A Comprehensive Evaluation of the Performance of Prediction Algorithms on Clinically Relevant Missense Variants.

The rapid integration of genomic technologies in clinical diagnostics has resulted in the detection of a multitude of missense variants whose clinical significance is often unknown. As a result, a plethora of computational tools have been developed to facilitate variant interpretation. However, choosing an appropriate software from such a broad range of tools can be challenging; therefore, systematic benchmarking with high-quality, independent datasets is critical. Using three independent benchmarking datasets compiled from the ClinVar database, we evaluated the performance of ten widely used prediction algorithms with missense variants from 21 clinically relevant genes, including BRCA1 and BRCA2. A fourth dataset consisting of 1053 missense variants was also used to investigate the impact of type 1 circularity on their performance. The performance of the prediction algorithms varied widely across datasets. Based on Matthews Correlation Coefficient and Area Under the Curve, SNPs&GO and PMut consistently displayed an overall above-average performance across the datasets. Most of the tools demonstrated greater sensitivity and negative predictive values at the expense of lower specificity and positive predictive values. We also demonstrated that type 1 circularity significantly impacts the performance of these tools and, if not accounted for, may confound the selection of the best performing algorithms.

A series of xanthenes inhibiting Rad6 function and Rad6-Rad18 interaction in the PCNA ubiquitination cascade.

Ubiquitination of proliferating cell nuclear antigen (PCNA) triggers pathways of DNA damage tolerance, including mutagenic translesion DNA synthesis, and comprises a cascade of reactions involving the E1 ubiquitin-activating enzyme Uba1, the E2 ubiquitin-conjugating enzyme Rad6, and the E3 ubiquitin ligase Rad18. We report here the discovery of a series of xanthenes that inhibit PCNA ubiquitination, Rad6∼ubiquitin thioester formation, and the Rad6-Rad18 interaction. Structure-activity relationship experiments across multiple assays reveal chemical and structural features important for different activities along the pathway to PCNA ubiquitination. The compounds that inhibit these processes are all a subset of the xanthen-3-ones we tested. These small molecules thus represent first-in-class probes of Rad6 function and the association of Rad6 and Rad18, the latter being a new inhibitory activity discovered for a small molecule, in the PCNA ubiquitination cascade and potential therapeutic agents to contain cancer progression.

The Medicago truncatula IEF Gene Is Crucial for the Progression of Bacterial Infection During Symbiosis.

Legumes are able to meet their nitrogen need by establishing nitrogen-fixing symbiosis with rhizobia. Nitrogen fixation is performed by rhizobia, which has been converted to bacteroids, in newly formed organs, the root nodules. In the model legume Medicago truncatula, nodule cells are invaded by rhizobia through transcellular tubular structures called infection threads (ITs) that are initiated at the root hairs. Here, we describe a novel M. truncatula early symbiotic mutant identified as infection-related epidermal factor (ief), in which the formation of ITs is blocked in the root hair cells and only nodule primordia are formed. We show that the function of MtIEF is crucial for the bacterial infection in the root epidermis but not required for the nodule organogenesis. The IEF gene that appears to have been recruited for a symbiotic function after the duplication of a flower-specific gene is activated by the ERN1-branch of the Nod factor signal transduction pathway and independent of the NIN activity. The expression of MtIEF is induced transiently in the root epidermal cells by the rhizobium partner or Nod factors. Although its expression was not detectable at later stages of symbiosis, complementation experiments indicate that MtIEF is also required for the proper invasion of the nodule cells by rhizobia. The gene encodes an intracellular protein of unknown function possessing a coiled-coil motif and a plant-specific DUF761 domain. The IEF protein interacts with RPG, another symbiotic protein essential for normal IT development, suggesting that combined action of these proteins plays a role in nodule infection.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The Rad5 Helicase and RING Domains Contribute to Genome Stability through their Independent Catalytic Activities.

