Chimeric Mutants Research Articles

Differentiation in detoxification gene complements, including neofunctionalization of duplicated cytochrome P450 genes, between lineages of cotton bollworm, Helicoverpa armigera.

Here we investigate the evolutionary dynamics of five enzyme superfamilies (CYPs, GSTs, UGTs, CCEs and ABCs) involved in detoxification in Helicoverpa armigera. The reference assembly for an African isolate of the major lineages, H. a. armigera, has 373 genes in the five superfamilies. Most of its CYPs, GSTs, UGTs and CCEs and a few of its ABCs occur in blocks and most of the clustered genes are in subfamilies specifically implicated in detoxification. Most of the genes have orthologues in the reference genome for the Oceania lineage, H. a. conferta. However, clustered orthologues and subfamilies specifically implicated in detoxification show greater sequence divergence and less constraint on non-synonymous differences between the two assemblies than do other members of the five superfamilies. Two duplicated CYPs, which were found in the H. a. armigera but not H. a. conferta reference genome, were also missing in 16 Chinese populations spanning two different lineages of H. a. armigera. The enzyme produced by one of these duplicates has higher activity against esfenvalerate than a previously described chimeric CYP mutant conferring pyrethroid resistance. Various transposable elements were found in the introns of most detoxification genes, generating diverse gene structures. Extensive resequencing data for the Chinese H. a. armigera and H. a. conferta lineages also revealed complex copy number polymorphisms in 17 CCE001s in a cluster also implicated in pyrethroid metabolism, with substantial haplotype differences between all three lineages. Our results suggest that cotton bollworm has a versatile complement of detoxification genes which are evolving in diverse ways across its range.

Effects of carbohydrate binding module of pullulanase type I on the raw starch rearrangement by enhancing the hydrolysis activity

Starch, a versatile ingredient used in various industries, often requires modification for improvements in its physicochemical and functional properties for utilization. Although carbohydrate-binding modules (CBMs) are non-catalytic domains that recognize and bind to the substrate without any enzymatic activity, CBMs are commonly found in the starch-modifying enzyme, pullulanase (PulA). Herein, we identified two PulAs from Deinococcus radiodurans and Deinococcus wulumuqiensis that differed in activity only by CBM composition. Structural comparison analysis showed unique substrate-binding coordination of CBMb1. The analysis with a deletion mutant (ΔCBMb1-DrPulA), and a chimeric mutant (CBMb1-DwPulA) revealed that CBMb1 can improve amylopectin but not pullulan hydrolysis. Moreover, CBMb1 introduction increased the hydrolysis activity of raw waxy corn starch by 60% which leads to the formation of distinct α-glucan microparticles (GMP). GMP produced using CBMb1-DwPulA exhibited B-type crystallinity, similar to that produced by DwPulA. However, a relatively high proportion of short-length maltooligosaccharides was observed in the GMP produced by CBMb1-DwPulA compared with that produced by DwPulA. This contributed to the formation of smaller particles, high colloidal dispersion stability, and increased the absolute value of the zeta potential. Altogether, our findings showed that Deinococcus sp.-derived CBMs can be used to customize GMPs by changing the debranching pattern.

Rationalization design, soluble expression and PEG modification of highly active recombinant human-porcine uricase mutant protein

Uric acid is the end product of purine metabolism in humans due to inactivation of the uricase determined by the mutated uricase gene. Uricase catalyzes the conversion of uric acid into water-soluble allantoin that is easily excreted by the kidneys. Hyperuricemia occurs when the serum concentration of uric acid exceeds its solubility (7 mg/dL). However, modifications to improve the uricase activity is under development for treating the hyperuricemia. Here we designed 7 types of human-porcine chimeric uricase by multiple sequence comparisons and targeted mutagenesis. An optimal human-porcine chimeric uricase mutant (uricase-10) with both high activity (6.33 U/mg) and high homology (91.45 %) was determined by enzyme activity measurement. The engineering uricase was further modified with PEGylation to improve the stability of recombinant protein drugs and reduce immunogenicity, uricase-10 could be more suitable for the treatment of gout and hyperuricemia theoretically.

