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1.
Mol Cell ; 79(2): 342-358.e12, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32645368

RESUMO

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.


Assuntos
Calcineurina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Biotinilação , Centrossomo/metabolismo , Simulação por Computador , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Monoéster Fosfórico Hidrolases/química , Fosforilação , Mapas de Interação de Proteínas , Proteoma/metabolismo , Receptor Notch1/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
2.
Biochemistry ; 62(11): 1594-1607, 2023 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-37224425

RESUMO

The ERM (ezrin, radixin, and moesin) family of proteins and the related protein merlin participate in scaffolding and signaling events at the cell cortex. The proteins share an N-terminal FERM [band four-point-one (4.1) ERM] domain composed of three subdomains (F1, F2, and F3) with binding sites for short linear peptide motifs. By screening the FERM domains of the ERMs and merlin against a phage library that displays peptides representing the intrinsically disordered regions of the human proteome, we identified a large number of novel ligands. We determined the affinities for the ERM and merlin FERM domains interacting with 18 peptides and validated interactions with full-length proteins through pull-down experiments. The majority of the peptides contained an apparent Yx[FILV] motif; others show alternative motifs. We defined distinct binding sites for two types of similar but distinct binding motifs (YxV and FYDF) using a combination of Rosetta FlexPepDock computational peptide docking protocols and mutational analysis. We provide a detailed molecular understanding of how the two types of peptides with distinct motifs bind to different sites on the moesin FERM phosphotyrosine binding-like subdomain and uncover interdependencies between the different types of ligands. The study expands the motif-based interactomes of the ERMs and merlin and suggests that the FERM domain acts as a switchable interaction hub.


Assuntos
Domínios FERM , Neurofibromina 2 , Humanos , Neurofibromina 2/genética , Neurofibromina 2/química , Neurofibromina 2/metabolismo , Ligantes , Estrutura Terciária de Proteína , Peptídeos
3.
Nucleic Acids Res ; 49(8): 4371-4385, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33744975

RESUMO

Higher-order chromatin structure undergoes striking changes in response to various developmental and environmental signals, causing distinct cell types to adopt specific chromatin organization. High throughput chromatin conformation capture (Hi-C) allows studying higher-order chromatin structure; however, this technique requires substantial amounts of starting material, which has limited the establishment of cell type-specific higher-order chromatin structure in plants. To overcome this limitation, we established a protocol that is applicable to a limited amount of nuclei by combining the INTACT (isolation of nuclei tagged in specific cell types) method and Hi-C (INT-Hi-C). Using this INT-Hi-C protocol, we generated Hi-C data from INTACT purified endosperm and leaf nuclei. Our INT-Hi-C data from leaf accurately reiterated chromatin interaction patterns derived from conventional leaf Hi-C data. We found that the higher-order chromatin organization of mixed leaf tissues and endosperm differs and that DNA methylation and repressive histone marks positively correlate with the chromatin compaction level. We furthermore found that self-looped interacting genes have increased expression in leaves and endosperm and that interacting intergenic regions negatively impact on gene expression in the endosperm. Last, we identified several imprinted genes involved in long-range and trans interactions exclusively in endosperm. Our study provides evidence that the endosperm adopts a distinct higher-order chromatin structure that differs from other cell types in plants and that chromatin interactions influence transcriptional activity.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/química , Montagem e Desmontagem da Cromatina , Cromatina/química , Endosperma/química , Imageamento Tridimensional/métodos , Análise de Célula Única/métodos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/química , Metilação de DNA , DNA de Plantas/química , Regulação da Expressão Gênica de Plantas , Impressão Genômica , Histonas/química , Folhas de Planta/química , Folhas de Planta/genética , Conformação Proteica
4.
Opt Lett ; 47(19): 5132-5135, 2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36181204

RESUMO

We demonstrate a spectrally correlated photon-pair source at telecom wavelengths (spanning across the S-, C-, and L-bands), based on type-0 spontaneous parametric downconversion (SPDC) in a fiber-coupled Zn-indiffused MgO doped periodically poled lithium niobate (PPLN) ridge waveguide. Modal analysis of the waveguide performed through numerical finite element method (FEM) simulation indicates that device temperature can be used to dramatically vary and control the emission spectrum. Efficient photon-pair generation is measured over a broad wavelength range from ∼1520 - 1580 nm [full width at half maximum (FWHM) > 45 nm] with a coincidence-to-accidental ratio (CAR) as high as ∼668 and spectral brightness ∼2.5 × 107 pairs/s/mW/nm. Such sources can be employed in wavelength division multiplexed (WDM) quantum key distribution (QKD) over existing fiber-optic networks.

