that contribute to genome integrity outside canonical ICL repair (e.g. PCNA and CSB), and these may play a role in regulating SNM1As function, subcellular localisation, and post-translational modification state. These data also provide further insight into other DNA repair pathways to which SNM1A may contribute. This review aims to discuss all aspects of the exonuclease, SNM1A, and its contribution to DNA damage tolerance.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Adutos de DNA/metabolismo , Reparo do DNA , Exodesoxirribonucleases/metabolismo , Animais , Proteínas de Ciclo Celular/química , DNA/efeitos dos fármacos , DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/química , Humanos , Conformação Proteica
10.
Life Sci Alliance ; 3(7)2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32467316

RESUMO

The cohesin subunit STAG2 has emerged as a recurrently inactivated tumor suppressor in human cancers. Using candidate approaches, recent studies have revealed a synthetic lethal interaction between STAG2 and its paralog STAG1 To systematically probe genetic vulnerabilities in the absence of STAG2, we have performed genome-wide CRISPR screens in isogenic cell lines and identified STAG1 as the most prominent and selective dependency of STAG2-deficient cells. Using an inducible degron system, we show that chemical genetic degradation of STAG1 protein results in the loss of sister chromatid cohesion and rapid cell death in STAG2-deficient cells, while sparing STAG2-wild-type cells. Biochemical assays and X-ray crystallography identify STAG1 regions that interact with the RAD21 subunit of the cohesin complex. STAG1 mutations that abrogate this interaction selectively compromise the viability of STAG2-deficient cells. Our work highlights the degradation of STAG1 and inhibition of its interaction with RAD21 as promising therapeutic strategies. These findings lay the groundwork for the development of STAG1-directed small molecules to exploit synthetic lethality in STAG2-mutated tumors.

11.
ACS Med Chem Lett ; 11(3): 340-345, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32184967

RESUMO

Inhibitors based on a 3-acylaminoindazole scaffold were synthesized to yield potent dual AAK1/BMP2K inhibitors. Optimization furnished a small molecule chemical probe (SGC-AAK1-1, 25) that is potent and selective for AAK1/BMP2K over other NAK family members, demonstrates narrow activity in a kinome-wide screen, and is functionally active in cells. This inhibitor represents one of the best available small molecule tools to study the functions of AAK1 and BMP2K.

12.
J Biol Chem ; 295(10): 2948-2958, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31914405

RESUMO

Forkhead box N1 (FOXN1) is a member of the forkhead box family of transcription factors and plays an important role in thymic epithelial cell differentiation and development. FOXN1 mutations in humans and mice give rise to the "nude" phenotype, which is marked by athymia. FOXN1 belongs to a subset of the FOX family that recognizes an alternative forkhead-like (FHL) consensus sequence (GACGC) that is different from the more widely recognized forkhead (FKH) sequence RYAAAYA (where R is purine, and Y is pyrimidine). Here, we present the FOXN1 structure in complex with DNA containing an FHL motif at 1.6 Å resolution, in which the DNA sequence is recognized by a mixture of direct and water-mediated contacts provided by residues in an α-helix inserted in the DNA major groove (the recognition helix). Comparisons with the structure of other FOX family members revealed that the FKH and FHL DNA sequences are bound in two distinct modes, with partially different registers for the protein DNA contacts. We identified a single alternative rotamer within the recognition helix itself as an important determinant of DNA specificity and found protein sequence features in the recognition helix that could be used to predict the specificity of other FOX family members. Finally, we demonstrate that the C-terminal region of FOXN1 is required for high-affinity DNA binding and that FOXN1 has a significantly reduced affinity for DNA that contains 5'-methylcytosine, which may have implications for the role of FOXN1 in thymic involution.