Structural differences between the closely related RNA helicases, UAP56 and URH49, fashion distinct functional apo-complexes

mRNA export is an essential pathway for the regulation of gene expression. In humans, closely related RNA helicases, UAP56 and URH49, shape selective mRNA export pathways through the formation of distinct complexes, known as apo-TREX and apo-AREX complexes, and their subsequent remodeling into similar ATP-bound complexes. Therefore, defining the unidentified components of the apo-AREX complex and elucidating the molecular mechanisms underlying the formation of distinct apo-complexes is key to understanding their functional divergence. In this study, we identify additional apo-AREX components physically and functionally associated with URH49. Furthermore, by comparing the structures of UAP56 and URH49 and performing an integrated analysis of their chimeric mutants, we exhibit unique structural features that would contribute to the formation of their respective complexes. This study provides insights into the specific structural and functional diversification of these two helicases that diverged from the common ancestral gene Sub2.

Force transmission by retrograde actin flow-induced dynamic molecular stretching of Talin

Force transmission at integrin-based adhesions is important for cell migration and mechanosensing. Talin is an essential focal adhesion (FA) protein that links F-actin to integrins. F-actin constantly moves on FAs, yet how Talin simultaneously maintains the connection to F-actin and transmits forces to integrins remains unclear. Here we show a critical role of dynamic Talin unfolding in force transmission. Using single-molecule speckle microscopy, we found that the majority of Talin are bound only to either F-actin or the substrate, whereas 4.1% of Talin is linked to both structures via elastic transient clutch. By reconstituting Talin knockdown cells with Talin chimeric mutants, in which the Talin rod subdomains are replaced with the stretchable β-spectrin repeats, we show that the stretchable property is critical for force transmission. Simulations suggest that unfolding of the Talin rod subdomains increases in the linkage duration and work at FAs. This study elucidates a force transmission mechanism, in which stochastic molecular stretching bridges two cellular structures moving at different speeds.

The replicase protein nsp2 of Chinese highly pathogenic porcine reproductive and respiratory virus is involved in protective immunity by promoting viral clearance

The genome segment for replicase protein nsp2 represents the fastest evolving region of porcine reproductive and respiratory syndrome virus (PRRSV), and our previous studies have shown that the PRRSV nsp2 genetic variation contributes to poor cross-neutralization. By using in vitro antibody absorption assay, here we show that the papain-like protease 2 (PLP2) domain of nsp2 is a target of neutralizing antibodies. This was further verified by cross-neutralization assay with a series of inter-lineage chimeric mutants between the Chinese highly pathogenic PRRSV (HP-PRRSV) strain JXwn06 and the low virulent NADC30-like strain CHsx1401 (lineage 1). The role of nsp2 in protective immunity was subsequently tested in a one-month SPF piglet model by immunizing the piglets with CHsx1401 or its derivatives carrying JXwn06 structural protein region (SP) alone (CHsx1401-SPJX) or in combination with PLP2 region (CHsx1401-SPplp2JX), or the whole nsp2 region (CHsx1401-SPnsp2JX), followed by challenge with JXwn06 at 42 days post immunization, a time point when the viremia was undetectable. All chimera groups were protected from the challenge by JXwn06, whereas the group CHsx1401 failed to provide beneficial protection. Interestingly, the group CHsx1401-SPnsp2JX, but not CHsx1401-SPplp2JX, showed the lowest lung microscopic lesions and viral tissue load. Significantly, the vaccine virus CHsx1401-SPnsp2JX was undetectable in the examined tissues, and so was for the challenge virus except for one piglet, highlighting an important role of HP-PRRSV nsp2 in promoting viral clearance. The findings provide insight into the mechanisms underlying the protective immunity against PRRSV and have important implications in PRRSV vaccine development.

Structural, Functional, and Mutational Studies of a Potent Subtilisin Inhibitor from Budgett's Frog, Lepidobatrachus laevis.

