Search bioRxiv⌕ Search

Biology subjects

Baumann, U.

Publications and source records attributed to Baumann, U..

16 recordsLinked to original sources

TeTIR domains define a new class of animal TIR-like NADase effectors in STAND proteins

TIR domains are found in diverse immune signaling proteins and, in several systems, function as NAD+-cleaving enzymes causing or signaling cell death. In plants, TIR domains occur as effector domains in TNL immune receptors, where they are coupled to a central STAND ATPase and a C-terminal repeat domain. Here, we identify TeTIR (TEP1 extended TIR), a previously unrecognized family of TIR-like domains associated with NACHT-type ATPases and C-terminal WD40 or TPR repeat domains. The TeTIR domain of human TEP1 exhibits robust NADase activity comparable to SARM1-TIR, making TEP1 the only other NADase-active TIR-like protein identified in mammals. NADase activity is conserved in TEP1 homologs from diverse eukaryotes. The crystal structure of TEP1 TeTIR reveals an extended TIR-like fold with additional secondary-structure elements and a distinctive RWG-containing loop. Molecular dynamics simulations indicate that the catalytic glutamate contributes to catalysis and the conformational equilibrium of the active-site region, promoting formation of a substrate-accessible state. Structure-guided mutagenesis identifies conserved residues contributing to NAD+ recognition and catalysis, including features distinct from canonical TIR NADases. Expression of isolated TEP1 TeTIR causes NAD+ depletion and cell death in a catalytic-glutamate-dependent manner. TeTIRs thus define a distinct family of animal TIR-like NADases embedded in NACHT-type STAND architectures.

biochemistry↗

DUCs are C2 domain containing plant-specific deubiquitinases stabilizing endocytic cargo at the plasma membrane

Deubiquitinases (DUBs) remove ubiquitin modifications from proteins in a substrate- or linkage-selective manner and regulate numerous cell-biological processes, including endocytosis. By performing homology-agnostic bioinformatical screens for undescribed deubiquitinase classes, we identified the plant-specific DUC (deubiquitinase with C2) family, whose members are highly selective for cleaving K63-linked ubiquitin chains. The crystal structure of the Arabidopsis member AtDUC1 reveals that DUC enzymes display a papain-like fold with a characteristic DUB-like active site, despite the apparent absence of sequence similarity to other deubiquitinase families. The K63 linkage specificity is attributed to the recognition of both distal and proximal ubiquitin units via highly conserved contact residues. Arabidopsis DUC members localize to the plasma membrane by virtue of their lipid-binding C2-like domains. In protoplast experiments, all three Arabidopsis DUC enzymes demonstrate the capacity to stabilize an endocytotic model cargo at the plasma membrane, a process that requires both the C2-mediated membrane association and catalytic DUB activity.

biochemistry↗

A prebiotic-postbiotic combination supports dietary carbohydrate-targeting functional properties in a fiber-deprived microbiota

Dietary fiber deprivation compromises gut mucosal barrier integrity by promoting microbial degradation of host mucus, a process linked to various gut-related auto immune diseases. While postbiotics are considered safer alternatives to fiber for susceptible patients, their mechanistic ef- fects on a fiber-deprived gut remain poorly understood. Here, we demonstrate in a mouse model that a fermented postbiotic, alone or in combination with a prebiotic and aloe vera, counteracted the increase of detrimental properties of the microbiota on a fiber-free diet. The supplement regimen reshaped the gut microbiota, counteracting the expansion of key mucin-degrading bac- teria, including Akkermansia muciniphila and Parabacteroides goldsteinii. Metatranscriptomic analysis revealed this compositional change corresponded to a community-wide functional pivot away from expressing mucinolytic enzymes, such as sialidases, and towards utilizing alternative substrates. These microbial shifts recapitulated the effects of dietary fiber reintroduction and translated to direct host benefits, including sustentation of the colonic mucus layer and attenu- ation of diet-induced type III immune cytokine expression. Our findings provide a mechanistic rationale for using postbiotics to functionally replace dietary fiber, offering a promising strategy to support gut homeostasis in contexts where fiber intake is limited.

