Biology subjects

Fenske, R.

Publications and source records attributed to Fenske, R..

9 recordsLinked to original sources

bioRxiv2022

PRC2 direct transfer from G-quadruplex RNA to dsDNA: Implications for RNA-binding chromatin modifiers

The chromatin-modifying enzyme, Polycomb Repressive Complex 2 (PRC2), deposits the H3K27me3 epigenetic mark to negatively regulate expression at numerous target genes, and this activity has been implicated in embryonic development, cell differentiation, and various cancers. A biological role for RNA binding in regulating PRC2 histone methyltransferase activity is generally accepted, but the nature and mechanism of this relationship remains an area of active investigation. Notably, many in vitro studies demonstrate that RNA inhibits PRC2 activity on nucleosomes through mutually antagonistic binding, while some in vivo studies indicate that PRC2s RNA-binding activity is critical for facilitating its biological function(s). Here we use biochemical, biophysical, and computational approaches to interrogate PRC2s RNA and DNA binding kinetics. Our findings demonstrate that PRC2-polynucleotide dissociation rates are dependent on the concentration of free ligand, indicating the potential for direct transfer between ligands without a free-enzyme intermediate. Direct transfer explains the variation in dissociation kinetics reported previously, allows reconciliation of prior in vitro and in vivo studies, and expands the potential mechanisms of RNA-mediated PRC2 regulation. Moreover, simulations indicate that such a direct transfer mechanism could be obligatory for RNA to recruit proteins to chromatin. SignificanceStudies of PRC2 in vitro indicate that RNA inhibits its histone methyltransferase (HMTase) activity through mutually antagonistic binding with nucleosomes, but some in vivo studies paradoxically suggest that RNA binding is necessary to facilitate chromatin occupancy and HMTase activity. Our findings unveil a protein-intrinsic mechanism for directly exchanging RNA and DNA/nucleosome in PRC2s binding site(s), which reconciles these prior findings by allowing antagonistic or synergistic RNA-mediated regulation dependent on RNA-nucleosome proximity. Furthermore, there is an increasing awareness that multiple chromatin-associated proteins exhibit regulatory RNA binding activity, and our findings indicate this "direct transfer" mechanism may be generally required for such regulation.

biophysics↗

bioRxiv2022

RNA- and DNA-binding proteins generally exhibit direct transfer of polynucleotides: Implications for target site search

We previously demonstrated that the PRC2 chromatin-modifying enzyme exhibits the ability to directly transfer between RNA and DNA without a free-enzyme intermediate state. Simulations suggested that such a direct transfer mechanism may be generally necessary for RNA to recruit proteins to chromatin, but the prevalence of direct transfer capability is unknown. Herein, we used fluorescence polarization assays and observed direct transfer for several well-characterized nucleic acid-binding proteins: three-prime repair exonuclease 1 (TREX1), heterogeneous nuclear ribonucleoprotein U, Fem-3-binding factor 2, and MS2 bacteriophage coat protein. For TREX1, the direct transfer mechanism was additionally interrogated by single molecule assays, and the data suggest that direct transfer occurs through an unstable ternary intermediate with partially associated ligands. Generally, direct transfer could allow many DNA- and RNA-binding proteins to conduct a one-dimensional search for their target sites. Furthermore, presumably long-lived protein-polynucleotide complexes might instead be readily replaced by other protein-polynucleotide complexes in vivo. SignificanceClassically, the lifetime of a protein-ligand complex is presumed to be an intrinsic property, unaffected by competitor molecules in free solution. By contrast, a few oligomeric nucleic acid binding proteins have been observed to exchange competing ligands in their binding sites, and consequently their lifetimes decrease with competitor concentration. Our findings indicate that this "direct transfer" is a more general property of nucleic acid binding proteins. This suggests that many DNA- and RNA-binding proteins can reduce the dimensionality of their search for their target sites by intramolecular direct transfer to nucleosome DNA, instead of relying entirely on three-dimensional diffusion, and it suggests that their mean complex lifetimes in vivo can be regulated by the concentration of free ligand molecules.

biophysics↗

bioRxiv2021

Autophagy promotes photomorphogenesis during seedling development in Arabidopsis in carbon limiting conditions

Autophagy is a conserved catabolic process that plays an essential role under nutrient starvation condition and influences different developmental processes. We observed that seedlings of autophagy mutants (atg2, atg5, atg7, and atg9) germinated in the dark showed delayed chloroplast development following illumination. The delayed chloroplast development was characterized by a decrease in photosynthetic and chlorophyll biosynthetic proteins, lower chlorophyll content, reduced chloroplast size, and increased levels of proteins involved in lipid biosynthesis. Confirming the biological impact of these differences, photosynthetic performance was impaired in autophagy mutants 12h post illumination. We investigated if the delayed chloroplast development could be explained by lower lipid import to the chloroplast or lower triglyceride (TAG) turnover. We observed that the limitations in the chloroplast lipid import imposed by trigalactosyldiacylglycerol1 are unlikely to explain the delay in photomorphogenesis. However, we found that lower TAG mobility in the triacylglycerol lipase mutant sugardependent1 significantly affected photomorphogenesis. Moreover, we showed that lower levels of carbon resources exacerbated the delay in photomorphogenesis whereas higher levels of carbon resources had an opposite effect. This work provides evidence that autophagic process operate during de-etiolation in a manner that contributes to photomorphogenesis through increasing lipid turnover to physically or energetically sustain photomorphogenesis.