Subtilases play a significant role in microbial pathogen infections by degrading the host proteins. Subtilisin inhibitors are crucial in fighting against these harmful microorganisms. LL-TIL, from skin secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays demonstrated that LL-TIL acts as a slow-tight binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition constants of 91 pM and 2.4 nM, respectively. The solution structures of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type fold with a canonical conformation of a reactive site loop (RSL). The structure of the LL-TIL-subtilisin complex and molecular dynamics (MD) simulations provided an in-depth view of the structural basis of inhibition. NMR relaxation data and molecular dynamics simulations indicated a rigid conformation of RSL, which does not alter significantly upon subtilisin binding. The energy calculation for subtilisin inhibition predicted Ile31 as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL broadened the inhibitory profile and attenuated subtilisin inhibition by 2 orders of magnitude. These results provide a template to engineer more specific and potent TIL-type subtilisin inhibitors.

CRISPR/Cas9-mediated disruption of CjACOS5 confers no-pollen formation on sugi trees (Cryptomeria japonica D. Don)

Sugi (Cryptomeria japonica D. Don) is an economically important coniferous tree in Japan. However, abundant sugi pollen grains are dispersed and transported by the wind each spring and cause a severe pollen allergy syndrome (Japanese cedar pollinosis). The use of pollen-free sugi that cannot produce pollen has been thought as a countermeasure to Japanese cedar pollinosis. The sugi CjACOS5 gene is an ortholog of Arabidopsis ACOS5 and rice OsACOS12, which encode an acyl-CoA synthetase that is involved in the synthesis of sporopollenin in pollen walls. To generate pollen-free sugi, we mutated CjACOS5 using the CRISPR/Cas9 system. As a result of sugi transformation mediated by Agrobacterium tumefaciens harboring the CjACOS5-targeted CRISPR/Cas9 vector, 1 bp-deleted homo biallelic mutant lines were obtained. Chimeric mutant lines harboring both mutant and wild-type CjACOS5 genes were also generated. The homo biallelic mutant lines had no-pollen in male strobili, whereas chimeric mutant lines had male strobili with or without pollen grains. Our results suggest that CjACOS5 is essential for the production of pollen in sugi and that its disruption is useful for the generation of pollen-free sugi. In addition to conventional transgenic technology, genome editing technology, including CRISPR/Cas9, can confer new traits on sugi.

The Genetic Variation of Porcine Reproductive and Respiratory Syndrome Virus Replicase Protein nsp2 Modulates Viral Virulence and Persistence.

Fast evolution in the field of the replicase nsp2 represents a most prominent feature of porcine reproductive and respiratory syndrome virus (PRRSV). Here, we determined its biological significance in viral pathogenesis by constructing interlineage chimeric mutants between the Chinese highly pathogenic PRRSV (HP-PRRSV) strain JXwn06 (lineage 8) and the low-virulent NADC30-like strain CHsx1401 (lineage 1). Replacement with nsp2 from JXwn06 was surprisingly lethal to the backbone virus CHsx1401, but combined substitution with the structural protein-coding region (SP) gave rise to viable virus CHsx1401-SPnsp2JX. Meanwhile, a derivative carrying only the SP region (CHsx1401-SPJX) served as a control. Subsequent animal experiments revealed that acquisition of SP alone (CHsx1401-SPJX) did not allow CHsx1401 to gain much virulence, but additional swapping of HP-PRRSV nsp2 (CHsx1401-SPnsp2JX) enabled CHsx1401 to acquire some properties of HP-PRRSV, exemplified by prolonged high fever, microscopic lung hemorrhage, and a significant increase in proinflammatory cytokines in the acute stage. Consistent with this was the transcriptomic analysis of persistently infected secondary lymphoid tissues that revealed a much stronger induction of host cellular immune responses in this group and identified several core immune genes (e.g., TLR4, IL-1β, MPO, etc.) regulated by HP-PRRSV nsp2. Interestingly, immune activation status in the individual groups correlated well with the rate of viremia clearance and viral tissue load reduction. Overall, the above results suggest that the Chinese HP-PRRSV nsp2 is a critical virulence regulator and highlight the importance of nsp2 genetic variation in modulating PRRSV virulence and persistence via immune modulation. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has been a major threat to the world swine industry. In the field, rapid genetic variations (e.g., deletion, mutation, recombination, etc.) within the nsp2 region present an intriguing conundrum to PRRSV biology and pathogenesis. By making chimeric mutants, here, we show that the Chinese highly pathogenic PRRSV (HP-PRRSV) nsp2 is a virulence factor and a much stronger inducer of host immune responses (e.g., inflammation) than its counterpart, currently epidemic, NADC30-like strains. Differences in the ability to modulate host immunity provide insight into the mechanisms of why NADC30-like strains and their derivatives are rising to be the dominant viruses, whereas the Chinese HP-PRRSV strains gradually give away center stage in the field. Our results have important implications in understanding PRRSV evolution, interlineage recombination, and persistence.

