Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

Novel assay format for total anti-adeno-associated virus antibody detection with low capsid consumption and built-in specificity control.

Aim: To develop an assay format for detection of total anti-adeno-associated virus 2 (AAV2) antibodies with low capsid material consumption. Methods: An immune complex (IC) assay format was developed. The format is based on the formation of ICs in solution and their subsequent detection using an anti-AAV2 antibody for capture and an antibody against the study species IgG for detection. Results: The feasibility of the IC assay for detection of preexisting and treatment-emergent anti-AAV2 antibodies was demonstrated in cynomolgus monkey and human serum samples, including samples from a preclinical study with AAV2-based therapies. Conclusion: The presented IC assay is an easy-to-perform total anti-AAV2 antibody assay that requires a small amount of unlabeled capsid material and provides an intrinsic specificity control.

Regulation of developmental gatekeeping and cell fate transition by the calpain protease DEK1 in Physcomitrium patens.

Calpains are cysteine proteases that control cell fate transitions whose loss of function causes severe, pleiotropic phenotypes in eukaryotes. Although mainly considered as modulatory proteases, human calpain targets are directed to the N-end rule degradation pathway. Several such targets are transcription factors, hinting at a gene-regulatory role. Here, we analyze the gene-regulatory networks of the moss Physcomitrium patens and characterize the regulons that are misregulated in mutants of the calpain DEFECTIVE KERNEL1 (DEK1). Predicted cleavage patterns of the regulatory hierarchies in five DEK1-controlled subnetworks are consistent with a pleiotropic and regulatory role during cell fate transitions targeting multiple functions. Network structure suggests DEK1-gated sequential transitions between cell fates in 2D-to-3D development. Our method combines comprehensive phenotyping, transcriptomics and data science to dissect phenotypic traits, and our model explains the protease function as a switch gatekeeping cell fate transitions potentially also beyond plant development.

DPH1 Gene Mutations Identify a Candidate SAM Pocket in Radical Enzyme Dph1•Dph2 for Diphthamide Synthesis on EF2.

In eukaryotes, the Dph1•Dph2 dimer is a non-canonical radical SAM enzyme. Using iron-sulfur (FeS) clusters, it cleaves the cosubstrate S-adenosyl-methionine (SAM) to form a 3-amino-3-carboxy-propyl (ACP) radical for the synthesis of diphthamide. The latter decorates a histidine residue on elongation factor 2 (EF2) conserved from archaea to yeast and humans and is important for accurate mRNA translation and protein synthesis. Guided by evidence from archaeal orthologues, we searched for a putative SAM-binding pocket in Dph1•Dph2 from Saccharomyces cerevisiae. We predict an SAM-binding pocket near the FeS cluster domain that is conserved across eukaryotes in Dph1 but not Dph2. Site-directed DPH1 mutagenesis and functional characterization through assay diagnostics for the loss of diphthamide reveal that the SAM pocket is essential for synthesis of the décor on EF2 in vivo. Further evidence from structural modeling suggests particularly critical residues close to the methionine moiety of SAM. Presumably, they facilitate a geometry specific for SAM cleavage and ACP radical formation that distinguishes Dph1•Dph2 from classical radical SAM enzymes, which generate canonical 5'-deoxyadenosyl (dAdo) radicals.

DPH1 and DPH2 variants that confer susceptibility to diphthamide deficiency syndrome in human cells and yeast models.