Genomic stability is compromised by DNA damage that obstructs replication. Rad5 plays a prominent role in DNA damage bypass processes that evolved to ensure the continuation of stalled replication. Like its human orthologs, the HLTF and SHPRH tumor suppressors, yeast Rad5 has a RING domain that supports ubiquitin ligase activity promoting PCNA polyubiquitylation and a helicase domain that in the case of HLTF and Rad5 was shown to exhibit an ATPase-linked replication fork reversal activity. The RING domain is embedded in the helicase domain, confusing their separate investigation and the understanding of the exact role of Rad5 in DNA damage bypass. Particularly, it is still debated whether the helicase domain plays a catalytic or a non-enzymatic role during error-free damage bypass and whether it facilitates a function separately from the RING domain. In this study, through in vivo and in vitro characterization of domain-specific mutants, we delineate the contributions of the two domains to Rad5 function. Yeast genetic experiments and whole-genome sequencing complemented with biochemical assays demonstrate that the ubiquitin ligase and the ATPase-linked activities of Rad5 exhibit independent catalytic activities in facilitating separate pathways during error-free lesion bypass. Our results also provide important insights into the mutagenic role of Rad5 and indicate its tripartite contribution to DNA damage tolerance.

The Combination of Single-Cell and Next-Generation Sequencing Can Reveal Mosaicism for BRCA2 Mutations and the Fine Molecular Details of Tumorigenesis.

Germline mutations in the BRCA1 and BRCA2 genes are responsible for hereditary breast and ovarian cancer syndrome. Germline and somatic BRCA1/2 mutations may define therapeutic targets and refine cancer treatment options. However, routine BRCA diagnostic approaches cannot reveal the exact time and origin of BRCA1/2 mutation formation, and thus, the fine details of their contribution to tumor progression remain less clear. Here, we establish a diagnostic pipeline using high-resolution microscopy and laser microcapture microscopy to test for BRCA1/2 mutations in the tumor at the single-cell level, followed by deep next-generation sequencing of various tissues from the patient. To demonstrate the power of our approach, here, we describe a detailed single-cell-level analysis of an ovarian cancer patient we found to exhibit constitutional somatic mosaicism of a pathogenic BRCA2 mutation. Employing next-generation sequencing, BRCA2 c.7795G>T, p.(Glu2599Ter) was detected in 78% of reads in DNA extracted from ovarian cancer tissue and 25% of reads in DNA derived from peripheral blood, which differs significantly from the expected 50% of a hereditary mutation. The BRCA2 mutation was subsequently observed at 17-20% levels in the normal ovarian and buccal tissue of the patient. Together, our findings suggest that this mutation occurred early in embryonic development. Characterization of the mosaic mutation at the single-cell level contributes to a better understanding of BRCA mutation formation and supports the concept that the combination of single-cell and next-generation sequencing methods is advantageous over traditional mutational analysis methods. This study is the first to characterize constitutional mosaicism down to the single-cell level, and it demonstrates that BRCA2 mosaicism occurring early during embryogenesis can drive tumorigenesis in ovarian cancer.

Robust high-throughput assays to assess discrete steps in ubiquitination and related cascades.

BACKGROUND: Ubiquitination and ubiquitin-like protein post-translational modifications play an enormous number of roles in cellular processes. These modifications are constituted of multistep reaction cascades. Readily implementable and robust methods to evaluate each step of the overall process, while presently limited, are critical to the understanding and modulation of the reaction sequence at any desired level, both in terms of basic research and potential therapeutic drug discovery and development. RESULTS: We developed multiple robust and reliable high-throughput assays to interrogate each of the sequential discrete steps in the reaction cascade leading to protein ubiquitination. As models for the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, the E3 ubiquitin ligase, and their ultimate substrate of ubiquitination in a cascade, we examined Uba1, Rad6, Rad18, and proliferating cell nuclear antigen (PCNA), respectively, in reconstituted systems. Identification of inhibitors of this pathway holds promise in cancer therapy since PCNA ubiquitination plays a central role in DNA damage tolerance and resulting mutagenesis. The luminescence-based assays we developed allow for the quantitative determination of the degree of formation of ubiquitin thioester conjugate intermediates with both E1 and E2 proteins, autoubiquitination of the E3 protein involved, and ubiquitination of the final substrate. Thus, all covalent adducts along the cascade can be individually probed. We tested previously identified inhibitors of this ubiquitination cascade, finding generally good correspondence between compound potency trends determined by more traditional low-throughput methods and the present high-throughput ones. CONCLUSIONS: These approaches are readily adaptable to other E1, E2, and E3 systems, and their substrates in both ubiquitination and ubiquitin-like post-translational modification cascades.