3′ UTR is critical for viral RNA accumulation of jasmine virus H

Jasmine virus H (JaVH) is a newly reported viral pathogen of jasmine in China and USA. To study the viral gene function and pathogenic mechanism, a full-length infectious clone of JaVH (pXT-JaVHFJ) was constructed under the control of the cauliflower mosaic virus 35S promoter. pXT-JaVHFJ induced a systemic infection in Nicotiana benthamiana plants by Agro-infiltration, which demonstrated that pXT-JaVHFJ was biologically active. Jasmine showed yellow spots after rubbing with total RNA extracted from Agro-infiltrated N. benthamiana, indicating that JaVH was highly associated with yellow mosaic symptoms observed on jasmine. To investigate the occurrence and mutations of the virus, jasmine samples were collected from eight provinces of China and were tested for JaVH. The samples that were tested positive for JaVH were used to determine the complete genome sequences. They were comprised of 3867 or 3868 nucleotides and their genome organizations resembled that we previous reported for JaVH-FJ. Phylogenetic analyses and sequence comparisons suggest that the eight virus isolates were close isolates of JaVH-FJ and the isolate from Jilin Province was most closely related to JaVH-FJ with 99.2% nucleotide identity over the entire genome and 99.7% identity of coat protein. Further comparative analyses of JaVH-FJ and JaVH-JL revealed additional nucleotide differences in the 3′-untranslated region (3′ UTR). An infectious clone of JaVH-JL and chimeric mutants containing JaVH-FJ or JaVH-JL 3′ UTRs were then constructed for further study. The differential accumulation of JaVH with distinct 3′ UTR suggested that the 3′ UTR of JaVH plays a crucial role in viral RNA accumulation.

SARS-CoV-2 evolution influences GBP and IFITM sensitivity

SARS-CoV-2 spike requires proteolytic processing for viral entry. A polybasic furin-cleavage site (FCS) in spike, and evolution toward an optimized FCS by dominant variants of concern (VOCs), are linked to enhanced infectivity and transmission. Here we show interferon-inducible restriction factors Guanylate-binding proteins (GBP) 2 and 5 interfere with furin-mediated spike cleavage and inhibit the infectivity of early-lineage isolates Wuhan-Hu-1 and VIC. By contrast, VOCs Alpha and Delta escape restriction by GBP2/5 that we map to the spike substitution D614G present in these VOCs. Despite inhibition of spike cleavage, these viruses remained sensitive to plasma membrane IFITM1, but not endosomal IFITM2 and 3, consistent with a preference for TMPRSS2-dependent plasma membrane entry. Strikingly, we find that Omicron is unique among VOCs, being sensitive to restriction factors GBP2/5, and also IFITM1, 2, and 3. Using chimeric spike mutants, we map the Omicron phenotype and show that the S1 domain determines Omicron's sensitivity to GBP2/5, whereas the S2' domain determines its sensitivity to endosomal IFITM2/3 and preferential use of TMPRSS2-independent entry. We propose that evolution of SARS-CoV-2 for the D614G substitution has allowed for escape from GBP restriction factors, but the selective pressures on Omicron for spike changes that mediate antibody escape, and altered tropism, have come at the expense of increased sensitivity to innate immune restriction factors that target virus entry.