5.
Plant Biotechnol J ; 15(9): 1163-1174, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28182326

RESUMO

Secondary cell wall (SCW) biosynthesis is an important stage of the cotton fibre development, and its transcriptional regulation is poorly understood. We selected the Gossypium hirsutum GDSL (GhGDSL) lipase/hydrolase gene (CotAD_74480), which is expressed during SCW biosynthesis (19 through to 25 days postanthesis; DPA), for study. T1 -transgenic cotton lines expressing the ß-glucuronidase (gus) reporter under the control of a 1026-bp promoter fragment of GhGDSL (PGhGDSL ) showed 19 DPA stage-specific increase in GUS expression. 5' deletion indicated that the 194-bp fragment between -788 and -594 relative to the transcription start site was essential for this stage-specific expression. Site-directed mutagenesis of eight transcription factor binding sites within PGhGDSL demonstrated that the MYB1AT motif (AAACCA) at -603/-598 was critical for the 19 DPA-specific reporter gene expressions. Yeast one-hybrid (Y1H) analysis identified nine proteins, including GhMYB1 (CotAD_64719) that bound to the PGhGDSL promoter. Further, Y1H experiments using the 5' promoter deletions and individually mutated promoter motifs indicated that GhMYB1 interacted with PGhGDSL at MYB1AT sequence. GhMYB1 was expressed specifically in fibre from 19 DPA, overlapping with the sharp rise in GhGDSL expression, indicating that it could regulate GhGDSL during fibre development. Analysis of genes co-expressed with GhMYB1 showed that it potentially regulates a number of other 19-25 DPA-specific genes in networks including those functioning in the cell wall and precursor synthesis, but not the major polysaccharide and protein components of the fibre SCW. GhGDSL and its promoter are therefore potential tools for the improvement of cotton fibre quality traits.


Assuntos
Fibra de Algodão , Regulação da Expressão Gênica de Plantas/genética , Gossypium/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Sítios de Ligação , Parede Celular/metabolismo , Glucuronidase , Gossypium/enzimologia , Gossypium/crescimento & desenvolvimento , Hidrolases/genética , Hidrolases/metabolismo , Lipase/genética , Lipase/metabolismo , Mutagênese Sítio-Dirigida , Fenótipo , Proteínas de Plantas/genética , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Int J Biol Macromol ; 263(Pt 2): 130455, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38417748

RESUMO

Rv1176c of Mycobacterium tuberculosis H37Rv belongs to the PadR-s1 subfamily of the PadR family of protein. Rv1176c forms a stable dimer in solution. Its stability is characterized by a thermal melting transition temperature (Tm) of 39.4 °C. The crystal structure of Rv1176c was determined at a resolution of 2.94 Å, with two monomers in the asymmetric unit. Each monomer has a characteristic N-terminal winged-helix-turn-helix DNA-binding domain. Rv1176c C-terminal is a coiled-coil dimerization domain formed of α-helices α5 to α7. In the Rv1176c dimer, there is domain-swapping of the C-terminal domain in comparison to other PadR homologs. In the dimer, there is a long inter-subunit tunnel in which different ligands can bind. Rv1176c was found to bind to the promoter region of its own gene with high specificity. M. smegmatis MC2 155 genome lacks homolog of Rv1176c. Therefore, it was used as a surrogate to characterize the functional role of Rv1176c. Expression of Rv1176c in M. smegmatis MC2 155 cells imparted enhanced tolerance towards oxidative stress. Rv1176c expressing M. smegmatis MC2 155 cells exhibited enhanced intracellular survival in J774A.1 murine macrophage cells. Overall, our studies demonstrate Rv1176c to be a PadR-s1 subfamily transcription factor that can moderate the effect of oxidative stress.


