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Aminoacylation by tRNA synthetase is a crucial part of protein synthesis and is widely recognized as a therapeutic target for drug development. Unlike the arginyl‐tRNA synthetases (ArgRSs) reported previously, here, we report an ArgRS of Leishmania donovani (LdArgRS) that can follow the canonical two‐step aminoacylation process. Since a previously uncharacterized insertion region is present within its catalytic domain, we implemented the splicing by overlap extension PCR (SOE–PCR) method to create a deletion mutant (ΔIns‐LdArgRS) devoid of this region to investigate its function. Notably, the purified LdArgRS and ΔIns‐LdArgRS exhibited different oligomeric states along with variations in their enzymatic activity. The full‐length protein showed better catalytic efficiency than ΔIns‐LdArgRS, and the insertion region was identified as the tRNA binding domain. In addition, a benzothiazolo‐coumarin derivative (Comp‐7j) possessing high pharmacokinetic properties was recognized as a competitive and more specific inhibitor of LdArgRS than its human counterpart. Removal of the insertion region altered the mode of inhibition for ΔIns‐LdArgRS and caused a reduction in the inhibitor's binding affinity. Both purified proteins depicted variances in the secondary structural content upon ligand binding and thus, thermostability. Apart from the trypanosomatid‐specific insertion and Rossmann fold motif, LdArgRS revealed typical structural characteristics of ArgRSs, and Comp‐7j was found to bind within the ATP binding pocket. Furthermore, the placement of tRNAArg near the insertion region enhanced the stability and compactness of LdArgRS compared to other ligands. This study thus reports a unique ArgRS with respect to catalytic as well as structural properties, which can be considered a plausible drug target for the derivation of novel anti‐leishmanial agents. [ABSTRACT FROM AUTHOR]

Summary: N6‐methyladenonsine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) and plays critical roles in mRNA processing and metabolism. However, perturbation of individual m6A modification to reveal its function and the phenotypic effects is still lacking in plants. Here, we describe the construction and characterization of programmable m6A editing tools by fusing the m6A writers, the core catalytic domain of the MTA and MTB complex, and the AlkB homologue 5 (ALKBH5) eraser, to catalytically dead Cas13a (dCas13a) to edit individual m6A sites on mRNAs. We demonstrated that our m6A editors could efficiently and specifically deposit and remove m6A modifications on specific RNA transcripts in both Nicotiana benthamiana and Arabidopsis thaliana. Moreover, we found that targeting SHORT‐ROOT (SHR) transcripts with a methylation editor could significantly increase its m6A levels with limited off‐target effects and promote its degradation. This leads to a boost in plant growth with enlarged leaves and roots, increased plant height, plant biomass, and total grain weight in Arabidopsis. Collectively, these findings suggest that our programmable m6A editing tools can be applied to study the functions of individual m6A modifications in plants, and may also have potential applications for future crop improvement. [ABSTRACT FROM AUTHOR]

The Byr2 kinase of fission yeast Schizosaccharomyces pombe is recruited to the membrane with the assistance of Ras1. Byr2 is also negatively regulated by 14-3-3 proteins encoded by rad24 and rad25. We conducted domain and mutational analysis of Byr2 to determine which region is critical for its binding to 14-3-3 proteins. Rad24 and Rad25 bound to both the Ras interaction domain in the N-terminus and to the C-terminal catalytic domain of Byr2. When amino acid residues S87 and T94 of the Ras-interacting domain of Byr2 were mutated to alanine, Rad24 could no longer bind to Byr2. S402, S566, S650, and S654 mutations in the C-terminal domain of Byr2 also abolished its interaction with Rad24 and Rad25. More than three mutations in the C-terminal domain were required to abolish completely its interaction with 14-3-3 protein, suggesting that multiple residues are involved in this interaction. Expression of the N-terminal domain of Byr2 in wild-type cells lowered the mating ratio, because it likely blocked the interaction of Byr2 with Ste4 and Ras1, whereas expression of the catalytic domain of Byr2 increased the mating ratio as a result of freeing from intramolecular regulation by the N-terminal domain of Byr2. The S87A and T94A mutations of Byr2 increased the mating ratio and attenuated inhibition of Byr2 by Rad24; therefore, these two amino acids are critical for its regulation by Rad24. S566 of Byr2 is critical for activity of Byr2 but not for its interaction with 14-3-3 proteins. In this study, we show that 14-3-3 proteins interact with two separate domains in Byr2 as negative regulators. [ABSTRACT FROM AUTHOR]