The autosomal-recessive diphthamide deficiency syndrome presents as intellectual disability with developmental abnormalities, seizures, craniofacial and additional morphological phenotypes. It is caused by reduced activity of proteins that synthesize diphthamide on human translation elongation factor 2. Diphthamide synthesis requires seven proteins (DPH1-DPH7), with clinical deficiency described for DPH1, DPH2 and DPH5. A limited set of variant alleles from syndromic patients has been functionally analyzed, but databases (gnomAD) list additional so far uncharacterized variants in human DPH1 and DPH2. Because DPH enzymes are conserved among eukaryotes, their functionality can be assessed in yeast and mammalian cells. Our experimental assessment of known and uncharacterized DPH1 and DPH2 missense alleles showed that six variants are tolerated despite inter-species conservation. Ten additional human DPH1 (G113R, A114T, H132P, H132R, S136R, C137F, L138P, Y152C, S221P, H240R) and two DPH2 (H105P, C341Y) variants showed reduced functionality and hence are deficiency-susceptibility alleles. Some variants locate close to the active enzyme center and may affect catalysis, while others may impact on enzyme activation. In sum, our study has identified functionally compromised alleles of DPH1 and DPH2 genes that likely cause diphthamide deficiency syndrome.

Targeted chain-exchange-mediated reconstitution of a split type-I cytokine for conditional immunotherapy.

Antibody-cytokine fusions targeted against tumor-associated antigens (TAAs) are promising cancer immunotherapy agents, with many such molecules currently undergoing clinical trials. However, due to the limited number of tumor-specific targets, on-target off-tumor effects can lead to systemic toxicity. Additionally, targeted cytokines can be scavenged by cytokine receptors on peripheral cells, decreasing tumor penetration. This study aims at overcoming these issues by engineering a platform for targeted conditionally active type I cytokines. Building on our previously reported PACE (Prodrug-Activating Chain Exchange) platform, we split the type I cytokine interleukin-4 (IL-4) to create two inactive IL-4 prodrugs, and fused these split IL-4 counterparts to the C-termini of antibody-like molecules that undergo proximity-induced chain exchange. In doing so, we developed IL-4 prodrugs that preferentially reconstitute into active IL-4 on target cells. We demonstrate that pre-assembled split IL-4 (without additional inactivation) retains activity and present two different strategies of splitting and inactivating IL-4. Using an IL-4 responsive cell-line, we show that IL-4 prodrugs are targeted to TAAs on target cells and regain activity upon chain exchange, primarily in a cis-activation setting. Furthermore, we demonstrate that split IL-4 complementation is also possible in a trans-activation setting, which opens up the possibility for activation of immune cells in the tumor vicinity. We demonstrate that targeted on-cell prodrug conversion is more efficient than nonspecific activation in-solution. Due to the structural similarity between IL-4 and other type I cytokines relevant in cancer immunotherapy such as IL-2, IL-15, and IL-21, cytokine-PACE may be expanded to develop a variety of targeted conditionally active cytokines for cancer immunotherapy.

The mosaic oat genome gives insights into a uniquely healthy cereal crop.

Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.

A novel DPH5-related diphthamide-deficiency syndrome causing embryonic lethality or profound neurodevelopmental disorder.

PURPOSE: Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs). METHODS: Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants. RESULTS: DPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2. CONCLUSION: We provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.

Prodrug-Activating Chain Exchange (PACE) converts targeted prodrug derivatives to functional bi- or multispecific antibodies.

Driven by the potential to broaden the target space of conventional monospecific antibodies, the field of multi-specific antibody derivatives is growing rapidly. The production and screening of these artificial proteins entails a high combinatorial complexity. Antibody-domain exchange was previously shown to be a versatile strategy to produce bispecific antibodies in a robust and efficient manner. Here, we show that the domain exchange reaction to generate hybrid antibodies also functions under physiological conditions. Accordingly, we modified the exchange partners for use in therapeutic applications, in which two inactive prodrugs convert into a product with additional functionalities. We exemplarily show the feasibility for generating active T cell bispecific antibodies from two inactive prodrugs, which per se do not activate T cells alone. The two complementary prodrugs harbor antigen-targeting Fabs and non-functional anti-CD3 Fvs fused to IgG-CH3 domains engineered to drive chain-exchange reactions between them. Importantly, Prodrug-Activating Chain Exchange (PACE) could be an attractive option to conditionally activate therapeutics at the target site. Several examples are provided that demonstrate the efficacy of PACE as a new principle of cancer immunotherapy in vitro and in a human xenograft model.

The Barley and Wheat Pan-Genomes.