Assuntos
Mycobacterium tuberculosis , Animais , Camundongos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Cristalografia por Raios X , Fatores de Transcrição/genética
7.
Sci Rep ; 13(1): 15101, 2023 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-37699996

RESUMO

Over the past decade, long non-coding RNA (lncRNA), which lacks protein-coding potential, has emerged as an essential regulator of the genome. The present study examined 13,599 lncRNAs in Arabidopsis thaliana, 11,565 in Oryza sativa, and 32,397 in Zea mays for their characteristic features and explored the associated genomic and epigenomic features. We found lncRNAs were distributed throughout the chromosomes and the Helitron family of transposable elements (TEs) enriched, while the terminal inverted repeat depleted in lncRNA transcribing regions. Our analyses determined that lncRNA transcribing regions show rare or weak signals for most epigenetic marks except for H3K9me2 and cytosine methylation in all three plant species. LncRNAs showed preferential localization in the nucleus and cytoplasm; however, the distribution ratio in the cytoplasm and nucleus varies among the studied plant species. We identified several conserved endogenous target mimic sites in the lncRNAs among the studied plants. We found 233, 301, and 273 unique miRNAs, potentially targeting the lncRNAs of A. thaliana, O. sativa, and Z. mays, respectively. Our study has revealed that miRNAs, which interact with lncRNAs, target genes that are involved in a diverse array of biological and molecular processes. The miRNA-targeted lncRNAs displayed a strong affinity for several transcription factors, including ERF and BBR-BPC, mutually present in all three plants, advocating their conserved functions. Overall, the present study showed that plant lncRNAs exhibit conserved genomic and epigenomic characteristics and potentially govern the growth and development of plants.


Assuntos
Arabidopsis , MicroRNAs , RNA Longo não Codificante , RNA Longo não Codificante/genética , Citoplasma , Elementos de DNA Transponíveis/genética , MicroRNAs/genética
8.
Int J Biol Macromol ; 223(Pt B): 1693-1704, 2022 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-36257367

RESUMO

The circadian clock is regulated by signaling networks that enhance a plant's ability to coordinate internal events with the external environment. In this study, we examine the rhythmic expression of long non-coding RNAs (lncRNAs) using multiple transcriptomes of Arabidopsis thaliana in the diel light cycle and integrated this information to have a better understanding of the functions of lncRNAs in regulating the circadian clock. We identified 968, 1050, and 998 lncRNAs at 8 h light, 16 h light and 8 h dark conditions, respectively. Among these, 423, 486, and 417 lncRNAs were uniquely present at 8 h light, 16 h light, and 8 h dark, respectively, whereas 334 lncRNAs were common under the three conditions. The specificity of identified lncRNAs under different light conditions was verified using qRT-PCR. The identified lncRNAs were less GC-rich and expressed at a significantly lower level than the mRNAs of protein-coding genes. In addition, we identified enriched motifs in lncRNA transcribing regions that were associated with light-responsive genes (SORLREP and SORLIP), flower development (AGAMOUS), and circadian clock (CCA1) under all three light conditions. We identified 10 and 12 different lncRNAs targeting different miRNAs with perfect and interrupted complementarity (endogenous target mimic). These predicted lncRNA-interacting miRNAs govern the function of a set of genes involved in the developmental process, reproductive structure development, gene silencing and transcription regulation. We demonstrated that the lncRNA transcribing regions were enriched for epigenetic marks such as H3.3, H3K4me2, H3K4me3, H4K16ac, H3K36ac, H3K56ac and depleted for heterochromatic (H3K9me2 and H3K27me1) and repressive (H3K27me3) histone modifications. Further, we found that hypermethylated genomic regions negatively correlated with lncRNA transcribing regions. Overall, our study showed that lncRNAs expressed corresponding to the diel light cycle are implicated in regulating the circadian rhythm and governing the developmental stage-specific growth.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Relógios Circadianos , RNA Longo não Codificante , Arabidopsis/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Relógios Circadianos/genética , Ritmo Circadiano/genética
9.
Cell Chem Biol ; 29(2): 339-350.e10, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-34324879

RESUMO

There are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/SpyCatcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures.


