Rational Development and Characterization of a Ubiquitin Variant with Selectivity for Ubiquitin C-Terminal Hydrolase L3

There is currently a lack of reliable methods and strategies to probe the deubiquitinating enzyme UCHL3. Current small molecules reported for this purpose display reduced potency and selectivity in cellular assays. To bridge this gap and provide an alternative approach to probe UCHL3, our group has carried out the rational design of ubiquitin-variant activity-based probes with selectivity for UCHL3 over the closely related UCHL1 and other DUBs. The approach successfully produced a triple-mutant ubiquitin variant activity-based probe, UbVQ40V/T66K/V70F-PRG, that was ultimately 20,000-fold more selective for UCHL3 over UCHL1 when assessed by rate of inactivation assays. This same variant was shown to selectively form covalent adducts with UCHL3 in MDA-MB-231 breast cancer cells and no reactivity toward other DUBs expressed. Overall, this study demonstrates the feasibility of the approach and also provides insight into how this approach may be applied to other DUB targets.

Transcriptome Analysis of Triple Mutant for OsMADS62, OsMADS63, and OsMADS68 Reveals the Downstream Regulatory Mechanism for Pollen Germination in Rice (Oryza sativa)

The MADS (MCM1-AGAMOUS-DEFFICIENS-SRF) gene family has a preserved domain called MADS-box that regulates downstream gene expression as a transcriptional factor. Reports have revealed three MADS genes in rice, OsMADS62, OsMADS63, and OsMADS68, which exhibits preferential expression in mature rice pollen grains. To better understand the transcriptional regulation of pollen germination and tube growth in rice, we generated the loss-of-function homozygous mutant of these three OsMADS genes using the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system in wild-type backgrounds. Results showed that the triple knockout (KO) mutant showed a complete sterile phenotype without pollen germination. Next, to determine downstream candidate genes that are transcriptionally regulated by the three OsMADS genes during pollen development, we proceeded with RNA-seq analysis by sampling the mature anther of the mutant and wild-type. Two hundred and seventy-four upregulated and 658 downregulated genes with preferential expressions in the anthers were selected. Furthermore, downregulated genes possessed cell wall modification, clathrin coat assembly, and cellular cell wall organization features. We also selected downregulated genes predicted to be directly regulated by three OsMADS genes through the analyses for promoter sequences. Thus, this study provides a molecular background for understanding pollen germination and tube growth mediated by OsMADS62, OsMADS63, and OsMADS68 with mature pollen preferred expression.

SARS-CoV-2 comes up with a perfect recipe for disaster: Each mutation plays a specific role in the evolution of variants of concern

COVID-19 pandemic has extended for close to two years with the continuous emergence of new variants. Mutations in the receptor binding domain (RBD) are of prime importance in dictating the SARS-CoV-2 spike protein function. By studying a series of single, double and triple RBD mutants, we have delineated the individual and collective effects of RBD mutations in a variant of concern (VOC) containing multiple mutations (Gamma variant; K417T/E484K/N501Y) on binding to angiotensin converting enzyme 2 (ACE2) receptor, antibody escape and protein stability. Our results show that each mutation in the VOC serves a distinct function that improves virus fitness landscape supporting its positive selection, even though individual mutations have deleterious effects that make them prone to negative selection. K417T contributes to increased expression, increased stability and escape from class 1 antibodies; however, it has decreased ACE2 binding. E484K contributes to escape from class 2 antibodies; however, it has decreased expression, decreased stability, and decreased ACE2 binding affinity. N501Y increases receptor binding affinity; however, it has decreased stability and decreased expression. But when these mutations come together, the deleterious effects are mitigated in the triple mutant due to the presence of compensatory effects, which improves the chances of selection of mutations together. These results show the implications of presence of multiple mutations on virus evolution and indicate the emergence of future SARS-CoV-2 variants with multiple mutations that enhance viral fitness on different fronts by balancing both positive and negative selection.

Machine learning guided design of high affinity ACE2 decoys for SARS-CoV-2 neutralization

A potential therapeutic candidate for neutralizing SARS-CoV-2 infection is engineering high- affinity soluble ACE2 decoy proteins to compete for binding of the viral spike (S) protein. Previously, a deep mutational scan of ACE2 was performed and has led to the identification of a triple mutant ACE2 variant, named ACE22.v.2.4, that exhibits nanomolar affinity binding to the RBD domain of S. Using a recently developed transfer learning algorithm, TLmutation, we sought to identified other ACE2 variants, namely double mutants, that may exhibit similar binding affinity with decreased mutational load. Upon training a TLmutation model on the effects of single mutations, we identified several ACE2 double mutants that bind to RBD with tighter affinity as compared to the wild type, most notably, L79V;N90D that binds RBD with similar affinity to ACE22.v.2.4. The successful experimental validation of the double mutants demonstrated the use transfer and supervised learning approaches for engineering protein-protein interactions and identifying high affinity ACE2 peptides for targeting SARS-CoV-2.