microbiology↗

A family of bacterial Josephin-like deubiquitinases with an unusual cleavage mode

Many intracellular bacteria secrete deubiquitinase (DUB) effectors into eukaryotic host cells to keep the bacterial surface or the enclosing vesicle membrane free of ubiquitin marks. Here, we describe a new family of bacterial DUBs that is structurally related to eukaryotic Josephins, but contains members that catalyze a unique destructive substrate deubiquitination. These ubiquitin C-terminal clippases (UCCs) cleave ubiquitin before the C-terminal diGly motif, thereby truncating the modifier and leaving a remnant on the substrate. By comparing the crystal structures of substrate-bound clippases and a closely related conventional DUB, we identified the factors causing the shift and found them conserved in other clippases, including one highly specific for M1-linked ubiquitin chains. This new enzyme class has great potential as tools to study the ubiquitin system, in particular aspects involving branched chains.

biochemistry↗

Non-prime- and Prime-side Profiling of Pro-Pro Endopeptidase Specificity Using Synthetic Combinatorial Peptide Libraries and Mass Spectrometry

A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline-proline bonds in proteins. Since a proteases function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in-depth characterization of PPEP prime-side specificity. However, PPEP specificity is also determined by the non-prime-side residues in the substrate. To gain a more complete insight into the determinants of PPEP specificity, we characterized the non-prime- and prime-side specificity of various PPEPs using a combination of synthetic combinatorial peptide libraries and mass spectrometry. With this approach, we deepened our understanding of the P3-P3 specificities of PPEP-1 and PPEP-2, while identifying PPEP-2s endogenous substrate as the most optimal substrate in our library data. Furthermore, by employing the library approach, we investigated the altered specificity of mutants of PPEP-1 and PPEP-2. Additionally, we characterized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displays a PPEP-1-like prime-side specificity, which was substantiated by the experimental data. Intriguingly, another putative PPEP from Clostridioides difficile, CD1597, did not display Pro-Pro endoproteolytic activity. Collectively, we characterized PPEP specificity in detail using our robust peptide library method and, together with additional structural information, provide more insight into the intricate mechanisms that govern protease specificity.

molecular biology↗

Impaired transitioning of the FXR ligand binding domain to an active state underlies a PFIC5 phenotype

Nuclear receptor farnesoid X receptor (FXR) acts as a key regulator of bile acid pool homeostasis and metabolism. Within the enterohepatic circulation, reabsorbed bile acids act as FXR agonists, which transcriptionally controls the synthesis and transport of bile acids. Binding occurs in the ligand binding domain (LBD), favoring a conformational change to the active state in which helix 12 interacts with the LBD to form an interaction surface for nuclear co-activators. The homozygous missense variant T296I, identified in a PFIC patient, is located close to the critical helix 12 interaction. Here, we identified reduced transcriptional activity of the variant protein on the downstream targets bile salt export pump (BSEP) and small heterodimer partner (SHP) in vitro, within the patients liver, and in iPSC-derived hepatic organoids. BSEP-dependent Tauro-DBD transport was impaired in T296I patient-derived organoids, but could be rescued via lipid nanoparticle-mediated FXR WT mRNA delivery, indicating the variant is responsible for the identified reduced BSEP expression. Using molecular dynamics simulations, we observed a reduced transitioning from the inactive to the active state for the T296I variant, indicating a molecular mechanism underlying the reduced activity. To our knowledge, this is the first study to describe the conformational change from an inactive to an active state of the FXR LBD. This might be useful for new therapeutic approaches targeting the activation of FXR.

physiology↗

The structural basis for deubiquitination by the fingerless USP-type effector TssM

