Vliv elicitinů na změny proteomu tabáku

Title in English: Elicitins impact on the proteome of tobacco Abstract in English: Cryptogein is a proteinaceous elicitor secreted by an oomycete Phytophthora cryptogea that can induce resistance to P. parasitica in tobacco plants. On the basis of previous computer modeling, a series of cryptogein mutants was prepared with altered abilities to bind sterols, phospholipids or both. The sterol binding and phospholipid transfer activities corresponded to expectations based on the structural data reported previously. Induction of synthesis of reactive oxygen species (ROS) in tobacco cells suspension and proteomic analysis of intercellular fluid changes in tobacco leaves triggered by these mutant elicitins were not proportional to their ability to bind or transfer sterols and phospholipids. However, changes in the intercellular proteome corresponded to transcription levels of defense genes and resistance to P. parasitica and structure-prediction of mutants did not reveal any significant changes in protein structure. These results suggest, contrary to previous proposals, that the sterol-binding ability of cryptogein and its mutants, and the associated conformational change in the -loop, might not be principal factors in either ROS production or resistance induction. Nevertheless results support importance of...Název česky: Vliv elicitinů na změny proteomu tabáku Abstrakt česky: Kryptogein je proteinový elicitor sekretovaný oomycetou Phytophthora cryptogea. V rostlinách tabáku je schopen indukovat rezistenci vůči P. parasitica. Na základě dříve provedeného počítačového modelování byly připraveny mutantní formy kryptogeinu s alterovanou schopností vázat steroly, fosfolipidy či obojí, přičemž schopnost vazby sterolů a transferu fosfolipidů jsme ověřili i experimentálně. Úroveň indukce syntézy reaktivních forem kyslíku (ROS) v suspenzi tabákových buněk a proteomických změn v mezibuněčné tekutině listů tabáku vyvolaných těmito mutantními elicitiny nebyla úměrná jejich schopnostem vázat či transportovat steroly a fosfolipidy. Změny v intercelulárním proteomu však odpovídaly úrovním transkripce obranných genů a rezistence vůči P. parasitica, přičemž nebyly predikovány významné změny ve struktuře připravených mutantních proteinů. Naše výsledky nejsou ve shodě s dřívějšími předpoklady a naznačují, že sterol-vazebné schopnosti kryptogeinu a jeho mutantů a s nimi asociované změny konformace -smyčky nemusí být zásadními faktory řídícími produkci ROS či indukci rezistence. Výsledky nicméně podporují význam -smyčky při interakci elicitinu s vysoce afinitním vazebným místem na cytoplasmatické membráně buněk tabáku.Department of BiochemistryKatedra biochemieFaculty of SciencePřírodovědecká fakult

An armadillo-domain protein participates in a telomerase interaction network

Key messageArabidopsis and human ARM protein interact with telomerase. Deregulated mRNA levels of DNA repair and ribosomal protein genes in an Arabidopsis arm mutant suggest non-telomeric ARM function. The human homolog ARMC6 interacts with hTRF2.AbstractTelomerase maintains telomeres and has proposed non-telomeric functions. We previously identified interaction of the C- terminal domain of Arabidopsis telomerase reverse transcriptase (AtTERT) with an armadillo/β-catenin-like repeat (ARM) containing protein. Here we explore protein– protein interactions of the ARM protein, AtTERT domains, POT1a, TRF-like family and SMH family proteins, and the chromatin remodeling protein CHR19 using bimolecular fluorescence complementation (BiFC), yeast two-hybrid (Y2H) analysis, and co- immunoprecipitation. The ARM protein interacts with both the N- and C-terminal domains of AtTERT in different cellular compartments. ARM interacts with CHR19 and TRF-like I family proteins that also bind AtTERT directly or through interaction with POT1a. The putative human ARM homolog co-precipitates telomerase activity and interacts with hTRF2 protein in vitro. Analysis of Arabidopsis arm mutants shows no obvious changes in telomere length or telomerase activity, suggesting that ARM is not essential for telomere maintenance. The observed interactions with telomerase and Myb-like domain proteins (TRF-like family I) may therefore reflect possible non- telomeric functions. Transcript levels of several DNA repair and ribosomal genes are affected in arm mutants, and ARM, likely in association with other proteins, suppressed expression of XRCC3 and RPSAA promoter constructs in luciferase reporter assays. In conclusion, ARM can participate in non-telomeric functions of telomerase, and can also perform its own telomerase-independent functions

Retromer and TBC1D5 maintain late endosomal RAB7 domains to enable amino acid–induced mTORC1 signaling