Endoplasmic reticulum protein BIK binds to and inhibits mitochondria-localized antiapoptotic proteins

The proapoptotic BCL-2 homology (BH3)-only endoplasmic reticulum (ER)-resident protein BCL-2 interacting killer (BIK) positively regulates mitochondrial outer membrane permeabilization, the point of no return in apoptosis. It is generally accepted that BIK functions at a distance from mitochondria by binding and sequestering antiapoptotic proteins at the ER, thereby promoting ER calcium release. Although BIK is predominantly localized to the ER, we detect by fluorescence lifetime imaging microscopy-FRET microscopy, BH3 region-dependent direct binding between BIK and mitochondria-localized chimeric mutants of the antiapoptotic proteins BCL-XL and BCL-2 in both baby mouse kidney (BMK) and MCF-7cells. Direct binding was accompanied by cell type-specific differential relocalization in response to coexpression of either BIK or one of its target binding partners, BCL-XL, when coexpressed in cells. In BMK cells with genetic deletion of both BAX and BAK (BMK-double KO), our data suggest that a fraction of BIK protein moves toward mitochondria in response to the expression of a mitochondria-localized BCL-XL mutant. In contrast, in MCF-7cells, our data suggest that BIK is localized at both ER and mitochondria-associated ER membranes and binds to the mitochondria-localized BCL-XL mutant via relocalization of BCL-XL to ER and mitochondria-associated ER membrane. Rather than functioning at a distance, our data suggest that BIK initiates mitochondrial outer membrane permeabilization via direct interactions with ER and mitochondria-localized antiapoptotic proteins, which occur via ER-mitochondria contact sites, and/or by relocalization of either BIK or antiapoptotic proteins in cells.

Subfunctionalization of a monolignol to a phytoalexin glucosyltransferase is accompanied by substrate inhibition

Uridine diphosphate-dependent glycosyltransferases (UGTs) mediate the glycosylation of plant metabolites, thereby altering their physicochemical properties and bioactivities. Plants possess numerous UGT genes, with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics, but the biological function and molecular basis of these phenomena are not fully understood. The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition (Ki) at 4 μM scopoletin, whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 μM. We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs. A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reduced scopoletin substrate inhibition, whereas its monolignol glycosylation activity was almost unaffected. Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation, leading to enhanced promiscuity, which was accompanied by substrate inhibition. Studies of 3D structures identified open and closed UGT conformers, allowing visualization of the dynamics of conformational changes that occur during catalysis. Previously postulated substrate access tunnels likely serve as drainage channels. The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release. Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions. The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.

The mechanisms of siRNA selection by plant Argonaute proteins triggering DNA methylation.

The model plant Arabidopsis thaliana encodes as many as ten Argonaute proteins (AGO1-10) with different functions. Each AGO selectively loads a set of small RNAs by recognizing their length and 5' nucleotide identity to properly regulate target genes. Previous studies showed that AGO4 and AGO6, key factors in DNA methylation, incorporate 24-nt small-interfering RNAs with 5' adenine (24A siRNAs). However, it has been unclear how these AGOs specifically load 24A siRNAs. Here, we biochemically investigated the siRNA preference of AGO4, AGO6 and their chimeric mutants. We found that AGO4 and AGO6 use distinct mechanisms to preferentially load 24A siRNAs. Moreover, we showed that the 5' A specificity of AGO4 and AGO6 is not determined by the previously known nucleotide specificity loop in the MID domain but rather by the coordination of the MID and PIWI domains. These findings advance our mechanistic understanding of how small RNAs are accurately sorted into different AGO proteins in plants.

The SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant.