Intracellular bacteria are threatened by ubiquitin-mediated autophagy, whenever the bacterial surface or enclosing membrane structures become targets of host ubiquitin ligases. As a countermeasure, many intracellular pathogens encode deubiquitinase (DUB) effectors to keep their surfaces free of ubiquitin modifications. Most bacterial DUBs belong to the OTU or CE-clan families. The betaproteobacteria B. pseudomallei and B. mallei, causative agents of melioidosis and glanders, respectively, encode the TssM effector, the only known bacterial DUB belonging to the USP class. TssM is much shorter than typical eukaryotic USP enzymes and lacks the canonical ubiquitin-recognition region. By solving the crystal structures of isolated TssM and its complex with ubiquitin, we found that TssM lacks the entire Fingers subdomain of the USP fold. Instead, the TssM family has evolved the functionally analog Littlefinger loop, which is located towards the end of the USP domain and recognizes different ubiquitin interfaces than those used by canonical USPs. The structures also revealed the presence of an N-terminal immunoglobulin-fold domain, which is able to form a strand-exchange dimer and might mediate TssM localization to the bacterial surface.

biochemistry↗

Structural polymorphisms within a common powdery mildew effector scaffold as a driver of co-evolution with cereal immune receptors

In plants, host-pathogen coevolution often manifests in reciprocal, adaptive genetic changes through variations in host nucleotide-binding leucine-rich repeat immune receptors (NLR) and virulence-promoting pathogen effectors. In grass powdery mildew (PM) fungi, an extreme expansion of a RNase-like effector family, termed RALPH, dominates the effector repertoire, with some members recognized as avirulence (AVR) effectors by cereal NLR receptors. We report the structures of the sequence-unrelated barley PM effectors AVRA6, AVRA7 and allelic AVRA10/AVRA22 variants, which are detected by highly sequence-related barley NLRs MLA6, MLA7, MLA10, and MLA22, and of wheat PM AVRPM2 detected by the unrelated wheat NLR PM2. The AVR effectors adopt a common scaffold, which is shared with the ribonuclease (RNase) T1/F1-family. We found striking variations in the number, position, and length of individual structural elements between RALPH AVRs, which is associated with a differentiation of RALPH effector subfamilies. We show that all RALPH AVRs tested have lost nuclease and synthetase activities of the RNase T1/F1- family and lack significant binding to RNA, implying that their virulence activities are associated with neo-functionalization events. Structure-guided mutagenesis identified six AVRA6 residues that are sufficient to turn a sequence-diverged member of the same RALPH subfamily into an effector specifically detected by MLA6. Similar structure-guided information for AVRA10 and AVRA22 indicates that MLA receptors detect largely distinct effector surface patches. Thus, coupling of sequence and structural polymorphisms within the RALPH scaffold of PMs facilitated escape from NLR recognition and potential acquisition of diverse virulence functions.

plant biology↗

HvSL1 and HvMADS16 promote stamen identity to restrict multiple ovary formation in barley

Correct floral development is a consequence of a sophisticated balance between environmental and molecular cues. Floral mutants provide insight into the main genetic determinants that integrate these cues, as well as providing opportunities to assess functional conservation across species. In this study, we characterize the barley (Hordeum vulgare) multiovary mutants mov2.g and mov1 and propose causative gene sequences: a C2H2 zinc-finger HvSL1 and a B-class gene HvMADS16, respectively. In the absence of HvSL1, flowers lack stamens but exhibit functional supernumerary carpels resulting in multiple seeds per floret when artificially pollinated. Deletion of HvMADS16 in mov1 causes homeotic conversion of lodicules and stamens into bract-like organs and carpels that contain non-functional ovules. Based on developmental, genetic, and molecular data we propose a model by which stamen specification in barley is defined by HvSL1 acting upstream of barley B-class genes, specifically the transcriptional up-regulation of HvMADS16. The present work identifies strong conservation of stamen formation pathways with rice, but also reveals intriguing species-specific differences. The findings lay the foundation for a better understanding of floral architecture in Triticeae, a key target for crop improvement. HighlightAnalysis of the barley multiovary mov1 and mov2 loci indicates that HvSL1 and HvMADS16 exhibit both unique and conserved roles in the specification and development of cereal flowers.