Assuntos
Proteínas Luminescentes/química , Oxirredutases/química , Peptídeos/química , Células Cultivadas , Escherichia coli/citologia , Humanos , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Oxirredutases/metabolismo , Peptídeos/metabolismo , Conformação Proteica , Solubilidade
10.
Elife ; 112022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35924897

RESUMO

Protein phosphatase 2A (PP2A) holoenzymes target broad substrates by recognizing short motifs via regulatory subunits. PP2A methylesterase 1 (PME-1) is a cancer-promoting enzyme and undergoes methylesterase activation upon binding to the PP2A core enzyme. Here, we showed that PME-1 readily demethylates different families of PP2A holoenzymes and blocks substrate recognition in vitro. The high-resolution cryoelectron microscopy structure of a PP2A-B56 holoenzyme-PME-1 complex reveals that PME-1 disordered regions, including a substrate-mimicking motif, tether to the B56 regulatory subunit at remote sites. They occupy the holoenzyme substrate-binding groove and allow large structural shifts in both holoenzyme and PME-1 to enable multipartite contacts at structured cores to activate the methylesterase. B56 interface mutations selectively block PME-1 activity toward PP2A-B56 holoenzymes and affect the methylation of a fraction of total cellular PP2A. The B56 interface mutations allow us to uncover B56-specific PME-1 functions in p53 signaling. Our studies reveal multiple mechanisms of PME-1 in suppressing holoenzyme functions and versatile PME-1 activities derived from coupling substrate-mimicking motifs to dynamic structured cores.


Assuntos
Proteína Fosfatase 2 , Microscopia Crioeletrônica , Desmetilação , Holoenzimas/metabolismo , Metilação , Proteína Fosfatase 2/metabolismo
11.
J Sep Sci ; 34(9): 1076-83, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21404442

RESUMO

The seminal plasma comprises secretions from various accessory sex glands. During fertilization spermatozoa undergo complex sequences of precisely timed events that are regulated by the activation of different intracellular signaling pathways. The precision and efficacy of these pathways are often influenced by the assembly and interactions of multiprotein complexes, thereby directing the flow of regulatory information. Our knowledge about these protein complexes present in human seminal plasma (HuSP) is limited. Here we report the identification and characterization of a native high molecular weight zinc-binding multiprotein complex from HuSP by utilizing 2-DE followed by MS. Twenty-six proteins representing isoforms and/or fragments of 11 different proteins were found to be assembled in this complex. Prostate-specific antigen, zinc α2-glycoprotein, prostatic acid phosphatase, and prolactin inducible protein were the major proteins of this complex. Dynamic light scattering experiments revealed changes in aggregation pattern accompanied with deviation from physiological pH and in presence of SDS. However, no significant changes were observed in the presence of physiological ligands such as zinc and fructose. The present study will be useful and contribute to guide the future studies performed for elucidation of biological significance of this native complex in HuSP.


Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Sêmen/química , Zinco/química , Eletroforese em Gel Bidimensional , Humanos , Masculino , Espectrometria de Massas , Peso Molecular , Mapeamento de Peptídeos
12.
Front Cell Dev Biol ; 9: 642737, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33748134

RESUMO

Developmentally programmed formation of DNA double-strand breaks (DSBs) by Spo11 initiates a recombination mechanism that promotes synapsis and the subsequent segregation of homologous chromosomes during meiosis. Although DSBs are induced to high levels in meiosis, their formation and repair are tightly regulated to minimize potentially dangerous consequences for genomic integrity. In S. cerevisiae, nine proteins participate with Spo11 in DSB formation, but their molecular functions have been challenging to define. Here, we describe our current view of the mechanism of meiotic DSB formation based on recent advances in the characterization of the structure and function of DSB proteins and discuss regulatory pathways in the light of recent models.