Retromer is an evolutionarily conserved multiprotein complex that orchestrates the endocytic recycling of integral membrane proteins. Here, we demonstrate that retromer is also required to maintain lysosomal amino acid signaling through mTORC1 across species. Without retromer, amino acids no longer stimulate mTORC1 translocation to the lysosomal membrane, which leads to a loss of mTORC1 activity and increased induction of autophagy. Mechanistically, we show that its effect on mTORC1 activity is not linked to retromer’s role in the recycling of transmembrane proteins. Instead, retromer cooperates with the RAB7-GAP TBC1D5 to restrict late endosomal RAB7 into microdomains that are spatially separated from the amino acid– sensing domains. Upon loss of retromer, RAB7 expands into the ragulator-decorated amino acid–sensing domains and interferes with RAG-GTPase and mTORC1 recruitment. Depletion of retromer in Caenorhabditis elegans reduces mTORC1 signaling and extends the lifespan of the worms, confirming an evolutionarily conserved and unexpected role for retromer in the regulation of mTORC1 activity and longevity

Elicitins impact on the proteome of tobacco

Title in English: Elicitins impact on the proteome of tobacco Abstract in English: Cryptogein is a proteinaceous elicitor secreted by an oomycete Phytophthora cryptogea that can induce resistance to P. parasitica in tobacco plants. On the basis of previous computer modeling, a series of cryptogein mutants was prepared with altered abilities to bind sterols, phospholipids or both. The sterol binding and phospholipid transfer activities corresponded to expectations based on the structural data reported previously. Induction of synthesis of reactive oxygen species (ROS) in tobacco cells suspension and proteomic analysis of intercellular fluid changes in tobacco leaves triggered by these mutant elicitins were not proportional to their ability to bind or transfer sterols and phospholipids. However, changes in the intercellular proteome corresponded to transcription levels of defense genes and resistance to P. parasitica and structure-prediction of mutants did not reveal any significant changes in protein structure. These results suggest, contrary to previous proposals, that the sterol-binding ability of cryptogein and its mutants, and the associated conformational change in the -loop, might not be principal factors in either ROS production or resistance induction. Nevertheless results support importance of..

Telomerase Interaction Partners–Insight from Plants

Telomerase, an essential enzyme that maintains chromosome ends, is important for genome integrity and organism development. Various hypotheses have been proposed in human, ciliate and yeast systems to explain the coordination of telomerase holoenzyme assembly and the timing of telomerase performance at telomeres during DNA replication or repair. However, a general model is still unclear, especially pathways connecting telomerase with proposed non-telomeric functions. To strengthen our understanding of telomerase function during its intracellular life, we report on interactions of several groups of proteins with the Arabidopsis telomerase protein subunit (AtTERT) and/or a component of telomerase holoenzyme, POT1a protein. Among these are the nucleosome assembly proteins (NAP) and the minichromosome maintenance (MCM) system, which reveal new insights into the telomerase interaction network with links to telomere chromatin assembly and replication. A targeted investigation of 176 candidate proteins demonstrated numerous interactions with nucleolar, transport and ribosomal proteins, as well as molecular chaperones, shedding light on interactions during telomerase biogenesis. We further identified protein domains responsible for binding and analyzed the subcellular localization of these interactions. Moreover, additional interaction networks of NAP proteins and the DOMINO1 protein were identified. Our data support an image of functional telomerase contacts with multiprotein complexes including chromatin remodeling and cell differentiation pathways

RagD auto-activating mutations impair MiT/TFE activity in kidney tubulopathy and cardiomyopathy syndrome

: Heterozygous mutations in the gene encoding RagD GTPase were shown to cause a novel autosomal dominant condition characterized by kidney tubulopathy and cardiomyopathy. We previously demonstrated that RagD, and its paralogue RagC, mediate a non-canonical mTORC1 signaling pathway that inhibits the activity of TFEB and TFE3, transcription factors of the MiT/TFE family and master regulators of lysosomal biogenesis and autophagy. Here we show that RagD mutations causing kidney tubulopathy and cardiomyopathy are "auto- activating", even in the absence of Folliculin, the GAP responsible for RagC/D activation, and cause constitutive phosphorylation of TFEB and TFE3 by mTORC1, without affecting the phosphorylation of "canonical" mTORC1 substrates, such as S6K. By using HeLa and HK-2 cell lines, human induced pluripotent stem cell-derived cardiomyocytes and patient-derived primary fibroblasts, we show that RRAGD auto-activating mutations lead to inhibition of TFEB and TFE3 nuclear translocation and transcriptional activity, which impairs the response to lysosomal and mitochondrial injury. These data suggest that inhibition of MiT/TFE factors plays a key role in kidney tubulopathy and cardiomyopathy syndrome