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1.
Plant Physiol ; 180(2): 1081-1100, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30890662

RESUMEN

Induction of heat shock proteins (HSPs) in response to heat stress (HS) is indispensable for conferring thermotolerance. Glc, a fundamental signaling and metabolic molecule, provides energy to stressed seedlings to combat stress. The recovery of stressed plants from detrimental HS in response to Glc is largely mediated by HSPs, but the mechanistic basis of this thermotolerance is not well defined. In this study, we show that Glc has a prominent role in providing thermotolerance. Glc-mediated thermotolerance involves HSP induction via the TARGET OF RAPAMYCIN (TOR)-E2Fa signaling module. Apart from HSPs, TOR-E2Fa also regulates the Arabidopsis (Arabidopsis thaliana) ortholog of human Hikeshi, named HIKESHI-LIKE PROTEIN1 (HLP1). Expression of proHLP1::GUS in the shoot apical meristem (SAM) after HS coincides with TOR-E2Fa expression, substantiating a role for TOR-E2Fa-HLP1 in providing thermotolerance. We also demonstrate that Glc along with heat could induce proliferation activity in the SAM after HS recovery, which was arrested by the TOR inhibitor AZD-8055. Molecular and physiological studies suggest that HS-activated heat stress transcription factor A1s also positively regulate HLP1 transcription, suggesting convergence of the Glc and HS signaling pathways. Loss of functional HLP1 causes HS hypersensitivity, whereas HLP1 overexpressors display increased thermotolerance. HLP1 binds to the promoters of Glc-regulated HS-responsive genes and promotes chromatin acetylation. In addition, Glc modifies the chromatin landscape at thermomemory-related loci by promoting H3K4 trimethylation (H3K4me3). Glc-primed accumulation of H3K4me3 at thermomemory-associated loci is mediated through HLP1. These findings reveal the novel function of Glc-regulated HLP1 in mediating thermotolerance/thermomemory response.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Glucosa/farmacología , Proteínas de Unión al ARN/metabolismo , Temperatura , Acetilación , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proliferación Celular/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Sitios Genéticos , Respuesta al Choque Térmico/efectos de los fármacos , Histonas/metabolismo , Mutación con Pérdida de Función , Meristema/efectos de los fármacos , Meristema/crecimiento & desarrollo , Desarrollo de la Planta/efectos de los fármacos , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacos
2.
J Biol Chem ; 293(34): 13134-13150, 2018 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-29945970

RESUMEN

The SNF1-related protein kinase 1 (SnRK1) is a heterotrimeric eukaryotic kinase that interacts with diverse proteins and regulates their activity in response to starvation and stress signals. Recently, the FCS-like zinc finger (FLZ) proteins were identified as a potential scaffold for SnRK1 in plants. However, the evolutionary and mechanistic aspect of this complex formation is currently unknown. Here, in silico analyses predicted that FLZ proteins possess conserved intrinsically disordered regions (IDRs) with a propensity for protein binding in the N and C termini across the plant lineage. We observed that the Arabidopsis FLZ proteins promiscuously interact with SnRK1 subunits, which formed different isoenzyme complexes. The FLZ domain was essential for mediating the interaction with SnRK1α subunits, whereas the IDRs in the N termini facilitated interactions with the ß and ßγ subunits of SnRK1. Furthermore, the IDRs in the N termini were important for mediating dimerization of different FLZ proteins. Of note, the interaction of FLZ with SnRK1 was confined to cytoplasmic foci, which colocalized with the endoplasmic reticulum. An evolutionary analysis revealed that in general, the IDR-rich regions are under more relaxed selection than the FLZ domain. In summary, the findings in our study reveal the structural details, origin, and evolution of a land plant-specific scaffold of SnRK1 formed by the coordinated actions of IDRs and structured regions in the FLZ proteins. We propose that the FLZ protein complex might be involved in providing flexibility, thus enhancing the binding repertoire of the SnRK1 hub in land plants.


Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Genoma de Planta , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Fosforilación , Filogenia , Unión Proteica , Conformación Proteica , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/genética , Dedos de Zinc
3.
Plant J ; 94(2): 232-245, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29406622

RESUMEN

SNF1-related protein kinase 1 (SnRK1) is a central regulator of plant growth during energy starvation. The FCS-like zinc finger (FLZ) proteins have recently been identified as adaptor proteins which facilitate the interaction of SnRK1 with other proteins. In this study, we found that two starvation-induced FLZ genes, FLZ6 and FLZ10, work as repressors of SnRK1 signalling. The reduced expression of these genes resulted in an increase in the level of SnRK1α1, which is the major catalytic subunit of SnRK1. This lead to a concomitant increase in phosphorylated protein and SnRK1 activity in the flz6 and flz10 mutants. FLZ6 and FLZ10 specifically interact with SnRK1α subunits in the cytoplasmic foci, which co-localized with the endoplasmic reticulum. In physiological assays, similar to the SnRK1α1 overexpression line, flz mutants showed compromised growth. Further, growth promotion in response to favourable growth conditions was found to be attenuated in the mutants. The enhanced SnRK1 activity in the mutants resulted in a reduction in the level of phosphorylated RIBOSOMAL S6 KINASE and the expression of E2Fa and its targets, indicating that TARGET OF RAPAMYCIN-dependent promotion of protein synthesis and cell cycle progression is impaired. Taken together, this study uncovers a plant-specific modulation of SnRK1 signalling.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Arabidopsis/fisiología , Proteínas de Unión al ADN/fisiología , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica de las Plantas , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal , Factores de Transcripción/fisiología
4.
Virus Genes ; 45(3): 488-98, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22872567

RESUMEN

Buffalopox virus (BPXV), a close variant of vaccinia virus (VACV) has emerged as a zoonotic pathogen. The host tropism of poxviruses is governed by host-range genes. Among the host-range genes: E3L, K3L, and C7L are essential for virus replication by preventing interferon resistance, whereas B5R is essential for spread of the virus and evasion from the host's immune response as in VACV. We report sequence analysis of host-range genes: E3L, K3L, C7L, and membrane protein gene (B5R) of BPXVs from buffalo, cattle, and human from recent outbreaks in India-their phylogenetic relationship with reference strain (BP4) and other Orthopoxviruses. BPXVs revealed a sequence homology with VACVs including zoonotic Brazilian VACV-like viruses. The aa sequences of E3L and K3L genes were 100 % similar in buffalo, cattle, and human isolates. However, four significant point mutations (I11K; N12K and S36F in C7L gene and D249G in B5R gene) were observed specific to buffalo isolate only. This signifies that different strains of BPXV were circulated during the outbreak. The mutations in C7L and B5R could play an important role in adaptation of BPXV in human and cattle which needs further functional studies. The strain of BPXV isolated from buffalo may not be adopted in human and cow. Various point mutations were observed in the host-range genes of reference strain (BPXV-BP4) which may be due to several passages of virus in cell culture. The phylogeny constructed based on concatenated gene sequences revealed that BPXVs are not as closely related to vaccine strain (Lister and Lister-derived strain-LC16m8), as hypothesized earlier, rather they are more closely related to reference strain (BPXV-BP4) and other vaccinia and vaccinia-like viruses such as Passatempo and Aracatuba viruses. The availability of information regarding host tropism determinants would allow us to understand molecular mechanism of species tropism of poxviruses which would be useful in unveiling new strategies to control zoonotic poxviral infections.