Assuntos
DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , DNA/química , Metilação de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Humanos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência
13.
Sci Rep ; 9(1): 16452, 2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31712618

RESUMO

Calcium/Calmodulin-dependent Protein Kinase Kinase 2 (CAMKK2) acts as a signaling hub, receiving signals from various regulatory pathways and decoding them via phosphorylation of downstream protein kinases - such as AMPK (AMP-activated protein kinase) and CAMK types I and IV. CAMKK2 relevance is highlighted by its constitutive activity being implicated in several human pathologies. However, at present, there are no selective small-molecule inhibitors available for this protein kinase. Moreover, CAMKK2 and its closest human homolog, CAMKK1, are thought to have overlapping biological roles. Here we present six new co-structures of potent ligands bound to CAMKK2 identified from a library of commercially-available kinase inhibitors. Enzyme assays confirmed that most of these compounds are equipotent inhibitors of both human CAMKKs and isothermal titration calorimetry (ITC) revealed that binding to some of these molecules to CAMKK2 is enthalpy driven. We expect our results to advance current efforts to discover small molecule kinase inhibitors selective to each human CAMKK.


Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/química , Inibidores de Proteínas Quinases/química , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Descoberta de Drogas , Humanos , Ligantes , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Recombinantes , Relação Estrutura-Atividade
14.
Front Plant Sci ; 10: 1105, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31620147

RESUMO

Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s-known as stress/ABA-activated protein kinases (SAPKs). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2 box interacts with the kinase domain αC helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2 box and ABA box. Mutations in ScSAPK8 SnRK2 box aimed at perturbing its interaction with the protein kinase domain reduced protein kinase activity in vitro. On the other hand, mutations to ScSAPK ABA box did not impact protein kinase activity but did alter the protein autophosphorylation pattern. Taken together, our results demonstrate that both SnRK2 and ABA boxes might play a role in sugarcane SnRK2 function.

15.
Elife ; 82019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31612854

RESUMO

Antibodies are a key resource in biomedical research yet there are no community-accepted standards to rigorously characterize their quality. Here we develop a procedure to validate pre-existing antibodies. Human cell lines with high expression of a target, determined through a proteomics database, are modified with CRISPR/Cas9 to knockout (KO) the corresponding gene. Commercial antibodies against the target are purchased and tested by immunoblot comparing parental and KO. Validated antibodies are used to definitively identify the most highly expressing cell lines, new KOs are generated if needed, and the lines are screened by immunoprecipitation and immunofluorescence. Selected antibodies are used for more intensive procedures such as immunohistochemistry. The pipeline is easy to implement and scalable. Application to the major ALS disease gene C9ORF72 identified high-quality antibodies revealing C9ORF72 localization to phagosomes/lysosomes. Antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over previously reported C9ORF72 properties.


Assuntos
Esclerose Amiotrófica Lateral/diagnóstico , Anticorpos Monoclonais/química , Proteína C9orf72/genética , Demência Frontotemporal/diagnóstico , Imuno-Histoquímica/normas , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/imunologia , Esclerose Amiotrófica Lateral/metabolismo , Animais , Anticorpos Monoclonais/classificação , Anticorpos Monoclonais/imunologia , Biomarcadores/metabolismo , Proteína C9orf72/imunologia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Demência Frontotemporal/genética , Demência Frontotemporal/imunologia , Demência Frontotemporal/metabolismo , Edição de Genes , Expressão Gênica , Células HEK293 , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Fagossomos/genética , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Células RAW 264.7
16.
ACS Med Chem Lett ; 10(9): 1266-1271, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31531195

RESUMO

Vaccinia-related kinases 1 and 2 (VRK1 and VRK2) are human Ser/Thr protein kinases associated with increased cell division and neurological disorders. Nevertheless, the cellular functions of these proteins are not fully understood. Despite their therapeutic potential, there are no potent and specific inhibitors available for VRK1 or VRK2. We report here the discovery and elaboration of an aminopyridine scaffold as a basis for VRK1 and VRK2 inhibitors. The most potent compound for VRK1 (26) displayed an IC50 value of 150 nM and was fairly selective in a panel of 48 human kinases (selectivity score S(50%) of 0.04). Differences in compound binding mode and substituent preferences between the two VRKs were identified by the structure-activity relationship combined with the crystallographic analysis of key compounds. We expect our results to serve as a starting point for the design of more specific and potent inhibitors against each of the two VRKs.

