Biology subjects

Holton, N.

Publications and source records attributed to Holton, N..

5 recordsLinked to original sources

bioRxiv2024

Novel interactions within the silent information regulator heterochromatin complex potentiate inter-subunit communication and gene repression

Organisms with smaller genomes often perform multiple functions using one multi-subunit protein complex. The S. cerevisiae Silent Information Regulator complex (SIRc) carries out all of the core functions of heterochromatin. SIR complexes first drive the initiation and spreading of histone deacetylation in an iterative manner. Subsequently, the same complexes are incorporated stably with nucleosomes, driving compaction and repression of the underlying chromatin domain. These two distinct functions of SIRc have each been characterized in much detail, but the mechanism by which the dynamic spreading state switches to stable compaction is not well-understood. This incomplete knowledge of potential intra-complex communication is partly due to a lack of structural information of the complex as a whole; only structures of fragments have been determined to date. Using cross-linking mass spectrometry in solution, we identified a novel inter-subunit interaction that physically connects the two states of SIRc. The Sir2 deacetylase makes direct interactions with the scaffolding subunit Sir4 through its coiled-coil domain, which also interacts with the Sir3 compaction/repression subunit. Within the hub of interactions are conserved residues in Sir2 that can sense deacetylation state, as well as amino acids that likely diverged and co-evolved to interact with Sir4, promoting species-specific functions. Mutation of this interaction hub disrupts heterochromatic repression, potentially by disrupting a conserved mechanism that communicates completion of deacetylation to switch to compaction. Our work highlights how a single multi-functional chromatin regulatory complex can stage a step-wise mechanism that requires a major transition in activities to achieve epigenetic gene repression.

molecular biology↗

bioRxiv2023

A GH81-type β-glucan-binding protein facilitates colonization by mutualistic fungi in barley

Cell walls are important interfaces of plant-fungal interactions. Host cell walls act as robust physical and chemical barriers against fungal invaders, making them an essential line of defense. Upon fungal colonization, plants deposit phenolics and callose at the sites of fungal penetration to reinforce their walls and prevent further fungal progression. Alterations in the composition of plant cell walls significantly impact host susceptibility. Furthermore, plants and fungi secrete glycan hydrolases acting on each others cell walls. These enzymes release a wide range of sugar oligomers into the apoplast, some of which trigger the activation of host immunity via host surface receptors. Recent characterization of cell walls from plant-colonizing fungi have emphasized the abundance of {beta}-glucans in different cell wall layers, which makes them suitable targets for recognition. To characterize host components involved in immunity against fungi, we performed a protein pull-down with the biotinylated {beta}-glucan laminarin. Thereby, we identified a glycoside hydrolase family 81-type glucan-binding protein (GBP) as the major {beta}-glucan interactor. Mutation of GBP1 and its only paralogue GBP2 in barley led to decreased colonization by the beneficial root endophytes Serendipita indica and S. vermifera, as well as the arbuscular mycorrhizal fungus Rhizophagus irregularis. The reduction of symbiotic colonization was accompanied by enhanced responses at the host cell wall. Moreover, GBP mutation in barley also increased resistance to fungal infections in roots and leaves by the hemibiotrophic pathogen Bipolaris sorokiniana and the obligate biotrophic pathogen Blumeria graminis f. sp. hordei, respectively. These results indicate that GBP1 is involved in the establishment of symbiotic associations with beneficial fungi, a role that has potentially been appropriated by barley-adapted pathogens. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/536646v1_figu1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@c47957org.highwire.dtl.DTLVardef@fa6727org.highwire.dtl.DTLVardef@18a54d2org.highwire.dtl.DTLVardef@c6b103_HPS_FORMAT_FIGEXP M_FIG C_FIG In BriefGBP1, a family 81 glycoside hydrolase, is an important {beta}-glucan interactor in barley. Mutation of GBP1 and its sole paralogue GBP2 leads to reduced colonization by beneficial root endophytes, AM fungi and pathogens, accompanied by enhanced responses at the plant cell wall. This indicates that GBP1 and {beta}-glucans are compatibility factors involved in the establishment of symbiotic associations with beneficial fungi, a role possibly hijacked by pathogens.

plant biology↗

bioRxiv2021

An unusual aspartic acid cluster in the reovirus attachment fiber σ1 mediates stability at low pH

The reovirus attachment protein {sigma}1 mediates cell attachment and receptor binding and is thought to undergo conformational changes during viral disassembly. {sigma}1 is a trimeric filamentous protein with an -helical coil-coiled Tail, a triple {beta}-spiral Body, and a globular Head. The Head domain features an unusual and conserved aspartic acid cluster at the trimer interface, which forms the only significant intra-trimer interactions in the Head, and must be protonated to allow trimer formation. Here we show that all domains of {sigma}1 are remarkably thermostable across a wide range of pH, even at the low pH of the stomach. Interestingly, we determine the optimal pH for stability to be between pH 5-6, a value close to the pH of the endosome and of the jejunum. The {sigma}1 Head is stable at acidic and neutral pH, but detrimerizes at basic pH. When Asp345 in the aspartic acid cluster is mutated to asparagine, the {sigma}1 Head loses stability at low pH and is more prone to detrimerize. Overall, the presence of the Body stabilizes the {sigma}1 Head. Our results confirm a role of the aspartic acid cluster as a pH-dependent molecular switch, and highlight its role in enhancing {sigma}1 stability at low pH.

microbiology↗

bioRxiv2020

The interaction of DNA repair factors ASCC2 and ASCC3 is affected by somatic cancer mutations

The ASCC3 subunit of the activating signal co-integrator complex is a dual-cassette Ski2-like nucleic acid helicase that provides single-stranded DNA for alkylation damage repair by the -ketoglutarate-dependent dioxygenase, AlkBH3. Other ASCC components integrate ASCC3/AlkBH3 into a complex DNA repair pathway. We mapped and structurally analyzed interacting ASCC2 and ASCC3 regions. The ASCC3 fragment comprises a central helical domain and terminal, extended arms that clasp the compact ASCC2 unit. ASCC2-ASCC3 interfaces are evolutionarily highly conserved and comprise a large number of residues affected by somatic cancer mutations. We quantified contributions of protein regions to the ASCC2-ASCC3 interaction, observing that changes found in cancers lead to reduced ASCC2-ASCC3 affinity. Functional dissection of ASCC3 revealed similar organization and regulation as in the spliceosomal RNA helicase, Brr2. Our results delineate functional regions in an important DNA repair complex and suggest possible molecular disease principles.

biochemistry↗

bioRxiv2019

A receptor for herbivore-associated molecular patterns mediates plant immunity

Plant-herbivore interactions are ubiquitous across nature and drive major agricultural losses. Inducible defense responses triggered through immune recognition aid in host plant protection; however, specific ligand-receptor pairs mediating the initial perception of herbivory remain unknown. Plants in the subtribe Phaseolinae detect herbivore-associated peptides in caterpillar oral secretions and the defined ligands are proteolytic fragments of chloroplastic ATP synthase termed inceptins. Using forward genetic mapping of inceptin-induced responses, we identify a cowpea (Vigna unguiculata) leucine-rich repeat receptor-like protein as an inceptin receptor (INR) sufficient for elicitor-induced responses and enhanced defense against armyworms (Spodoptera exigua). INR defines a receptor by which plants perceive herbivore-associated molecular patterns (HAMPs) and expands the paradigm of surface immune recognition to attack with mandibles.\n\nOne Sentence SummaryA plant cell surface receptor directly perceives peptides associated with caterpillar herbivory.

plant biology↗