The rarA gene as part of an expanded RecFOR recombination pathway: Negative epistasis and synthetic lethality with ruvB, recG, and recQ

The RarA protein, homologous to human WRNIP1 and yeast MgsA, is a AAA+ ATPase and one of the most highly conserved DNA repair proteins. With an apparent role in the repair of stalled or collapsed replication forks, the molecular function of this protein family remains obscure. Here, we demonstrate that RarA acts in late stages of recombinational DNA repair of post-replication gaps. A deletion of most of the rarA gene, when paired with a deletion of ruvB or ruvC, produces a growth defect, a strong synergistic increase in sensitivity to DNA damaging agents, cell elongation, and an increase in SOS induction. Except for SOS induction, these effects are all suppressed by inactivating recF, recO, or recJ, indicating that RarA, along with RuvB, acts downstream of RecA. SOS induction increases dramatically in a rarA ruvB recF/O triple mutant, suggesting the generation of large amounts of unrepaired ssDNA. The rarA ruvB defects are not suppressed (and in fact slightly increased) by recB inactivation, suggesting RarA acts primarily downstream of RecA in post-replication gaps rather than in double strand break repair. Inactivating rarA, ruvB and recG together is synthetically lethal, an outcome again suppressed by inactivation of recF, recO, or recJ. A rarA ruvB recQ triple deletion mutant is also inviable. Together, the results suggest the existence of multiple pathways, perhaps overlapping, for the resolution or reversal of recombination intermediates created by RecA protein in post-replication gaps within the broader RecF pathway. One of these paths involves RarA.

Exogenous Application of Low-Concentration Sugar Enhances Brassinosteroid Signaling for Skotomorphogenesis by Promoting BIN2 Degradation

In plants, seedling growth is subtly controlled by multiple environmental factors and endogenous phytohormones. The cross-talk between sugars and brassinosteroid (BR) signaling is known to regulate plant growth; however, the molecular mechanisms that coordinate hormone-dependent growth responses with exogenous sucrose in plants are incompletely understood. Skotomorphogenesis is a plant growth stage with rapid elongation of the hypocotyls. In the present study, we found that low-concentration sugars could improve skotomorphogenesis in a manner dependent on BR biosynthesis and TOR activation. However, accumulation of BZR1 in bzr1-1D mutant plants partially rescued the defects of skotomorphogenesis induced by the TOR inhibitor AZD, and these etiolated seedlings displayed a normal phenotype like that of wild-type seedlings in response to both sucrose and non-sucrose treatments, thereby indicating that accumulated BZR1 sustained, at least partially, the sucrose-promoted growth of etiolated seedlings (skotomorphogenesis). Moreover, genetic evidence based on a phenotypic analysis of bin2-3bil1bil2 triple-mutant and gain-of-function bin2–1 mutant plant indicated that BIN2 inactivation was conducive to skotomorphogenesis in the dark. Subsequent biochemical and molecular analyses enabled us to confirm that sucrose reduced BIN2 levels via the TOR–S6K2 pathway in etiolated seedlings. Combined with a determination of the cellulose content, our results indicated that sucrose-induced BIN2 degradation led to the accumulation of BZR1 and the enhancement of cellulose synthesis, thereby promoting skotomorphogenesis, and that BIN2 is the converging node that integrates sugar and BR signaling.

A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern

Saturation suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temperature of ~7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An additional derivative with three additional mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temperature exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 derivative showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qualitatively improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or additional components, and can be stored at room temperature. They are a useful modality to combat COVID-19, especially in remote and low-resource settings.