To unlock the genetic potential in crops, multi-genome comparisons are an essential tool. Decreasing costs and improved sequencing technologies have democratized plant genome sequencing and led to a vast increase in the amount of available reference sequences on the one hand and enabled the assembly of even the largest and most complex and repetitive crops genomes such as wheat and barley. These developments have led to the era of pan-genomics in recent years. Pan-genome projects enable the definition of the core and dispensable genome for various crop species as well as the analysis of structural and functional variation and hence offer unprecedented opportunities for exploring and utilizing the genetic basis of natural variation in crops. Comparing, analyzing, and visualizing these multiple reference genomes and their diversity requires powerful and specialized computational strategies and tools.

De Novo Genome Assembly of the Japanese Wheat Cultivar Norin 61 Highlights Functional Variation in Flowering Time and Fusarium-Resistant Genes in East Asian Genotypes.

Bread wheat is a major crop that has long been the focus of basic and breeding research. Assembly of its genome has been difficult because of its large size and allohexaploid nature (AABBDD genome). Following the first reported assembly of the genome of the experimental strain Chinese Spring (CS), the 10+ Wheat Genomes Project was launched to produce multiple assemblies of worldwide modern cultivars. The only Asian cultivar in the project is Norin 61, a representative Japanese cultivar adapted to grow across a broad latitudinal range, mostly characterized by a wet climate and a short growing season. Here, we characterize the key aspects of its chromosome-scale genome assembly spanning 15 Gb with a raw scaffold N50 of 22 Mb. Analysis of the repetitive elements identified chromosomal regions unique to Norin 61 that encompass a tandem array of the pathogenesis-related 13 family. We report novel copy-number variations in the B homeolog of the florigen gene FT1/VRN3, pseudogenization of its D homeolog and the association of its A homeologous alleles with the spring/winter growth habit. Furthermore, the Norin 61 genome carries typical East Asian functional variants different from CS, ranging from a single nucleotide to multi-Mb scale. Examples of such variation are the Fhb1 locus, which confers Fusarium head-blight resistance, Ppd-D1a, which confers early flowering, Glu-D1f for Asian noodle quality and Rht-D1b, which introduced semi-dwarfism during the green revolution. The adoption of Norin 61 as a reference assembly for functional and evolutionary studies will enable comprehensive characterization of the underexploited Asian bread wheat diversity.

DPH1 syndrome: two novel variants and structural and functional analyses of seven missense variants identified in syndromic patients.

DPH1 variants have been associated with an ultra-rare and severe neurodevelopmental disorder, mainly characterized by variable developmental delay, short stature, dysmorphic features, and sparse hair. We have identified four new patients (from two different families) carrying novel variants in DPH1, enriching the clinical delineation of the DPH1 syndrome. Using a diphtheria toxin ADP-ribosylation assay, we have analyzed the activity of seven identified variants and demonstrated compromised function for five of them [p.(Leu234Pro); p.(Ala411Argfs*91); p.(Leu164Pro); p.(Leu125Pro); and p.(Tyr112Cys)]. We have built a homology model of the human DPH1-DPH2 heterodimer and have performed molecular dynamics simulations to study the effect of these variants on the catalytic sites as well as on the interactions between subunits of the heterodimer. The results show correlation between loss of activity, reduced size of the opening to the catalytic site, and changes in the size of the catalytic site with clinical severity. This is the first report of functional tests of DPH1 variants associated with the DPH1 syndrome. We demonstrate that the in vitro assay for DPH1 protein activity, together with structural modeling, are useful tools for assessing the effect of the variants on DPH1 function and may be used for predicting patient outcomes and prognoses.

Intercomparison of the Radio-Chronometric Ages of Plutonium-Certified Reference Materials with Distinct Isotopic Compositions.