Asunto(s)
Especificidad del Huésped , Filogenia , Virus Vaccinia/aislamiento & purificación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Búfalos/virología , Bovinos/virología , Chlorocebus aethiops , ADN Viral/genética , Brotes de Enfermedades/veterinaria , Genes Virales , Humanos , India , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mutación Puntual , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Análisis de Secuencia de Proteína , Homología de Secuencia de Ácido Nucleico , Pase Seriado , Vaccinia/veterinaria , Vaccinia/virología , Virus Vaccinia/genética , Virus Vaccinia/fisiología , Células Vero , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral
5.
Plant Signal Behav ; 14(6): 1592535, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30871406

RESUMEN

The TARGET OF RAPAMYCIN-SNF1-RELATED PROTEIN KINASE 1 (TOR-SnRK1) arms race is a key regulator of plant growth in response to energy fluctuations and stress. Recently, we have identified that two members of the FCS-LIKE ZINC FINGER (FLZ) protein family, FLZ6 and 10, repress SnRK1 signaling and thereby involved in the activation of the TARGET OF RAPAMYCIN (TOR) signaling. In this study, we demonstrate that FLZ6 and 10 are also involved in the regulation of osmotic stress responses. Downregulation of FLZ6 and 10 results in enhanced expression of stress-responsive genes and better resilience towards osmotic stress at the seedling stage. These results indicate that FLZ6 and 10 are involved in the regulation of stress mitigation in plants through directly affecting SnRK1 signaling.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Osmorregulación/fisiología , Presión Osmótica/fisiología , Dedos de Zinc , Proteínas Adaptadoras Transductoras de Señales/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Mutación/genética
6.
Virology ; 519: 99-105, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29684630

RESUMEN

HIV-1 virus entry into target cells requires the envelope glycoprotein (Env) to first bind the primary receptor, CD4 and subsequently the co-receptor. Antibody access to the co-receptor binding site (CoRbs) in the pre-receptor-engaged state, prior to cell attachment, remains poorly understood. Here, we have demonstrated that for tier-1 Envs, the CoRbs is directly accessible to full-length CD4-induced (CD4i) antibodies even before primary receptor engagement, indicating that on these Envs the CoRbs site is either preformed or can conformationally sample post-CD4-bound state. Tier-2 and tier-3 Envs, which are resistant to full-length CD4i antibody, are neutralized by m36.4, a lower molecular mass of CD4i-directed domain antibody. In some tier-2 and tier-3 Envs, CoRbs is accessible to m36.4 even prior to cellular attachment in an Env-specific manner independent of their tier category. These data suggest differential structural arrangements of CoRbs and varied masking of ligand access to the CoRbs in different Env isolates.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Antígenos CD4/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , VIH-1/fisiología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Sitios de Unión , Antígenos CD4/metabolismo , Epítopos/inmunología , VIH-1/metabolismo , Humanos , Ligandos , Pruebas de Neutralización , Unión Proteica , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo
7.
Virology ; 510: 22-28, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28689085

RESUMEN

Efficient cleavage of HIV-1 Env gp160 into its constituent subunits correlates with selective binding to neutralizing antibodies and are the closest mimetic of native, functional Envs. This was first demonstrated with the clade B Env, JRFL. The correlation between efficient cleavage and selective binding to neutralizing antibodies is the guiding principle for immunogen design for HIV vaccine. We have recently reported that Envs 4-2.J41 (clade C) and JRCSF (clade B) are also efficiently cleaved and show similar properties. However, an efficiently cleaved, membrane-bound clade A Env suitable for genetic vaccination has not been directly demonstrated. Here we report that BG505 and a new clade A Env, QB726.70M.ENV.C4 (or A5) are efficiently cleaved on cell membrane. A5 shows desirable antigenic properties comparable with BG505 on cell surface. A5SOSIP in supernatant displays majority of bNAb binding epitopes. Thus, both BG505 and A5 Envs can be used in DNA prime-protein boost vaccination studies.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/inmunología , Antígenos VIH/metabolismo , Proteolisis , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Membrana Celular/metabolismo , Epítopos/inmunología , Unión Proteica
8.
PLoS One ; 10(3): e0122443, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25822521

RESUMEN

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Especificidad de Anticuerpos , VIH-1/inmunología , Proteolisis , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Vacunas contra el SIDA/inmunología , Antígenos CD4/química , Antígenos CD4/farmacología , Codón/genética , Epítopos/inmunología , Células HEK293 , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Mutación , Conformación Proteica , Solubilidad , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética
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