A Non-Canonical Function of Arabidopsis ERECTA Proteins in Gibberellin Signaling

The Arabidopsis ERECTA family (ERf) of leucine-rich repeat receptor-like kinases (LRR-RLKs), comprising ERECTA (ER), ERECTA-LIKE 1 (ERL1) and ERECTA-LIKE 2 (ERL2), control epidermal patterning, inflorescence architecture, stomata development, and hormonal signaling. Here we show that the er/erl1/erl2 triple mutant exhibits impaired gibberellin (GA) biosynthesis and perception alongside broad transcriptional changes. ERf proteins interact in the nucleus, via kinase domains, with the SWI3B subunit of the SWI/SNF chromatin remodeling complex (CRCs). The er/erl1/erl2 triple mutant exhibits reduced SWI3B protein level and affected nucleosomal chromatin structure. The ER kinase phosphorylates SWI3B in vitro, and the inactivation of all ERf proteins leads to the decreased phosphorylation of SWI3B protein in vivo. Correlation between DELLA overaccumulation and SWI3B proteasomal degradation together with the physical interaction of SWI3B with DELLA proteins explain the lack of RGA accumulation in the GA- and SWI3B-deficient erf mutant plants. Co-localization of ER and SWI3B on GID1 (GIBBERELLIN INSENSITIVE DWARF 1) DELLA target gene promoter regions and abolished SWI3B binding to GID1 promoters in er/erl1/erl2 plants supports the conclusion that ERf-SWI/SNF CRC interaction is important for transcriptional control of GA receptors. Thus, the involvement of ERf proteins in transcriptional control of gene expression, and observed similar features for human HER2 (Epidermal Growth Family Receptor-member), indicate an exciting target for further studies of evolutionarily conserved non-canonical functions of eukaryotic membrane receptors.ONE SENTENCE SUMMARYERECTA leucine-rich receptor-like kinase and SWI3B subunit of SWI/SNF chromatin remodeling complex cooperate in direct transcriptional control of GID1 genes in Arabidopsis.

Investigating molecular mechanisms of insecticide resistance in the Eastern Democratic Republic of the Congo

Background Malaria vector control in the Democratic Republic of the Congo is plagued by several major challenges, including inadequate infrastructure, lack of access to health care systems and preventative measures, and more recently the widespread emergence of insecticide resistance among Anopheles mosquitoes. Across 26 provinces, insecticide resistance has been reported from multiple sentinel sites. However, to date, investigation of molecular resistance mechanisms among Anopheles vector populations in DRC has been more limited. Methods Adult Anopheles gambiae sensu lato (s.l.) and Anopheles funestus s.l. were collected from two sites in Sud-Kivu province and one site in Haut-Uélé province and PCR-screened for the presence of 11 resistance mutations, to provide additional information on frequency of resistance mechanisms in the eastern DRC, and to critically evaluate the utility of these markers for prospective country-wide resistance monitoring. Results L1014F-kdr and L1014S-kdr were present in 75.9% and 56.7% of An. gambiae s.l. screened, respectively, with some individuals harbouring both resistant alleles. Across the three study sites, L43F-CYP4J5 allele frequency ranged from 0.42 to 0.52, with evidence for ongoing selection. G119S-ace1 was also identified in all sites but at lower levels. A triple mutant haplotype (comprising the point mutation CYP6P4-I236M, the insertion of a partial Zanzibar-like transposable element and duplication of CYP6AA1) was present at high frequencies. In An. funestus s.l. cis-regulatory polymorphisms in CYP6P9a and CYP6P9b were detected, with allele frequencies ranging from 0.82 to 0.98 and 0.65 to 0.83, respectively. Conclusions This study screened the most up-to-date panel of DNA-based resistance markers in An. gambiae s.l. and An. funestus s.l. from the eastern DRC, where resistance data is lacking. Several new candidate markers (CYP4J5, G119S-ace1, the triple mutant, CYP6P9a and CYP6P9b) were identified, which are diagnostic of resistance to major insecticide classes, and warrant future, larger-scale monitoring in the DRC to inform vector control decisions by the National Malaria Control Programme.

Tapetal 3-Ketoacyl-Coenzyme A Synthases Are Involved in Pollen Coat Lipid Accumulation for Pollen-Stigma Interaction in Arabidopsis

Pollen coat lipids form an outer barrier to protect pollen itself and play essential roles in pollen-stigma interaction. However, the precise molecular mechanisms underlying the production, deposition, regulation, and function of pollen coat lipids during anther development remain largely elusive. In lipid metabolism, 3-ketoacyl-coenzyme A synthases (KCS) are involved in fatty acid elongation or very-long-chain fatty acid (VLCFA) synthesis. In this study, we identified six members of the Arabidopsis KCS family expressed in anther. Among them, KCS7, KCS15, and KCS21 were expressed in tapetal cells at anther stages 8–10. Further analysis demonstrated that they act downstream of male sterility 1 (MS1), a regulator of late tapetum development. The kcs7/15/21 triple mutant is fertile. Both cellular observation and lipid staining showed pollen coat lipid was decreased in kcs7/15/21 triple mutant. After landing on stigma, the wild-type pollen grains were hydrated for about 5 min while the kcs7/15/21 triple mutant pollen took about 10 min to hydrate. Pollen tube growth of the triple mutant was also delayed. These results demonstrate that the tapetum-localized KCS proteins are involved in the accumulation of pollen coat lipid and reveal the roles of tapetal-derived pollen coat lipid for pollen-stigma interaction.