An intercomparison of the radio-chronometric ages of four distinct plutonium-certified reference materials varying in chemical form, isotopic composition, and period of production are presented. The cross-comparison of the different 234U/238Pu, 235U/239Pu, 236U/240Pu, and 241Am/241Pu model purification ages obtained at four independent analytical facilities covering a range of laboratory environments from bulk sample processing to clean facilities dedicated to nuclear forensic investigation of environmental samples enables a true assessment of the state-of-practice in "age dating capabilities" for nuclear materials. The analytical techniques evaluated used modern mass spectrometer instrumentation including thermal ionization mass spectrometers and inductively coupled plasma mass spectrometers for isotopic abundance measurements. Both multicollector and single collector instruments were utilized to generate the data presented here. Consensus values established in this study make it possible to use these isotopic standards as quality control standards for radio-chronometry applications. Results highlight the need for plutonium isotopic standards that are certified for 234U/238Pu, 235U/239Pu, 236U/240Pu, and 241Am/241Pu model purification ages as well as other multigenerational radio-chronometers such as 237Np/241Pu. Due to the capabilities of modern analytical instrumentation, analytical laboratories that focus on trace level analyses can obtain model ages with marginally larger uncertainties than laboratories that handle bulk samples. When isotope ratio measurement techniques like thermal ionization mass spectrometry and inductively coupled plasma mass spectrometry with comparable precision are utilized, model purification ages with similar uncertainties are obtained.

Interplay between reversible phosphorylation and irreversible ADP-ribosylation of eukaryotic translation elongation factor 2.

The functionality of eukaryotic translation elongation factor 2 (eEF2) is modulated by phosphorylation, eEF2 is simultaneously the molecular target of ADP-ribosylating toxins. We analyzed the interplay between phosphorylation and diphthamide-dependent ADP-ribosylation. Phosphorylation does not require diphthamide, eEF2 without it still becomes phosphorylated. ADP-ribosylation not only modifies the H715 diphthamide but also inhibits phosphorylation of S595 located in proximity to H715, and stimulates phosphorylation of T56. S595 can be phosphorylated by CDK2 and CDK1 which affects EEF2K-mediated T56-phosphorylation. Thus, ADP-ribosylation and S595-phosphorylation by kinases occur within the same vicinity and both trigger T56-phosphorylation. Diphthamide is surface-accessible permitting access to ADP-ribosylating enzymes, the adjacent S595 side chain extends into the interior. This orientation is incompatible with phosphorylation, neither allowing kinase access nor phosphate attachment. S595 phosphorylation must therefore be accompanied by structural alterations affecting the interface to ADP-ribosylating toxins. In agreement with that, replacement of S595 with Ala, Glu or Asp prevents ADP-ribosylation. Phosphorylation (starvation) as well as ADP-ribosylation (toxins) inhibit protein synthesis, both affect the S595/H715 region of eEF2, both trigger T57-phosphorylation eliciting similar transcriptional responses. Phosphorylation is short lived while ADP-ribosylation is stable. Thus, phosphorylation of the S595/H715 'modifier region' triggers transient interruption of translation while ADP-ribosylation arrests irreversibly.

Diphthamide affects selenoprotein expression: Diphthamide deficiency reduces selenocysteine incorporation, decreases selenite sensitivity and pre-disposes to oxidative stress.

The diphthamide modification of translation elongation factor 2 is highly conserved in eukaryotes and archaebacteria. Nevertheless, cells lacking diphthamide can carry out protein synthesis and are viable. We have analyzed the phenotypes of diphthamide deficient cells and found that diphthamide deficiency reduces selenocysteine incorporation into selenoproteins. Additional phenotypes resulting from diphthamide deficiency include altered tRNA-synthetase and selenoprotein transcript levels, hypersensitivity to oxidative stress and increased selenite tolerance. Diphthamide-eEF2 occupies the aminoacyl-tRNA translocation site at which UGA either stalls translation or decodes selenocysteine. Its position is in close proximity and mutually exclusive to the ribosomal binding site of release/recycling factor ABCE1, which harbors a redox-sensitive Fe-S cluster and, like diphthamide, is present in eukaryotes and archaea but not in eubacteria. Involvement of diphthamide in UGA-SECIS decoding may explain deregulated selenoprotein expression and as a consequence oxidative stress, NFkB activation and selenite tolerance in diphthamide deficient cells.