13.
Front Genet ; 12: 799805, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35069698

RESUMO

Stresses have been known to cause various responses like cellular physiology, gene regulation, and genome remodeling in the organism to cope and survive. Here, we assessed the impact of stress conditions on the chromatin-interactome network of Arabidopsis thaliana. We identified thousands of chromatin interactions in native as well as in salicylic acid treatment and high temperature conditions in a genome-wide fashion. Our analysis revealed the definite pattern of chromatin interactions and stress conditions could modulate the dynamics of chromatin interactions. We found the heterochromatic region of the genome actively involved in the chromatin interactions. We further observed that the establishment or loss of interactions in response to stress does not result in the global change in the expression profile of interacting genes; however, interacting regions (genes) containing motifs for known TFs showed either lower expression or no difference than non-interacting genes. The present study also revealed that interactions preferred among the same epigenetic state (ES) suggest interactions clustered the same ES together in the 3D space of the nucleus. Our analysis showed that stress conditions affect the dynamics of chromatin interactions among the chromatin loci and these interaction networks govern the folding principle of chromatin by bringing together similar epigenetic marks.

14.
Genome Biol ; 20(1): 41, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30791924

RESUMO

BACKGROUND: Imprinted genes are epigenetically modified during gametogenesis and maintain the established epigenetic signatures after fertilization, causing parental-specific gene expression. RESULTS: In this study, we show that imprinted paternally expressed genes (PEGs) in the Arabidopsis endosperm are marked by an epigenetic signature of Polycomb Repressive Complex2 (PRC2)-mediated H3K27me3 together with heterochromatic H3K9me2 and CHG methylation, which specifically mark the silenced maternal alleles of PEGs. The co-occurrence of H3K27me3 and H3K9me2 on defined loci in the endosperm drastically differs from the strict separation of both pathways in vegetative tissues, revealing tissue-specific employment of repressive epigenetic pathways in plants. Based on the presence of this epigenetic signature on maternal alleles, we are able to predict known PEGs at high accuracy and identify several new PEGs that we confirm using INTACT-based transcriptomes generated in this study. CONCLUSIONS: The presence of the three repressive epigenetic marks, H3K27me3, H3K9me2, and CHG methylation on the maternal alleles in the endosperm serves as a specific epigenetic signature that allows prediction of genes with parental-specific gene expression. Our study reveals that there are substantially more PEGs than previously identified, indicating that paternal-specific gene expression is of higher functional relevance than currently estimated. The combined activity of PRC2-mediated H3K27me3 together with the heterochromatic H3K9me3 has also been reported to silence the maternal Xist locus in mammalian preimplantation embryos, suggesting convergent employment of both pathways during the evolution of genomic imprinting.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Impressão Genômica , Proteínas Repressoras/metabolismo , Arabidopsis/metabolismo , Endosperma/metabolismo , Complexo Repressor Polycomb 2
15.
Genome Biol ; 20(1): 182, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477159

RESUMO

Following publication of the original article [1], the authors reported that Additional file 4, "Table S5. Parent-of-origin RNAseq dataset of 4 DAP INTACT-purified endosperm of Col × Ler reciprocal crosses" had the following error.

16.
Sci Rep ; 8(1): 3620, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29483524

RESUMO

Cotton fiber is a specialized unicellular structure useful for the study of cellular differentiation and development. Heat shock proteins (HSPs) have been shown to be involved in various developmental processes. Microarray data analysis of five Gossypium hirsutum genotypes revealed high transcript levels of GhHSP90 and GhHSP70 genes at different stages of fiber development, indicating their importance in the process. Further, we identified 26 and 55 members of HSP90 and HSP70 gene families in G. hirsutum. The treatment of specific inhibitors novobiocin (Nov; HSP90) and pifithrin/2-phenylethynesulfonamide (Pif; HSP70) in in-vitro cultured ovules resulted in a fewer number of fiber initials and retardation in fiber elongation. The molecular chaperone assay using bacterially expressed recombinant GhHSP90-7 and GhHSP70-8 proteins further confirmed the specificity of inhibitors. HSP inhibition disturbs the H2O2 balance that leads to the generation of oxidative stress, which consequently results in autophagy in the epidermal layer of the cotton ovule. Transmission electron microscopy (TEM) of inhibitor-treated ovule also corroborates autophagosome formation along with disrupted mitochondrial cristae. The perturbations in transcript profile of HSP inhibited ovules show differential regulation of different stress and fiber development-related genes and pathways. Altogether, our results indicate that HSP90 and HSP70 families play a crucial role in cotton fiber differentiation and development by maintaining cellular homeostasis.