developmental biology↗

TIR-catalyzed ADP-ribosylation reactions produce signaling molecules for plant immunity

Plant pathogen-activated immune signaling by nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/Interleukin-1 receptor (TIR) domain converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TIR-encoded NADases produce signaling molecules to promote exclusive EDS1-PAD4 and EDS1-SAG101 interactions with helper NLR sub-classes. Here we show that TIR-containing proteins catalyze adenosine diphosphate (ADP)-ribosylation of adenosine triphosphate (ATP) and ADP ribose (ADPR) via ADPR polymerase-like and NADase activity, forming ADP-ribosylated ATP (ADPr-ATP) and ADPr-ADPR (di-ADPR), respectively. Specific binding of di-ADPR or ADPr-ATP allosterically promotes EDS1-SAG101 interaction with helper NLR N requirement gene 1A (NRG1A) in vitro and in planta. Our data reveal an enzymatic activity of TIRs that enables specific activation of the EDS1-SAG101-NRG1 immunity branch.

plant biology↗

GFAT2 and AMDHD2 act in tandem to control the hexosamine biosynthetic pathway

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, cancer, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFAT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM) and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to pinpoint new HBP regulators. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFAT2 instead of the more common paralog GFAT1. Compared with GFAT1, GFAT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 loss-of-function resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFAT2 activity was also observed in other mESCs and, consistently, the GFAT2/GFAT1 ratio decreased with differentiation of mouse and human embryonic stem cells. Together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFAT2 for metabolite entry into the HBP.

biochemistry↗

Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for all glycosylation processes in mammals. It modulates the ER stress response, is implicated in cancer and diabetes, and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the first and rate-limiting HP enzyme. GFAT-1 activity is modulated through UDP-GlcNAc feedback inhibition and by kinase signaling, including Ser205 phosphorylation by protein kinase A (PKA). The consequence and molecular mechanism of GFAT-1 phosphorylation, however, remains poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans, leading to ER stress resistance. In human GFAT-1, the R203H substitution interfered with both UDP-GlcNAc inhibition and PKA-mediated Ser205 phosphorylation. Of note, Ser205 phosphorylation had two discernible effects: It lowered baseline GFAT-1 activity and abolished UDP-GlcNAc feedback inhibition. Thus, GFAT-1 phosphorylation by PKA uncoupled the feedback loop of the HP and, depending on UDP-GlcNAc availability, phosphorylation by PKA results in lower or enhanced GFAT-1 activity in vivo. Mechanistically, our data indicate that the relative positioning of the two GFAT-1 domains might be affected by phosphorylation and we propose a model how Ser205 phosphorylation modulates the activity and feedback inhibition of GFAT-1.

biochemistry↗

AtCIPK16 Mediates Salt Stress Potentially Through Phytohormones and Transcription Factors

Soil salinity causes large productivity losses for agriculture worldwide. "Next-generation crops" that can tolerate salt stress are required for the sustainability of global food production. Previous research in Arabidopsis thaliana aimed at uncovering novel factors underpinning improved plant salinity tolerance identified the protein kinase AtCIPK16. Overexpression of AtCIPK16 enhanced shoot Na+ exclusion and increased biomass in both Arabidopsis and barley. Here, a comparative transcriptomic study on Arabidopsis lines expressing AtCIPK16 was conducted in the presence and absence of salt stress, using an RNA-Seq approach, complemented by AtCIPK16 interaction and localisation studies. We are now able to provide evidence for AtCIPK16 activity in the nucleus. Moreover, the results manifest the involvement of a transcription factor, AtTZF1, phytohormones and the ability to quickly reach homeostasis as components important for improving salinity tolerance in transgenics overexpressing AtCIPK16. Furthermore, we suggest the possibility of both biotic and abiotic tolerance through AtCIPK16, and propose a model for the salt tolerance pathway elicited through AtCIPK16.