plant biology↗

bioRxiv2021

In vivo homopropargylglycine incorporation enables nascent protein tagging, isolation and characterisation from Arabidopsis thaliana

Determining which proteins are actively synthesised at a given point in time and extracting them for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables BONCAT (Bio-Orthogonal Non-Canonical Amino acid Tagging) of proteins being synthesised in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met-sites throughout protein AA sequences and correlation with independent studies of protein labelling with 15N verified the data. We provide evidence that HPG-based BONCAT tags nascent plant proteins more efficiently than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that AHAs induction of Met metabolism and greater inhibition of cell growth rate than HPG likely limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for determining which plant proteins are being synthesised at a given time point and enriches new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of PTMs in plants.

plant biology↗

bioRxiv2020

The Arabidopsis mitochondrial dicarboxylate carrier 2 maintains leaf metabolic homeostasis by uniting malate import and citrate export

Malate is the major substrate for respiratory oxidative phosphorylation in illuminated leaves. In the mitochondria malate is converted to citrate either for replenishing tricarboxylic acid (TCA) cycle with carbon, or to be exported as substrate for cytosolic biosynthetic pathways or for storage in the vacuole. In this study, we show that DIC2 functions as a mitochondrial malate/citrate carrier in vivo in Arabidopsis. DIC2 knockout (dic2-1) results in growth retardation that can only be restored by expressing DIC2 but not its closest homologs DIC1 or DIC3, indicating that their substrate preferences are not identical. Malate uptake by non-energised dic2-1 mitochondria is reduced but can be restored in fully energised mitochondria by altering fumarate and pyruvate/oxaloacetate transport. A reduced citrate export but an increased citrate accumulation in substrate-fed, energised dic2-1 mitochondria suggest that DIC2 facilitates the export of citrate from the matrix. Consistent with this, metabolic defects in response to a sudden dark shift or prolonged darkness could be observed in dic2-1 leaves, including altered malate, citrate and 2-oxoglutarate utilisation. There was no alteration in TCA cycle metabolite pools and NAD redox state at night; however, isotopic glucose tracing reveals a reduction in citrate labelling in dic2-1 which resulted in a diversion of flux towards glutamine, as well as the removal of excess malate via asparagine and threonine synthesis. Overall, these observations indicate that DIC2 is responsible in vivo for mitochondrial malate import and citrate export which coordinate carbon metabolism between the mitochondrial matrix and the other cell compartments. SIGNIFICANCE STATEMENTMitochondria are pivotal for plant metabolism. One of their central functions is to provide carbon intermediates for the synthesis of critical building blocks, such as amino acids. Malate import and citrate export are two of the most recognised and specialised features of the mitochondrial role in the plant cellular metabolic network, yet the possibility that a single carrier would unite both functions has not been considered. Here, we have demonstrated that DIC2 preferentially fulfils these two functions in Arabidopsis thaliana in vivo, making it a bifunctional gateway for two major metabolite fluxes into and out of the mitochondrial matrix in the plant cell. Our results highlight the significance of DIC2 in cooperation with other mitochondrial carriers in maintaining metabolic balance even under challenging environmental conditions.

plant biology↗

bioRxiv2020

Coordination and assembly of protein complexes encoded across mitochondrial and nuclear genomes is assisted by CLPP2 in Arabidopsis thaliana

Protein homeostasis in eukaryotic organelles and their progenitor prokaryotes is regulated by a series of proteases including the caseinolytic protease (CLPP). CLPP has essential roles in chloroplast biogenesis and maintenance, but the significance of the plant mitochondrial CLPP remains unknown and factors that aid coordination of nuclear and mitochondrial encoded subunits for complex assembly in mitochondria await discovery. We generated knock-out lines of the single gene for the mitochondrial CLP protease subunit, CLPP2, in Arabidopsis thaliana. Mutants had higher abundance of transcripts from mitochondrial genes encoding OXPHOS protein complexes, while transcripts for nuclear genes encoding other subunits of the same complexes showed no change in abundance. In contrast, the protein abundance of specific nuclear-encoded subunits in OXPHOS complexes I and V increased in CLPP2 knockouts, without accumulation of mitochondrial-encoded counterparts in the same complex. Protein complexes mainly or entirely encoded in the nucleus were unaffected. Analysis of protein import, assembly and function of Complex I revealed that while function was retained, protein homeostasis was disrupted through decreased assembly, leading to accumulation of soluble subcomplexes of nuclear-encoded subunits. Therefore, CLPP2 contributes to the mitochondrial protein degradation network through supporting coordination and assembly of protein complexes encoded across mitochondrial and nuclear genomes. One sentence summaryCLPP contributes to the mitochondrial protein degradation network through supporting coordination and assembly of protein complexes encoded across mitochondrial and nuclear genomes.