Assuntos
Gossypium/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Benzotiazóis/farmacologia , Fibra de Algodão , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Gossypium/efeitos dos fármacos , Novobiocina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Sulfonamidas/farmacologia , Tolueno/análogos & derivados , Tolueno/farmacologia
17.
FEBS J ; 284(3): 485-498, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28002650

RESUMO

The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide-binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 µm through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full-length proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PD-derived, SLiM-based PPIs. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Biblioteca de Peptídeos , Peptídeos/química , Proteoma/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Modelos Moleculares , Peptídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
18.
Cell Discov ; 3: 17027, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28884018

RESUMO

Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase; it forms diverse heterotrimeric holoenzymes that counteract kinase actions. Using a peptidome that tiles the disordered regions of the human proteome, we identified proteins containing [LMFI]xx[ILV]xEx motifs that serve as interaction sites for B'-family PP2A regulatory subunits and holoenzymes. The B'-binding motifs have important roles in substrate recognition and in competitive inhibition of substrate binding. With more than 100 novel ligands identified, we confirmed that the recently identified LxxIxEx B'α-binding motifs serve as common binding sites for B' subunits with minor variations, and that S/T phosphorylation or D/E residues at positions 2, 7, 8 and 9 of the motifs reinforce interactions. Hundreds of proteins in the human proteome harbor intrinsic or phosphorylation-responsive B'-interaction motifs, and localize at distinct cellular organelles, such as midbody, predicting kinase-facilitated recruitment of PP2A-B' holoenzymes for tight spatiotemporal control of phosphorylation at mitosis and cytokinesis. Moroever, Polo-like kinase 1-mediated phosphorylation of Cyk4/RACGAP1, a centralspindlin component at the midbody, facilitates binding of both RhoA guanine nucleotide exchange factor (epithelial cell transforming sequence 2 (Ect2)) and PP2A-B' that in turn dephosphorylates Cyk4 and disrupts Ect2 binding. This feedback signaling loop precisely controls RhoA activation and specifies a restricted region for cleavage furrow ingression. Our results provide a framework for further investigation of diverse signaling circuits formed by PP2A-B' holoenzymes in various cellular processes.

19.
J Biomol Struct Dyn ; 33(1): 147-57, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-24261636

RESUMO

SAP-1 is a 113 amino acid long single-chain protein which belongs to the type 2 cystatin gene family. In our previous study, we have purified SAP-1 from human seminal plasma and observed its cross-class inhibitory property. At this time, we report the interaction of SAP-1 with diverse proteases and its binding partners by CD-spectroscopic and molecular docking methods. The circular dichroism (CD) spectroscopic studies demonstrate that the conformation of SAP-1 is changed after its complexation with proteases, and the alterations in protein secondary structure are quantitatively calculated with increase of α-helices and reduction of ß-strand content. To get insight into the interactions between SAP-1 and proteases, we make an effort to model the three-dimensional structure of SAP-1 by molecular modeling and verify its stability and viability through molecular dynamics simulations and finally complexed with different proteases using ClusPro 2.0 Server. A high degree of shape complementarity is examined within the complexes, stabilized by a number of hydrogen bonds (HBs) and hydrophobic interactions. Using HB analyses in different protein complexes, we have identified a series of key residues that may be involved in the interactions between SAP-1 and proteases. These findings will assist to understand the mechanism of inhibition of SAP-1 for different proteases and provide intimation for further research.


Assuntos
Dicroísmo Circular/métodos , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Cistatinas Salivares/química , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Cistatinas Salivares/genética , Cistatinas Salivares/metabolismo , Homologia de Sequência de Aminoácidos
20.
Chem Biol Drug Des ; 85(3): 404-10, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25147059

RESUMO

SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (KD = 158 nm). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity.


Assuntos
Heparina/metabolismo , Serina Endopeptidases/metabolismo , Sítios de Ligação , Heparina/química , Humanos , Cinética , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Serina Endopeptidases/química , Serina Endopeptidases/isolamento & purificação , Ressonância de Plasmônio de Superfície
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