Recent studies have revealed the unique virological characteristics of Omicron, particularly those of its spike protein, such as less cleavage efficacy in cells, reduced ACE2 binding affinity, and poor fusogenicity. However, it remains unclear which mutation(s) determine these three virological characteristics of Omicron spike. Here, we show that these characteristics of the Omicron spike protein are determined by its receptor-binding domain. Of interest, molecular phylogenetic analysis revealed that acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidated that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue of another protomer in the spike trimer, conferring the attenuated cleavage efficiency and fusogenicity of Omicron spike. Our data shed light on the evolutionary events underlying the emergence of Omicron at the molecular level.

Identification of viral genes involved in pepper mottle virus replication and symptom development in Nicotiana benthamiana.

Pepper mottle virus (PepMoV) infects primarily Capsicum species, including pepper and bell pepper which are important vegetable and spice crops in Korea. We have previously collected 13 PepMoV isolates from nine regions comprising five provinces, causing different symptoms on inoculated indicator host plants in Korea. To further identify the responsible symptom determinant(s) and explore viral protein functions of PepMoV, two out of 13 isolates, including 134 and 205136, were used in this study. Isolate 134 causes necrosis and yellowing, while 205136 causes severe mottle and yellowing symptoms on Nicotiana benthamiana. All chimeric and site-directed mutants contain the PepMoV 134 genome as a backbone with specific regions switched for those from counterparts of PepMoV 205136. Effects of all mutants compared with 134 after inoculation onto N. benthamiana by agroinfiltration. Results from our study provide direct evidence that the helper component-proteinase (HC-Pro) and the nuclear inclusion protein b (NIb)-coat protein (CP) regions are involved in virus accumulation and symptom determinants. In addition, we mapped to amino acid residues tyrosine, glycine, and leucine at position 360, 385, and 527, respectively, in the HC-Pro region participate in faster viral accumulation or movement in the plant. The residue valine at position 2773 of NIb plays an essential role in isolate 134 symptom development. As part of this study, we seek to gain insight into viral factors involved in the PepMoV infection cycle and a better understanding of plant-virus interactions. These findings complement the insufficiency of the gene function study of the PepMoV virus and provide a novel perspective for the protein function study of the Potyvirus.

CRISPR/Cas9-mediated genome editing of RsGL1a and RsGL1b in radish (Raphanus sativus L.).

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool widely used for genome editing in various organisms, including plants. It introduces and facilitates the study of rare genetic mutations in a short time and is a potent tool to assist in plant molecular breeding. Radish (Raphanus sativus L.) is an important Brassicaceae vegetable cultivated and consumed worldwide. However, the application of the CRISPR/Cas9 system is limited by the absence of an efficient transformation system in radish. This study aimed to establish a CRISPR/Cas9 system in radish employing the Agrobacterium-mediated genetic transformation system reported recently. For this purpose, we performed genome editing using the CRISPR/Cas9 system targeting the GLABRA1 (GL1) orthologs, RsGL1a and RsGL1b, that induces leaf trichome formation in radish. A Cas9/single guide RNA (sgRNA) vector with a common sgRNA corresponding to RsGL1a and RsGL1b was transferred. A total of eight T0 plants were analyzed, of which six (editing efficiency 75%) had a mutated RsGL1a, five (62.5%) had a mutated RsGL1b, and five showed mutations in both RsGL1a and RsGL1b. Most mutations in T0 plants were short (<3 bp) deletions or insertions, causing frameshift mutations that might produce non-functional proteins. Chimeric mutations were detected in several T0 generation plants. In the T1 generation, the hairless phenotype was observed only in plants with knockout mutations in both RsGL1a and RsGL1b. The majority of mutant alleles in T0 plants, with the exception of the chimeric mutant plants detected, were stably inherited in the T1 generation. In conclusion, we successfully knocked out RsGL1a and RsGL1b using the CRISPR/Cas9 system and demonstrated that both RsGL1a and RsGL1b independently contribute to the induction of leaf trichome formation in radish. In this study, genome-edited plants without T-DNA, which are useful as breeding material, were obtained. The findings prove the feasibility of genome editing in radish using a CRISPR/Cas9 system that could accelerate its molecular breeding to improve agronomically desirable traits.