Multilevel structure-activity profiling reveals multiple green tea compound families that each modulate ubiquitin-activating enzyme and ubiquitination by a distinct mechanism.

We developed and implemented a reconstituted system to screen for modulators of the ubiquitination of proliferating cell nuclear antigen, a process that activates pathways of DNA damage tolerance and drug resistance. We identified the primary putatively health-beneficial green tea polyphenol epigallocatechin gallate (EGCG) and certain related small molecules as potent inhibitors of ubiquitination. EGCG directly and reversibly targets the ubiquitin-activating enzyme Uba1, blocking formation of the Uba1~ubiquitin thioester conjugate and thus ubiquitination and in the cell. Structure-activity relationship profiles across multiple biochemical and cellular assays for a battery of EGCG analogues revealed distinct chemical and mechanism-of-action clusters of molecules, with catechin gallates, alkyl gallates, and myricetin potently inhibiting ubiquitination. This study defines a number of related though distinct first-in-class inhibitors of ubiquitination, each series with its own unique activity pattern and mechanistic signature.

NAD1 Controls Defense-Like Responses in Medicago truncatula Symbiotic Nitrogen Fixing Nodules Following Rhizobial Colonization in a BacA-Independent Manner.

Legumes form endosymbiotic interaction with host compatible rhizobia, resulting in the development of nitrogen-fixing root nodules. Within symbiotic nodules, rhizobia are intracellularly accommodated in plant-derived membrane compartments, termed symbiosomes. In mature nodule, the massively colonized cells tolerate the existence of rhizobia without manifestation of visible defense responses, indicating the suppression of plant immunity in the nodule in the favur of the symbiotic partner. Medicago truncatulaDNF2 (defective in nitrogen fixation 2) and NAD1 (nodules with activated defense 1) genes are essential for the control of plant defense during the colonization of the nitrogen-fixing nodule and are required for bacteroid persistence. The previously identified nodule-specific NAD1 gene encodes a protein of unknown function. Herein, we present the analysis of novel NAD1 mutant alleles to better understand the function of NAD1 in the repression of immune responses in symbiotic nodules. By exploiting the advantage of plant double and rhizobial mutants defective in establishing nitrogen-fixing symbiotic interaction, we show that NAD1 functions following the release of rhizobia from the infection threads and colonization of nodule cells. The suppression of plant defense is self-dependent of the differentiation status of the rhizobia. The corresponding phenotype of nad1 and dnf2 mutants and the similarity in the induction of defense-associated genes in both mutants suggest that NAD1 and DNF2 operate close together in the same pathway controlling defense responses in symbiotic nodules.

Atypical transcriptional regulation and role of a new toxin-antitoxin-like module and its effect on the lipid composition of Bradyrhizobium japonicum.

A toxin-antitoxin (TA)-like system (designated as bat/bto genes) was identified in Bradyrhizobium japonicum, based on sequence homology and similarities in organization and size to known TA systems. Deletion of the bat/bto module resulted in pleiotropic alterations in cell morphology and metabolism. The generation time of the mutant was considerably decreased in rich media. Atomic force microscopy revealed the modified shape (shorter and wider) and softness of mutant cells. The synthesis of phosphatidylcholine was completely blocked in the mutant bacteria, and vaccenic acid, the predominant fatty acid of membranes of the wild-type cell, was replaced by palmitic acid in the mutant membranes. The mutant bacteria synthesized incomplete lipopolysaccharide molecules. Remarkable changes in the membrane lipid composition may explain the observed morphological alterations and growth properties of the mutant bacteria. The overlapping promoter region of bat/bto and glpD (coding for the aerobic sn-glycerol-3-phosphate dehydrogenase) genes suggests a complex regulation and the involvement of bat/bto in the control of main metabolic pathways and an important role in the maintenance of a normal physiological state of B. japonicum. These data reveal new aspects of the role of TA systems in bacteria.

LIN, a novel type of U-box/WD40 protein, controls early infection by rhizobia in legumes.

