RESUMEN
Ubiquitination is a versatile posttranslational modification that elicits signaling roles to impact on various cellular processes and disease states. The versatility is a result of the complexity of ubiquitin conjugates, ranging from a single ubiquitin monomer to polymers with different length and linkage types. Recent studies have revealed the abundant existence of branched ubiquitin chains in which one ubiquitin molecule is connected to two or more ubiquitin moieties in the same ubiquitin polymer. Compared to the homotypic ubiquitin chain, the branched chain is recognized or processed differently by readers and erasers of the ubiquitin system, respectively, resulting in a qualitative or quantitative alteration of the functional output. Furthermore, certain types of branched ubiquitination are induced by cellular stresses, implicating their important physiological role in stress adaption. In addition, the current chemical methodologies of solid phase peptide synthesis and expanding genetic code approach have been developed to synthesize different architectures of branched ubiquitin chains. The synthesized branched ubiquitin chains have shown their significance in understanding the topologies and binding partners of the branched chains. Here, we discuss the recent progresses on the detection, functional characterization and synthesis of branched ubiquitin chains as well as the future perspectives of this emerging field.
Asunto(s)
Polímeros/química , Ubiquitina/química , Ubiquitinación , Animales , Humanos , Espectrometría de Masas , Péptidos/química , Fosforilación , Complejo de la Endopetidasa Proteasomal/química , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
Ubiquitination and SUMOylation of protein are crucial for various biological responses. The recent unraveling of cross-talk between SUMO and ubiquitin (Ub) has shown the pressing needs to develop the platform for the synthesis of Ub tagged SUMO2 dimers to decipher its biological functions. Still, the platforms for facile synthesis of dimers under native condition are less explored and remain major challenges. Here, we have developed the platform that can expeditiously synthesize all eight Ub tagged SUMO2 and SUMOylated proteins under native condition. Expanding genetic code (EGC) method was employed to incorporate Se-alkylselenocysteine at lysine positions. Oxidative selenoxide elimination generates the electrophilic center, dehydroalanine, which upon Michael addition with C-terminal modified ubiquitin, a nucleophile, yield Ub tagged SUMO2. The dimers were further interrogated with USP7, a SUMO2 deubiquitinase, which is involved in DNA repair, to understand specificity toward the Ub tagged SUMO2 dimer. Our results have shown that the C-terminal domain of USP7 is crucial for USP7 efficiency and selectivity for the Ub tagged SUMO2 dimer.
Asunto(s)
Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Peptidasa Específica de Ubiquitina 7/metabolismo , Ubiquitina/metabolismo , Humanos , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Especificidad por Sustrato , Sumoilación , Ubiquitina/química , Peptidasa Específica de Ubiquitina 7/química , UbiquitinaciónRESUMEN
Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA have been evolved to generate genetically encoded noncanonical amino acids (ncAAs). Use of tryptophan (Trp) analogues with pyrrole ring modification for their spatial and polarity tuning in enzyme activity and substrate specificity is still limited. Herein, we report the application of an evolved PylRS, FOWRS2, for efficient incorporation of five Trp analogues into the deubiquitinase USP30 to decipher the role of W475 for diubiquitin selectivity. Structures of the five FOWRS-C/Trp analogue complexes at 1.7-2.5 Å resolution showed multiple ncAA binding modes. The W475 near the USP30 active site was replaced with Trp analogues, and the effect on the activity as well as the selectivity toward diubiquitin linkage types was examined. It was found that the Trp analogue with a formyl group attached to the nitrogen atom of the indole ring led to an improved activity of USP30 likely due to enhanced polar interactions and that another Trp analogue, 3-benzothienyl-l-alanine, induced a unique K6-specificity. Collectively, genetically encoded noncanonical Trp analogues by evolved PylRS·tRNACUAPyl pair unravel the spatial role of USP30-W475 in its diubiquitin selectivity.
Asunto(s)
Proteínas Mitocondriales/química , Tioléster Hidrolasas/química , Triptófano/análogos & derivados , Triptófano/química , Aminoacil-ARNt Sintetasas/química , Proteínas Arqueales/química , Dominio Catalítico , Humanos , Methanosarcina/enzimología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/metabolismo , Triptófano/metabolismoRESUMEN
Microtubules regulate eukaryotic cell functions, which have tremendous implication in tumor progression. Thus, the design of novel approaches for controlling microtubule function is extremely important. In this manuscript, a novel tetrapeptide Ser-Leu-Arg-Pro (SLRP) has been designed and synthesized from a small peptide library consisting of 14 tetrapeptides, which perturbs microtubule function through interaction in the "anchor region". We have studied the role of peptides on microtubule function on a chemically functionalized 2D platform. Interestingly, we have found that SLRP binds with tubulin and inhibits the kinesin-driven microtubule motility on a kinesin-immobilized chemically functionalized 2D platform. Further, this peptide modulator interacts with intracellular tubulin/microtubule and depolymerizes the microtubule networks. These interesting findings of perturbation of microtubule function both on engineered platforms and inside the cell by this small peptide modulator inspired us to study the effect of this tetrapeptide on cancer cell proliferation. We found that the novel tetrapeptide modulator causes moderate cytotoxicity to the human breast cancer cell (MCF-7 cell), induces the apoptotic death of MCF-7 cell, and activates the tumor suppressor proteins p53 and cyclin-dependent kinase inhibitor 1 (p21). To the best of our knowledge, this is the shortest peptide discovered, which perturbs microtubule function both on an engineered 2D platform and inside the cell.
