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1.
Nat Commun ; 14(1): 1576, 2023 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-36949076

RESUMO

Trypanosoma brucei is a protozoan parasite that causes human African trypanosomiasis. Its major surface antigen VSG is expressed from subtelomeric loci in a strictly monoallelic manner. We previously showed that the telomere protein TbRAP1 binds dsDNA through its 737RKRRR741 patch to silence VSGs globally. How TbRAP1 permits expression of the single active VSG is unknown. Through NMR structural analysis, we unexpectedly identify an RNA Recognition Motif (RRM) in TbRAP1, which is unprecedented for RAP1 homologs. Assisted by the 737RKRRR741 patch, TbRAP1 RRM recognizes consensus sequences of VSG 3'UTRs in vitro and binds the active VSG RNA in vivo. Mutating conserved RRM residues abolishes the RNA binding activity, significantly decreases the active VSG RNA level, and derepresses silent VSGs. The competition between TbRAP1's RNA and dsDNA binding activities suggests a VSG monoallelic expression mechanism in which the active VSG's abundant RNA antagonizes TbRAP1's silencing effect, thereby sustaining its full-level expression.


Assuntos
Trypanosoma brucei brucei , Tripanossomíase Africana , Animais , Humanos , Glicoproteínas Variantes de Superfície de Trypanosoma/genética , Motivo de Reconhecimento de RNA , Trypanosoma brucei brucei/metabolismo , RNA/genética , RNA/metabolismo
2.
Elife ; 82019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31486770

RESUMO

WW domain tandem-containing proteins such as KIBRA, YAP, and MAGI play critical roles in cell growth and polarity via binding to and positioning target proteins in specific subcellular regions. An immense disparity exists between promiscuity of WW domain-mediated target bindings and specific roles of WW domain proteins in cell growth regulation. Here, we discovered that WW domain tandems of KIBRA and MAGI, but not YAP, bind to specific target proteins with extremely high affinity and exquisite sequence specificity. Via systematic structural biology and biochemistry approaches, we decoded the target binding rules of WW domain tandems from cell growth regulatory proteins and uncovered a list of previously unknown WW tandem binding proteins including ß-Dystroglycan, JCAD, and PTPN21. The WW tandem-mediated target recognition mechanisms elucidated here can guide functional studies of WW domain proteins in cell growth and polarity as well as in other cellular processes including neuronal synaptic signaling.


Assuntos
Polaridade Celular , Proliferação de Células , Transporte Proteico , Proteínas/metabolismo , Domínios WW , Animais , Encéfalo/crescimento & desenvolvimento , Camundongos , Ligação Proteica , Mapas de Interação de Proteínas , Proteínas/química
3.
Neuron ; 104(3): 529-543.e6, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492534

RESUMO

Transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs) modulate AMPAR synaptic trafficking and transmission via disc-large (DLG) subfamily of membrane-associated guanylate kinases (MAGUKs). Despite extensive studies, the molecular mechanism governing specific TARP/MAGUK interaction remains elusive. Using stargazin and PSD-95 as the representatives, we discover that the entire tail of stargazin (Stg_CT) is required for binding to PSD-95. The PDZ binding motif (PBM) and an Arg-rich motif upstream of PBM conserved in TARPs bind to multiple sites on PSD-95, thus resulting in a highly specific and multivalent stargazin/PSD-95 complex. Stargazin in complex with PSD-95 or PSD-95-assembled postsynaptic complexes form highly concentrated and dynamic condensates via phase separation, reminiscent of stargazin/PSD-95-mediated AMPAR synaptic clustering and trapping. Importantly, charge neutralization mutations in TARP_CT Arg-rich motif weakened TARP's condensation with PSD-95 and impaired TARP-mediated AMPAR synaptic transmission in mice hippocampal neurons. The TARP_CT/PSD-95 interaction mode may have implications for understanding clustering of other synaptic transmembrane proteins.