plant biology↗

The wheat Seven in Absentia gene is associated with increases in biomass and yield in hot climates

Wheat productivity is severely reduced by high temperatures. Breeding of heat tolerant cultivars can be achieved by identifying genes controlling physiological and agronomical traits with high temperature and using these to select superior genotypes, but no gene underlying genetic variation for heat tolerance has previously been described. We completed the positional cloning of qYDH.3BL, a quantitative trait locus (QTL) on bread wheat chromosome 3B associated with increased yield in hot and dry climates. The delimited genomic region contained 12 putative genes and a sequence variant in the promoter region of one gene - seven in absentia, TaSINA. This was associated with the QTLs effects on early vigour, plant biomass and yield components in two distinct wheat populations grown under various growth conditions. Near isogenic lines carrying the positive allele at qYDH.3BL under-expressed TaSINA and had increased vigour and water use efficiency early in development, as well as increased biomass, grain number and grain weight following heat stress. A survey of worldwide distribution indicated that the positive allele became widespread from the 1950s through the CIMMYT wheat breeding programme but, to date, has been selected only in breeding programmes in Mexico and Australia.\n\nSignificance statementWheat is the worlds most widely grown crop and a staple of human diet. Even brief episodes of high temperature in the growing season cause severe yield reductions. Finding and deploying genes for heat stress tolerance in new varieties is a priority for food security with climate change. We narrowed a genetic locus to a small genomic region where genetic variation was present only in one gene that showed clear differences of expression and improved yield and physiology under stress in the populations. Using diagnostic markers to track the positive haplotype in nearly 750 accessions, we found many regions where the allele could be used in breeding programmes to increase wheats heat tolerance.

genetics↗

LNISKS: Reference-free mutation identification for large and complex crop genomes

Mutation discovery is often key to the identification of genes responsible for major phenotypic traits. In the context of bulked segregant analysis, common reference-based computational approaches are not always suitable as they rely on a genome assembly which may be incomplete or highly divergent from the studied accession. Reference-free methods based on short sequences of length k (k-mers), such as NIKS, exploit redundancy of information across pools of recombinant genomes. Building on concepts from NIKS we introduce LNISKS, a mutation discovery method which is suited for large and repetitive crop genomes. In our experiments, it rapidly and with high confidence, identified mutations from over 700 Gbp of bread wheat genomic sequence data. LNISKS is publicly available at https://github.com/rsuchecki/LNISKS.

bioinformatics↗

Optical and physical mapping with local finishing enables megabase-scale resolution of agronomically important regions in the wheat genome

BackgroundNumerous scaffold-level sequences for wheat are now being released and, in this context, we report on a strategy for improving the overall assembly to a level comparable to that of the human genome.\n\nResultsUsing chromosome 7A of wheat as a model, sequence-finished megabase scale sections of this chromosome were established by combining a new independent assembly based on a BAC-based physical map, BAC pool paired end sequencing, chromosome arm specific mate-pair sequencing and Bionano optical mapping with the IWGSC RefSeq v1.0 sequence and its underlying raw data. The combined assembly results in 18 super-scaffolds across the chromosome. The value of finished genome regions is demonstrated for two approximately 2.5 Mb regions associated with yield and the grain quality phenotype of fructan carbohydrate grain levels. In addition, the 50 Mb centromere region analysis incorporates cytological data highlighting the importance of non-sequence data in the assembly of this complex genome region.\n\nConclusionsSufficient genome sequence information is shown to be now available for the wheat community to produce sequence-finished releases of each chromosome of the reference genome. The high-level completion identified that an array of seven fructosyl transferase genes underpins grain quality and yield attributes are affected by five f-box-only-protein-ubiquitin ligase domain and four root-specific lipid transfer domain genes. The completed sequence also includes the centromere.

genomics↗