Impact of transposable elements on genome structure and evolution in bread wheat.

BACKGROUND: Transposable elements (TEs) are major components of large plant genomes and main drivers of genome evolution. The most recent assembly of hexaploid bread wheat recovered the highly repetitive TE space in an almost complete chromosomal context and enabled a detailed view into the dynamics of TEs in the A, B, and D subgenomes. RESULTS: The overall TE content is very similar between the A, B, and D subgenomes, although we find no evidence for bursts of TE amplification after the polyploidization events. Despite the near-complete turnover of TEs since the subgenome lineages diverged from a common ancestor, 76% of TE families are still present in similar proportions in each subgenome. Moreover, spacing between syntenic genes is also conserved, even though syntenic TEs have been replaced by new insertions over time, suggesting that distances between genes, but not sequences, are under evolutionary constraints. The TE composition of the immediate gene vicinity differs from the core intergenic regions. We find the same TE families to be enriched or depleted near genes in all three subgenomes. Evaluations at the subfamily level of timed long terminal repeat-retrotransposon insertions highlight the independent evolution of the diploid A, B, and D lineages before polyploidization and cases of concerted proliferation in the AB tetraploid. CONCLUSIONS: Even though the intergenic space is changed by the TE turnover, an unexpected preservation is observed between the A, B, and D subgenomes for features like TE family proportions, gene spacing, and TE enrichment near genes.

Hidden variation in polyploid wheat drives local adaptation.

Wheat has been domesticated into a large number of agricultural environments and has the ability to adapt to diverse environments. To understand this process, we survey genotype, repeat content, and DNA methylation across a bread wheat landrace collection representing global genetic diversity. We identify independent variation in methylation, genotype, and transposon copy number. We show that these, so far unexploited, sources of variation have had a significant impact on the wheat genome and that ancestral methylation states become preferentially "hard coded" as single nucleotide polymorphisms (SNPs) via 5-methylcytosine deamination. These mechanisms also drive local adaption, impacting important traits such as heading date and salt tolerance. Methylation and transposon diversity could therefore be used alongside SNP-based markers for breeding.

Methodology for the Preparation and Validation of Plutonium Age Dating Materials.

The present work describes a method for the preparation and validation of plutonium age dating reference materials. The test samples prepared in this context could be used to validate experimental protocols for determining the production date of plutonium via the 234U/238Pu, 235U/239Pu, 236U/240Pu, and 241Am/241Pu chronometers. The starting material was prepared using reactor-grade plutonium, which was purified using a dedicated method to guarantee high Pu recovery, while maximizing U and Am separation efficiencies. The U and Am separation factors were determined by the addition of high-amounts of 233U and 243Am spikes and their remeasurement in the final product. The prepared material is intended for quality control and assessment of method performance in nuclear forensics and safeguards.

Disruption of diphthamide synthesis genes and resulting toxin resistance as a robust technology for quantifying and optimizing CRISPR/Cas9-mediated gene editing.

We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30-50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16-18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.

Wild emmer genome architecture and diversity elucidate wheat evolution and domestication.

Wheat (Triticum spp.) is one of the founder crops that likely drove the Neolithic transition to sedentary agrarian societies in the Fertile Crescent more than 10,000 years ago. Identifying genetic modifications underlying wheat's domestication requires knowledge about the genome of its allo-tetraploid progenitor, wild emmer (T. turgidum ssp. dicoccoides). We report a 10.1-gigabase assembly of the 14 chromosomes of wild tetraploid wheat, as well as analyses of gene content, genome architecture, and genetic diversity. With this fully assembled polyploid wheat genome, we identified the causal mutations in Brittle Rachis 1 (TtBtr1) genes controlling shattering, a key domestication trait. A study of genomic diversity among wild and domesticated accessions revealed genomic regions bearing the signature of selection under domestication. This reference assembly will serve as a resource for accelerating the genome-assisted improvement of modern wheat varieties.