Assuntos
Canais de Cálcio/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Transmissão Sináptica , Animais , Guanilato Quinases/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Densidade Pós-Sináptica/metabolismo , Transporte Proteico
4.
Cell Rep ; 26(8): 2064-2077.e7, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30784589

RESUMO

Kibra is a synaptic scaffold protein regulating learning and memory. Alterations of Kibra-encoding gene WWC1 cause various neuronal disorders, including Alzheimer's disease and Tourette syndrome. However, the molecular mechanism underlying Kibra's function in neurons is poorly understood. Here we discover that Kibra, via its N-terminal WW12 tandem domains, binds to a postsynaptic density enriched protein, Dendrin, with a nanomolar dissociation constant. On the basis of the structure of Kibra WW12 in complex with Dendrin PY motifs, we developed a potent peptide inhibitor capable of specifically blocking the binding between Kibra and Dendrin in neurons. Systematic administration of the inhibitory peptide attenuated excitatory synaptic transmission, completely blocked long-term potentiation induction, and impaired spatial learning and memory. A Kibra mutation found in Tourette syndrome patients causes defects in binding to Dendrin. Thus, Kibra can modulate spatial learning and memory via binding to Dendrin.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/química , Fosfoproteínas/genética , Densidade Pós-Sináptica/metabolismo , Ligação Proteica , Aprendizagem Espacial
5.
J Am Soc Nephrol ; 29(9): 2362-2371, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30006415

RESUMO

BACKGROUND: The slit diaphragm is a specialized adhesion junction between opposing podocytes, establishing the final filtration barrier that prevents passage of proteins from the capillary lumen into the urinary space. Nephrin, the key structural and signaling adhesion molecule expressed in the slit diaphragm, contains an evolutionally conserved, atypical PDZ-binding motif (PBM) reported to bind to a variety of proteins in the slit diaphragm. Several mutations in NPHS1 (the gene encoding nephrin) that result in nephrin lacking an intact PBM are associated with glomerular diseases. However, the molecular basis of nephrin-PBM-mediated protein complexes is still unclear. METHODS: Using a combination of biochemic, biophysic, and cell biologic approaches, we systematically investigated the interactions between nephrin-PBM and PDZ domain-containing proteins in the slit diaphragm. RESULTS: We found that nephrin-PBM specifically binds to one member of the membrane-associated guanylate kinase family of scaffolding proteins, MAGI1, but not to another, MAGI2. The complex structure of MAGI1-PDZ3/nephrin-PBM reveals that the Gly at the -3 position of nephrin-PBM is the determining feature for MAGI1-PDZ3 recognition, which sharply contrasts with the typical PDZ/PBM binding mode. A single gain-of-function mutation within MAGI2 enabled nephrin-PBM binding. In addition, using our structural analysis, we developed a highly efficient inhibitory peptide capable of specifically blocking the nephrin/MAGI1 interaction. CONCLUSIONS: MAGI1 interacts with nephrin-PBM with exquisite specificity. A newly developed, potent inhibitory peptide that blocks this interaction may be useful for future functional investigations in vivo. Our findings also provide possible explanations for the diseases caused by NPHS1 mutations.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas de Membrana/metabolismo , Síndrome Nefrótica/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Transporte/genética , Moléculas de Adesão Celular , Comunicação Celular , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas do Citoesqueleto , Guanilato Quinases/metabolismo , Humanos , Glomérulos Renais/metabolismo , Podócitos/metabolismo , Pesquisa Qualitativa , Sensibilidade e Especificidade , Transdução de Sinais/genética
6.
Bioconjug Chem ; 28(8): 2199-2205, 2017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28700219

RESUMO

A short, flexible, and unstructured peptide tag that has versatile and facile use in protein labeling applications is highly desirable. Here, we report an 11-residue peptide tag with an internal cysteine (a W-tag, derived from a Comm PY peptide motif that is known to bind with Nedd4 WW3* domain) that can be installed at different regions of the target protein without compromising its covalent reactivity with the reactive label (a 35-residue synthetic Nedd4 WW3* domain derivative). This versatility is explained by the unique structural features of the reaction. NMR analysis reveals that both the W-tag peptide and reactive Nedd4 WW3* protein are unstructured before they encounter each other. The binding interaction of the two induces noticeable structural changes and promotes global folding. Consequently, the reactive cysteine residue at W-tag and the electrophilic chloroacetyl group at Nedd4 WW3* domain are positioned to be in close proximity, inducing an intermolecular covalent cross-linking. The covalent linkage in turn stabilizes the folding of the protein complex. This unique multistep mechanism renders this labeling reaction amenable to different sites of the proteins of interest: installation of the tag at N- and C-termini, in the flexible linker region, in the loop region, and the extracellular terminus of target proteins exhibited comparable reactivity. This work therefore represents the first proximity-induced cysteine reaction based on the unique binding features of WW domains that demonstrates unprecedented versatility.


Assuntos
Oligopeptídeos/química , Oligopeptídeos/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Cisteína/química , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato
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