Structures and gating mechanisms of human bestrophin anion channels

Bestrophin-1 (Best1) and bestrophin-2 (Best2) are two members of the bestrophin family of calcium (Ca2+)-activated chloride (Cl−) channels with critical involvement in ocular physiology and direct pathological relevance. Here, we report cryo-EM structures of wild-type human Best1 and Best2 in various states at up to 1.8 Å resolution. Ca2+-bound Best1 structures illustrate partially open conformations at the two Ca2+-dependent gates of the channels, in contrast to the fully open conformations observed in Ca2+-bound Best2, which is in accord with the significantly smaller currents conducted by Best1 in electrophysiological recordings. Comparison of the closed and open states reveals a C-terminal auto-inhibitory segment (AS), which constricts the channel concentrically by wrapping around the channel periphery in an inter-protomer manner and must be released to allow channel opening. Our results demonstrate that removing the AS from Best1 and Best2 results in truncation mutants with similar activities, while swapping the AS between Best1 and Best2 results in chimeric mutants with swapped activities, underlying a key role of the AS in determining paralog specificity among bestrophins.

Gibbin mesodermal regulation patterns epithelial development.

Proper ectodermal patterning during human development requires previously identified transcription factors such as GATA3 and p63, as well as positional signalling from regional mesoderm1-6. However, the mechanism by which ectoderm and mesoderm factors act to stably pattern gene expression and lineage commitment remains unclear. Here we identify the protein Gibbin,encoded by the Xia-Gibbs AT-hook DNA-binding-motif-containing 1 (AHDC1) disease gene7-9, as a key regulator of early epithelial morphogenesis. We find that enhancer- or promoter-bound Gibbin interacts with dozens of sequence-specific zinc-finger transcription factors and methyl-CpG-binding proteins to regulate the expression of mesoderm genes. The loss of Gibbin causes an increase in DNA methylation at GATA3-dependent mesodermal genes, resulting in a loss of signalling between developing dermal and epidermal cell types. Notably, Gibbin-mutant human embryonic stem-cell-derived skin organoids lack dermal maturation, resulting in p63-expressing basal cells that possess defective keratinocyte stratification. In vivo chimeric CRISPR mouse mutants reveal a spectrum of Gibbin-dependent developmental patterning defects affecting craniofacial structure, abdominal wall closure and epidermal stratification that mirror patient phenotypes. Our results indicate that the patterning phenotypes seen in Xia-Gibbs and related syndromes derive from abnormal mesoderm maturation as a result of gene-specific DNA methylation decisions.

Development of chimeric forms of the matrix metalloproteinase 2 collagen binding domain as artificial membrane binding proteins for targeting stem cells to cartilage lesions in osteoarthritic joints

Targeting stem cells to cartilage lesions has the potential to enhance engraftment and chondrogenesis. Denatured type II collagen fibrils (gelatin) are exposed in lesions at the surface of osteoarthritic articular cartilage and are therefore ideal target sites. We have designed and investigated chimeric mutants of the three modules of the MMP-2 collagen binding domain (CBD) as potential ligands for stem cell targeting. We expressed full-length CBD for the first time and used it to identify the most important amino acid residues for binding to gelatin. Module 2 of CBD had the highest affinity binding to both Type I and Type II gelatin, whereas module 1 showed specificity for type II gelatin and module 3 for type I gelatin. We went on to generate chimeric forms of CBD consisting of three repeats of module 1 (111), module 2 (222) or module 3 (333). 111 lacked solubility and could not be further characterised. However 222 was found to bind to type II gelatin 14 times better than CBD, suggesting it would be optimal for attachment to cartilage lesions, whilst 333 was found to bind to type I gelatin 12 times better than CBD, suggesting it would be optimal for attachment to lesions in type I collagen-rich tissues. We coated 222 onto the external membrane of Mesenchymal Stem Cells and demonstrated higher attachment of the coated cells to type II gelatin than uncoated cells. We conclude that the three modules of CBD each have specific biological properties that can be exploited for targeting stem cells to cartilage lesions and other pathological sites.