Dependent Pathway
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2021 ◽  
Alonso J Pardal ◽  
Andrew J Bowman

Core histones package chromosomal DNA and regulate genomic transactions, with their import and deposition involving a dedicated repertoire of molecular chaperones. Histones H3 and H4 have been predominantly characterised as obligate heterodimers, however, recent findings have alluded to the existence of a significant pool of monomeric histone H3 in the nucleoplasm. Using a combination of in vitro and in vivo experiments, here we show that monomeric H3 and H4 use an Importin 5 (Imp5) dependent pathway for their nuclear import, distinct from Importin 4 (Imp4) previously described for H3-H4 dimers. Using mutants that disrupt the histone fold, we show monomeric H3 loses its interaction with Imp4, but retains interactions with Imp5 and the chaperone NASP. H4 monomeric mutants similarly bind Imp5 and not Imp4, however, they lose interaction with NASP, retaining their interaction with the HAT1-RBBP7 complex instead. In vitro experiments revealed that Imp5 and NASP are mutually exclusive in their binding, suggesting a facilitated hand-off mechanism. Furthermore, new H3 accumulates rapidly in a NASP-bound complex after nuclear translocation. NASP can assemble into three distinct co-chaperoning complexes, including a novel complex containing NASP, H3 and the putative ubiquitin ligase UBR7, a NASP-H3-H4-RBBP7 subcomplex and the previously characterised NASP-H3-H4-ASF1-HAT1-RBBP7 multi-chaperoning complex. Here we propose an alternative import pathway and folding mechanism for monomeric H3 and H4 that involves Imp5, rather than Imp4, and hands off to nuclear chaperones NASP, RBBP7 and HAT1.

2021 ◽  
Gerardo Acosta Garcia ◽  
Jean Philippe Vielle Calzada ◽  
Jesus Agustin Badillo-Corona ◽  

In the ovule of flowering plants, the establishment of the haploid generation occurs when a somatic cell differentiates into a Megaspore Mother Cell (MMC) and initiates meiosis. As most flowering plants, Arabidopsis thaliana undergoes a monosporic type of gametogenesis; three meiotically derived cells degenerate without further division, and a single one, the functional megaspore (FM), divides mitotically to form the female gametophyte. In Arabidopsis, the ARGONAUTE4 clade proteins are involved in the control of megasporogenesis. In particular, mutations in ARGONAUTE9 (AGO9) lead to the ectopic differentiation of gametic precursors that can give rise female gametophytes. However, the genetic basis and molecular mechanisms that control monosporic gametogenesis remain largely unknown. Here, we show that Arabidopsis plants carrying loss-of-function mutations in the AGO9-interacting miR822a give rise to extranumerary surviving megaspores that acquire a FM identity and divide without giving rise to differentiated female gametophytes. The overexpression of three miR822a target genes encoding Cysteine/Histidine-Rich C1 domain proteins (DC1) phenocopy mir822a plants. The miR822a targets are overexpressed in ago9 mutant ovules, confirming that miR822a acts through an AGO9-dependent pathway to negatively regulate DC1 domain proteins. Our results identify a new role of miRNAs in the most prevalent form of female gametogenesis in flowering plants

2021 ◽  
Michael D. Paxhia ◽  
Diana M. Downs

Microbial metabolism is often considered modular, but metabolic engineering studies have shown that transferring pathways, or modules, between organisms is not always straightforward. The Thi5-dependent pathway(s) for synthesis of the pyrimidine moiety of thiamine from Saccharomyces cerevisiae and Legionella pneumophila functioned differently when incorporated into the metabolic network of Salmonella enterica . Function of Thi5 from Saccharomyces cerevisiae ( Sc Thi5) required modification of the underlying metabolic network, while Lp Thi5 functioned with the native network. Here we probe the metabolic requirements for heterologous function of Sc Thi5 and report a strong genetic and physiological evidence for a connection between alpha-ketoglutarate (αKG) levels and Sc Thi5 function. The connection was built with two classes of genetic suppressors linked to metabolic flux or metabolite pool changes. Further, direct modulation of nitrogen assimilation through nutritional or genetic modification implicated αKG levels in Thi5 function. Exogenous pyridoxal similarly improved Sc Thi5 function in S. enterica . Finally, directly increasing αKG and PLP with supplementation improved function of both Sc Thi5 and relevant variants of Thi5 from Legionella pneumophila ( Lp Thi5). The data herein suggest structural differences between Sc Thi5 and Lp Thi5 impact their level of function in vivo and implicate αKG in supporting function of the Thi5 pathway when placed in the heterologous metabolic network of S. enterica . IMPORTANCE Thiamine biosynthesis is a model metabolic node that has been used to extend our understanding of metabolic network structure and individual enzyme function. The requirements for in vivo function of the Thi5-dependent pathway found in Legionella and yeast are poorly characterized. Here we suggest that αKG modulates function of the Thi5 pathway in S. enterica and provide evidence that structural variation between Sc Thi5 and Lp Thi5 contribute to their functional differences in a Salmonella enterica host.