The formation of a nitrogen-fixing nodule requires the coordinated development of rhizobial colonization and nodule organogenesis. Based on its mutant phenotype, lumpy infections (lin), LIN functions at an early stage of the rhizobial symbiotic process, required for both infection thread growth in root hair cells and the further development of nodule primordia. We show that spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase is independent of LIN; thus, LIN is not necessary for nodule organogenesis. From this, we infer that LIN predominantly functions during rhizobial colonization and that the abortion of this process in lin mutants leads to a suppression of nodule development. Here, we identify the LIN gene in Medicago truncatula and Lotus japonicus, showing that it codes for a predicted E3 ubiquitin ligase containing a highly conserved U-box and WD40 repeat domains. Ubiquitin-mediated protein degradation is a universal mechanism to regulate many biological processes by eliminating rate-limiting enzymes and key components such as transcription factors. We propose that LIN is a regulator of the component(s) of the nodulation factor signal transduction pathway and that its function is required for correct temporal and spatial activity of the target protein(s).

The morphology of the NiO-Al2O3 catalyst.

The morphology of the NiO-Al2O3 catalyst, prepared by coprecipitation, impregnation, and mechanical powder mixing with ca. 5, 10, and 20 wt% nickel treated at 400 degrees, 700 degrees, and 1100 degrees C, was studied. Scanning electron microscopy is a useful technique for determining the manner of catalyst preparation for particle size estimation of catalyst components, whereas their mutual interactions become visible in the obtained images.

The katA catalase gene is regulated by OxyR in both free-living and symbiotic Sinorhizobium meliloti.

The characterization of an oxyR insertion mutant provides evidences that katA, which encodes the unique H2O2-inducible HPII catalase, is regulated by OxyR not only in free-living Sinorhizobium meliloti but also in symbiotic S. meliloti. Moreover, oxyR is expressed independently of exogenous H2O2 and downregulates its own expression in S. meliloti.

The typA gene is required for stress adaptation as well as for symbiosis of Sinorhizobium meliloti 1021 with certain Medicago truncatula lines.

In this article, we describe the typA gene of Sinorhizobium meliloti, the orthologue of typA/bipA genes found in a wide range of bacteria. We found that typA was required for survival of S. meliloti under certain stress conditions, such as growth at low temperature or low pH and in the presence of sodium dodecyl sulfate (SDS). The cold-sensitive phenotype of both Escherichia coli bipA and S. meliloti typA mutants were cross-complemented, indicating that the two genes are functionally equivalent. typA was indispensable for symbiosis on Medicago truncatula Jemalong and F83005.5 and contributes to the full efficiency of symbiosis on other host plant lines such as DZA315.16 or several cultivars of M. sativa. Hence, the symbiotic requirement for typA is host dependent. Interestingly, the symbiotic defect was different on Jemalong and F83005.5 plants, thus indicating that typA is required at a different stage of the symbiotic interaction.

Transcriptome analysis of Sinorhizobium meliloti during symbiosis.

BACKGROUND: Rhizobia induce the formation on specific legumes of new organs, the root nodules, as a result of an elaborated developmental program involving the two partners. In order to contribute to a more global view of the genetics underlying this plant-microbe symbiosis, we have mined the recently determined Sinorhizobium meliloti genome sequence for genes potentially relevant to symbiosis. We describe here the construction and use of dedicated nylon macroarrays to study simultaneously the expression of 200 of these genes in a variety of environmental conditions, pertinent to symbiosis. RESULTS: The expression of 214 S. meliloti genes was monitored under ten environmental conditions, including free-living aerobic and microaerobic conditions, addition of the plant symbiotic elicitor luteolin, and a variety of symbiotic conditions. Five new genes induced by luteolin have been identified as well as nine new genes induced in mature nitrogen-fixing bacteroids. A bacterial and a plant symbiotic mutant affected in nodule development have been found of particular interest to decipher gene expression at the intermediate stage of the symbiotic interaction. S. meliloti gene expression in the cultivated legume Medicago sativa (alfalfa) and the model plant M. truncatula were compared and a small number of differences was found. CONCLUSIONS: In addition to exploring conditions for a genome-wide transcriptome analysis of the model rhizobium S. meliloti, the present work has highlighted the differential expression of several classes of genes during symbiosis. These genes are related to invasion, oxidative stress protection, iron mobilization, and signaling, thus emphasizing possible common mechanisms between symbiosis and pathogenesis.