17.
Biophys Rev ; : 533-534, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31250316
18.
Structure ; 27(8): 1316-1325.e6, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31204252

RESUMO

Ubiquitylation, the posttranslational linkage of ubiquitin moieties to lysines in target proteins, helps regulate a myriad of biological processes. Ubiquitin, and sometimes ubiquitin-homology domains, are recognized by ubiquitin-binding domains, including CUE domains. CUE domains are thus generally thought to function by mediating interactions with ubiquitylated proteins. The chromatin remodeler, SMARCAD1, interacts with KAP1, a transcriptional corepressor. The SMARCAD1-KAP1 interaction is direct and involves the first SMARCAD1 CUE domain (CUE1) and the RBCC domain of KAP1. Here, we present a structural model of the KAP1 RBCC-SMARCAD1 CUE1 complex based on X-ray crystallography. Remarkably, CUE1, a canonical CUE domain, recognizes a cluster of exposed hydrophobic and surrounding charged/amphipathic residues on KAP1, which are presented in the context of a coiled-coil domain, not in a structure resembling ubiquitin. Together, these data suggest that CUE domains may have a wider function than simply recognizing ubiquitin and the ubiquitin-fold.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Proteína 28 com Motivo Tripartido/química , Proteína 28 com Motivo Tripartido/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Ubiquitina/metabolismo
19.
Pathology ; 51(3): 274-280, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30853107

RESUMO

Genetic defects on 6-pyruvoyl-tetrahydropterin synthase (PTPS) are the most prevalent cause of hyperphenylalaninaemia not due to phenylalanine hydrolyase deficiency (phenylketonuria). PTPS catalyses the second step of tetrahydrobiopterin (BH4) cofactor biosynthesis, and its deficiency represents the most common form of BH4 deficiency. Untreated PTPS deficiency results in depletion of the neurotransmitters dopamine, catecholamine and serotonin causing neurological symptoms. We archived reported missense variants of the PTS gene. Common in silico algorithms were used to predict the effects of such variants, and substantial proportions (up to 19%) of the variants were falsely classified as benign or uncertain. We have determined the crystal structure of the human PTPS hexamer, allowing another level of interpretation to understand the potential deleterious consequences of the variants from a structural perspective. The in silico and structure approaches appear to be complimentary and may provide new insights that are not available from each alone. Information from the protein structure suggested that the variants affecting amino acid residues required for interaction between monomeric subunits of the PTPS hexamer were those misclassified as benign by in silico algorithms. Our findings illustrate the important utility of 3D protein structure in interpretation of variants and also current limitations of in silico prediction algorithms. However, software to analyse mutation in the perspective of 3D protein structure is far less readily available than other in silico prediction tools.


Assuntos
Mutação , Fenilcetonúrias/genética , Fósforo-Oxigênio Liases/deficiência , Fósforo-Oxigênio Liases/genética , Humanos , Fenilcetonúrias/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Conformação Proteica
20.
Cell Rep ; 26(1): 79-93.e8, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30605688

RESUMO

ß-Catenin-dependent WNT signal transduction governs development, tissue homeostasis, and a vast array of human diseases. Signal propagation through a WNT-Frizzled/LRP receptor complex requires proteins necessary for clathrin-mediated endocytosis (CME). Paradoxically, CME also negatively regulates WNT signaling through internalization and degradation of the receptor complex. Here, using a gain-of-function screen of the human kinome, we report that the AP2 associated kinase 1 (AAK1), a known CME enhancer, inhibits WNT signaling. Reciprocally, AAK1 genetic silencing or its pharmacological inhibition using a potent and selective inhibitor activates WNT signaling. Mechanistically, we show that AAK1 promotes clearance of LRP6 from the plasma membrane to suppress the WNT pathway. Time-course experiments support a transcription-uncoupled, WNT-driven negative feedback loop; prolonged WNT treatment drives AAK1-dependent phosphorylation of AP2M1, clathrin-coated pit maturation, and endocytosis of LRP6. We propose that, following WNT receptor activation, increased AAK1 function and CME limits WNT signaling longevity.


Assuntos
Clatrina/metabolismo , Endocitose/fisiologia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Retroalimentação Fisiológica , Células HEK293 , Humanos , Masculino , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores
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