plant biology↗

bioRxiv2019

Differential expression of Ormdl genes in the islets of mice and humans with obesity

The orosomucoid-like proteins (Ormdl1-3) are emerging as the critical regulators of sphingolipid homeostasis, inflammation and ER stress. However, their roles in {beta}-cells and obesity remain unknown. Here, we showed that islets isolated from overweight/obese human donors displayed marginally reduced ORMDL1-2 expression while ORMDL3 expression was significantly reduced as compared to islets from lean donors. In contrast, Ormdl3 expression was significantly upregulated in the islets of leptin-deficient obese (ob/ob) mice compared to lean mice. We identified that the difference in expression of Ormdl3 between mouse and human islets was leptin-dependent, as treatment of ob/ob mice with leptin significantly reduced Ormld3 expression. Furthermore, Ormdl1-3 were significantly upregulated upon chemically-induced ER stress, but they showed differential responsiveness to cytokines in a {beta}-cell line. Knockdown of Ormdl3 substantially increased expression of apoptotic markers, which was rescued by a pharmacological inhibitor of ceramide synthase. Taken together we demonstrate leptin-dependent regulation of Ormdl3 expression in ob/ob islets, highlight the possible importance of {beta}-cell stress conditions in differential Ormdl expression and identify a critical role for Ormdl3 in {beta}-cell survival.

molecular biology↗

bioRxiv2019

A role for EP3 and its associated G protein, Gz, in negatively regulating beta-cell function and mass in the context of insulin resistance and obesity

When homozygous for the LeptinOb mutation (Ob), Black-and-Tan Brachyury (BTBR) mice become morbidly obese and severely insulin resistant, and by 10 weeks of age, frankly diabetic. Previous work has shown Prostaglandin EP3 Receptor (EP3) expression and activity is up-regulated in islets from BTBR-Ob mice as compared to lean controls, actively contributing to their beta-cell dysfunction. In this work, we aimed to test the impact of beta-cell-specific EP3 loss on the BTBR-Ob phenotype by crossing Ptger3 floxed mice with the Rat insulin promoter (RIP)-CreHerr driver strain. Instead, germline recombination of the floxed allele in the founder mouse - an event whose prevalence we identified as directly associated with underlying insulin resistance of the background strain - generated a full-body knockout. Full-body EP3 loss provided no diabetes protection to BTBR-Ob mice, but, unexpectedly, significantly worsened BTBR-lean insulin resistance and glucose tolerance. This in vivo phenotype was not associated with changes in beta-cell fractional area or markers of beta-cell replication ex vivo. Instead, EP3-null BTBR-lean islets had essentially uncontrolled insulin hypersecretion. The selective up-regulation of constitutively-active EP3 splice variants in islets from young, lean BTBR mice as compared to C57BL/6J, where no phenotype of EP3 loss has been observed, provides a potential explanation for the hypersecretion phenotype. In support of this, high islet EP3 expression in Balb/c females vs. Balb/c males was fully consistent with their sexually-dimorphic metabolic phenotype after loss of EP3-coupled Gz protein. Taken together, our findings provide a new dimension to the understanding of EP3 as a critical brake on insulin secretion. New and NoteworthyIslet Prostaglandin EP3 receptor (EP3) signaling is well-known as up-regulated in the pathophysiological conditions of type 2 diabetes, contributing to beta-cell dysfunction. Unexpected findings in mouse models of non-obese insulin sensitivity and resistance provide a new dimension to our understanding of EP3 as a key modulator of insulin secretion. A previously-unknown relationship between mouse insulin resistance and the penetrance of Rat insulin promoter-driven germline floxed allele recombination is critical to consider when creating beta-cell-specific knockouts. For Table of Contents Use Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/671289v3_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@15cf1d5org.highwire.dtl.DTLVardef@108f565org.highwire.dtl.DTLVardef@126e479org.highwire.dtl.DTLVardef@61fdf8_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2018

The macrocyclizing protease butelase 1 remains auto-catalytic and reveals the structural basis for ligase activity

Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform seed storage protein maturation upon cleavage dependent auto-activation in the low pH environment of storage vacuoles. AEPs have attracted attention for their macrocyclization reactions and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide binding region are the source of its efficient macrocyclization. All considered, it seems either butelase 1 has not fully specialized or a requirement for auto-catalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs.

biophysics↗