2021 ◽  
Vol 13 (1) ◽  
Jaedong Lee ◽  
Jeehyun Kwag

Abstract Background Accumulation of amyloid beta oligomers (AβO) in Alzheimer’s disease (AD) impairs hippocampal long-term potentiation (LTP), leading to memory deficits. Thus, identifying the molecular targets of AβO involved in LTP inhibition is critical for developing therapeutics for AD. Endocannabinoid (eCB) synthesis and release, a process collectively called eCB mobilization by hippocampal CA1 pyramidal cells, is known to facilitate LTP induction. eCB can be mobilized either by postsynaptic depolarization in an intracellular Ca2+ concentration ([Ca2+]i)-dependent pathway or by group 1 metabotropic glutamate receptor (mGluR) activation in a phospholipase Cβ (PLCβ)-dependent pathway. Moreover, group 1 mGluR activation during postsynaptic depolarization, which is likely to occur in vivo during memory processing, can cause synergistic enhancement of eCB (S-eCB) mobilization in a PLCβ-dependent pathway. Although AβO has been shown to disrupt [Ca2+]i-dependent eCB mobilization, the effect of AβO on PLCβ-dependent S-eCB mobilization and its association with LTP and hippocampus-dependent memory impairments in AD is unknown. Methods We used in vitro whole-cell patch-clamp recordings and western blot analyses to investigate the effect of AβO on PLCβ protein levels, PLCβ-dependent S-eCB mobilization, and spike-timing-dependent potentiation (tLTP) in AβO-treated rat hippocampal slices in vitro. In addition, we assessed the relationship between PLCβ protein levels and hippocampus-dependent memory impairment by performing a contextual fear memory task in vivo in the 5XFAD mouse model of AD. Results We found that AβO treatment in rat hippocampal slices in vitro decreased hippocampal PLCβ1 protein levels and disrupted S-eCB mobilization, as measured by western blot analysis and in vitro whole-cell patch-clamp recordings. This consequently led to the impairment of NMDA receptor (NMDAR)-mediated tLTP at CA3-CA1 excitatory synapses in AβO-treated rat hippocampal slices in vitro. Application of the PLCβ activator, m-3M3FBS, in rat hippocampal slices reinstated PLCβ1 protein levels to fully restore S-eCB mobilization and NMDAR-mediated tLTP. In addition, direct hippocampal injection of m-3M3FBS in 5XFAD mice reinstated PLCβ1 protein levels to those observed in wild type control mice and fully restored hippocampus-dependent contextual fear memory in vivo in 5XFAD mice. Conclusion We suggest that these results might be the consequence of memory impairment in AD by disrupting S-eCB mobilization. Therefore, we propose that PLCβ-dependent S-eCB mobilization could provide a new therapeutic strategy for treating memory deficits in AD.

Phytomedicine ◽  
2021 ◽  
Vol 92 ◽  
pp. 153737
Zhu Zhu ◽  
Liao Cui ◽  
Jing Yang ◽  
Chi Teng Vong ◽  
Yuanjia Hu ◽  

2021 ◽  
Vol 8 ◽  
Penglong Wu ◽  
Mingqi Cai ◽  
Jinbao Liu ◽  
Xuejun Wang

Background: Catecholamine surges and resultant excessive β-adrenergic stimulation occur in a broad spectrum of diseases. Excessive β-adrenergic stimulation causes cardiomyocyte necrosis, but the underlying mechanism remains obscure. Necroptosis, a major form of regulated necrosis mediated by RIPK3-centered pathways, is implicated in heart failure; however, it remains unknown whether excessive β-adrenergic stimulation-induced cardiac injury involves necroptosis. Hence, we conducted the present study to address these critical gaps.Methods and Results: Two consecutive daily injections of isoproterenol (ISO; 85 mg/kg, s.c.) or saline were administered to adult mixed-sex mice. At 24 h after the second ISO injection, cardiac area with Evans blue dye (EBD) uptake and myocardial protein levels of CD45, RIPK1, Ser166-phosphorylated RIPK1, RIPK3, and Ser345-phosphorylated MLKL (p-MLKL) were significantly greater, while Ser321-phosphorylated RIPK1 was significantly lower, in the ISO-treated than in saline-treated wild-type (WT) mice. The ISO-induced increase of EBD uptake was markedly less in RIPK3−/− mice compared with WT mice (p = 0.016). Pretreatment with the RIPK1-selective inhibitor necrostatin-1 diminished ISO-induced increases in RIPK3 and p-MLKL in WT mice and significantly attenuated ISO-induced increases of EBD uptake in WT but not RIPK3−/− mice.Conclusions: A large proportion of cardiomyocyte necrosis induced by excessive β-adrenergic stimulation belongs to necroptosis and is mediated by a RIPK1–RIPK3-dependent pathway, identifying RIPK1 and RIPK3 as potential therapeutic targets for catecholamine surges.

2021 ◽  
Donata Molisso ◽  
Mariangela Coppola ◽  
Martina Buonanno ◽  
Ilaria Di Lelio ◽  
Simona Maria Monti ◽  

Systemin (Sys) is an octadecapeptide which, upon wounding, is released from the carboxy terminus of its precursor, prosystemin(ProSys) to promote plant defenses. Recent findings on the disordered structure of ProSysprompted us to investigate a putative biological role of the whole precursor deprived of Sys peptide. We produced transgenic tomato plants expressing a truncated ProSys gene in which the exon coding for Sys was removed and compared their defense response with that induced by the exogenous application of the recombinant deleted ProSys[ProSys(1-178)].By combining protein structure analyses, transcriptomic analysis, gene expression profiling and bioassays with different pests we demonstrate that the truncated ProSys, that does not induce the endogenous ProSys gene, promotes defense barriers in tomato plants through a hormone independent defense pathway, likely associated with the production of oligogalacturonides (OGs). Both transgenic and plants treated with the recombinant protein showed the modulation of the expression of genes linked with defense responses and resulted protected against the lepidopteran pest Spodoptera littoralis and the fungus Botrytis cinerea. Our results suggest that the overall function of the wild type prosystemin is more complex than previously shown as it might activate at least two tomato defense pathways: the well-known Sys-dependent pathway connected with the induction of JA biosynthesis and the successive activation of a set of defense-related genes and the ProSys(1-178)-dependent pathway associated with OGs production leading to the OGs mediate plant immunity.

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