Asunto(s)
Diseño de Fármacos , Microtúbulos/metabolismo , Oligopéptidos/metabolismo , Tubulina (Proteína)/metabolismo , Apoptosis/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Simulación del Acoplamiento Molecular , Oligopéptidos/química , Oligopéptidos/farmacología , Unión Proteica , Conformación Proteica , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
An antimitotic cell penetrating octapeptide containing single Arg amino acid is discovered, which strongly binds with the exchangeable GTP/GDP binding site of tubulin, inhibits tubulin polymerization, reduces kinesin driven microtubule motility, activates apoptotic and mitotic check point proteins, induces apoptotic death and significantly inhibits the multicellular tumor spheroid growth of HeLa cells.
Asunto(s)
Antimitóticos/farmacología , Apoptosis/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Mitosis/efectos de los fármacos , Tubulina (Proteína)/química , Sitios de Unión , Genes cdc , Células HeLa , Humanos , Microtúbulos/efectos de los fármacos , PolimerizacionRESUMEN
Herein, we report a novel hexapeptide, derived from activity dependent neuroprotective protein (ADNP), that spontaneously self-assembles to form antiparallel ß-sheet structure and produces nanovesicles under physiological conditions. This peptide not only strongly binds with ß-tubulin in the taxol binding site but also binds with the microtubule lattice in vitro as well as in intracellular microtubule networks. Interestingly, it shows inhibition of amyloid fibril formation upon co-incubation with Aß peptide following an interesting mechanistic pathway and excellent neuroprotection in PC12 cells treated with anti-nerve growth factor (NGF). The potential of this hexapeptide opens up a new paradigm in design and development of novel therapeutics for AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Microtúbulos/metabolismo , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Fragmentos de Péptidos/metabolismo , Tubulina (Proteína)/metabolismo , Péptidos beta-Amiloides/química , Animales , Encéfalo , Supervivencia Celular , Dicroismo Circular , Evaluación Preclínica de Medicamentos , Cabras , Microscopía Confocal , Microscopía Electrónica de Transmisión , Microtúbulos/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fármacos Neuroprotectores/química , Oligopéptidos/química , Células PC12 , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Ratas , Espectroscopía Infrarroja por Transformada de Fourier , Tubulina (Proteína)/químicaRESUMEN
We report in this work that the Aß peptide directly interacts with tubulin close to the vinblastine and GTP/GDP binding site, inhibits the tubulin polymerization rate, induces tubulin aggregation, causes cell shrinking, enhances Mad2, BubR1, p53, and p21 activation in MCF7 cells and induces the apoptotic death of A549, HeLa and MCF7 cells.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Tubulina (Proteína)/metabolismo , Péptidos beta-Amiloides/química , Apoptosis , Sitios de Unión , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Células MCF-7 , Proteínas Mad2/metabolismo , Péptidos , Polimerizacion , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Tubulina (Proteína)/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
This communication describes the synthesis, structural investigation and tubulin binding of purine rare imino-tautomer based Ag(i) and Hg(ii)-carbene complexes. These complexes exhibit cytotoxicity through tubulin interaction by binding to a site close to the GTP binding site.
Asunto(s)
Complejos de Coordinación/farmacología , Mercurio/farmacología , Metano/análogos & derivados , Plata/farmacología , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/metabolismo , Adenina/análogos & derivados , Adenina/química , Línea Celular Tumoral , Supervivencia Celular , Complejos de Coordinación/química , Humanos , Mercurio/química , Metano/química , Metano/farmacología , Polimerizacion , Plata/química , Moduladores de Tubulina/químicaRESUMEN
An amyloid inhibitor octapeptide simultaneously forms amyloid type fibrous aggregates on its own and interacts with the microtubule lattice three times stronger than a Xenopus Microtubule Associated Protein (XMAP215).
Asunto(s)
Amiloide/antagonistas & inhibidores , Microtúbulos/fisiología , Secuencia de Aminoácidos , Movimiento Celular , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
A versatile method of dual chemical functionalization of graphene oxide (GO) with Tris-[nitrilotris(acetic acid)] (Tris-NTA) and biotin for cellular delivery of oligohistidine- and biotin-tagged biomolecules is reported. Orthogonally functionalized GO surfaces with Tris-NTA and biotin to obtain a dual-functionalized GO (DFGO) are prepared and characterized by various spectroscopic and microscopic techniques. Fluorescence microscopic images reveal that DFGO surfaces are capable of binding oligohistidine-tagged biomolecules/proteins and avidin/biotin-tagged biomolecules/proteins orthogonally. The DFGO nanoparticles are non-cytotoxic in nature and can deliver oligohistidine- and biotin-tagged biomolecules simultaneously into the cell.
Asunto(s)
Biotina/química , Portadores de Fármacos/síntesis química , Grafito/química , Histidina/química , Nanopartículas/química , Oligopéptidos/química , Avidina/química , Línea Celular Tumoral , Portadores de Fármacos/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Colorantes Fluorescentes , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Histidina/metabolismo , Humanos , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Ácido Nitrilotriacético/química , Oligopéptidos/metabolismo , Óxidos , Espectroscopía Infrarroja por Transformada de Fourier , Coloración y Etiquetado/métodos , Trometamina/análogos & derivados , Trometamina/químicaRESUMEN
MASK AND CAPTURE: Photodestruction of parts of a biotin-functionalised surface by shining UV light through a photomask produces a biotin micropattern. These micropatterns can selectively capture functional biotin-tagged biomolecules/proteins such as microtubules and molecular beacons.