Enzymes play a key role in natural mineralization, but their function is disabling in bone metabolism when patients suffer severe degenerations like hypophosphatasia. To regulate bone homeostasis, a self‐activating phosphorus‐rich system is proposed on calcium glycerophosphate (CaGP) modified polyurethane (PU) fibrous substrate, wherein ample enzyme catalytic domains are imported with intestinal alkaline phosphatase (IALP) chelating and activating by calcium in situ that is testified by chemical potential evaluation. Therefore, Ca‐bonding IALP catalyzes organophosphate to phosphate locally to achieve a calcified symbiotic state, regulating mineralization and accelerating osteoinduction in vitro and in vivo. The self‐activating enzyme system promotes early osteogenic differentiation and angiogenesis, resulting in rapid healing of cranial defect with a "long‐tail" effect compared to an enzyme‐assisted system. Transcriptomic and metabolomic analyses demonstrate positive associations with neovascularization and calcium/phosphorus metabolism, specifically upregulated glycerophospholipids and calcium ions in favor of osteogenic differentiation under cytokine‐free conditions. These results provide a potential enzyme‐activating therapy in bone homeostasis and regeneration. [ABSTRACT FROM AUTHOR]

One of the most recent advances in the genome editing field has been the addition of "TALE Base Editors", an innovative platform for cell therapy that relies on the deamination of cytidines within double strand DNA, leading to the formation of an uracil (U) intermediate. These molecular tools are fusions of transcription activator-like effector domains (TALE) for specific DNA sequence binding, split-DddA deaminase halves that will, upon catalytic domain reconstitution, initiate the conversion of a cytosine (C) to a thymine (T), and an uracil glycosylase inhibitor (UGI). We developed a high throughput screening strategy capable to probe key editing parameters in a precisely defined genomic context in cellulo, excluding or minimizing biases arising from different microenvironmental and/or epigenetic contexts. Here we aimed to further explore how target composition and TALEB architecture will impact the editing outcomes. We demonstrated how the nature of the linker between TALE array and split DddAtox head allows us to fine tune the editing window, also controlling possible bystander activity. Furthermore, we showed that both the TALEB architecture and spacer length separating the two TALE DNA binding regions impact the target TC editing dependence by the surrounding bases, leading to more restrictive or permissive editing profiles. [ABSTRACT FROM AUTHOR]

One of the most recent advances in the genome editing field has been the addition of "TALE Base Editors", an innovative platform for cell therapy that relies on the deamination of cytidines within double strand DNA, leading to the formation of an uracil (U) intermediate. These molecular tools are fusions of transcription activator-like effector domains (TALE) for specific DNA sequence binding, split-DddA deaminase halves that will, upon catalytic domain reconstitution, initiate the conversion of a cytosine (C) to a thymine (T), and an uracil glycosylase inhibitor (UGI). We developed a high throughput screening strategy capable to probe key editing parameters in a precisely defined genomic context in cellulo, excluding or minimizing biases arising from different microenvironmental and/or epigenetic contexts. Here we aimed to further explore how target composition and TALEB architecture will impact the editing outcomes. We demonstrated how the nature of the linker between TALE array and split DddAtox head allows us to fine tune the editing window, also controlling possible bystander activity. Furthermore, we showed that both the TALEB architecture and spacer length separating the two TALE DNA binding regions impact the target TC editing dependence by the surrounding bases, leading to more restrictive or permissive editing profiles. [ABSTRACT FROM AUTHOR]

YKL-40, also known as human cartilage glycoprotein-39 (HC-gp39) or CHI3L1, shares structural similarities with chitotriosidase (CHIT1), an active chitinase, but lacks chitinase activity. Despite being a biomarker for inflammatory disorders and cancer, the reasons for YKL-40's inert chitinase function have remained elusive. This study reveals that the loss of chitinase activity in YKL-40 has risen from multiple sequence modifications influencing its chitin affinity. Contrary to the common belief associating the lack of chitinase activity with amino acid substitutions in the catalytic motif, attempts to activate YKL-40 by creating two amino acid mutations in the catalytic motif (MT-YKL-40) proved ineffective. Subsequent exploration that included creating chimeras of MT-YKL-40 and CHIT1 catalytic domains (CatDs) identified key exons responsible for YKL-40 inactivation. Introducing YKL-40 exons 3, 6, or 8 into CHIT1 CatD resulted in chitinase inactivation. Conversely, incorporating CHIT1 exons 3, 6, and 8 into MT-YKL-40 led to its activation. Our recombinant proteins exhibited properly formed disulfide bonds, affirming a defined structure in active molecules. Biochemical and evolutionary analysis indicated that the reduced chitinase activity of MT-YKL-40 correlates with specific amino acids in exon 3. M61I and T69W substitutions in CHIT1 CatD diminished chitinase activity and increased chitin binding. Conversely, substituting I61 with M and W69 with T in MT-YKL-40 triggered chitinase activity while reducing the chitin-binding activity. Thus, W69 plays a crucial role in a unique subsite within YKL-40. These findings emphasize that YKL-40, though retaining the structural framework of a mammalian chitinase, has evolved to recognize chitin while surrendering chitinase activity. [ABSTRACT FROM AUTHOR]

During base excision repair (BER), the apurinic or apyrimidinic (AP) site serves as an intermediate product following base excision. In plants, APE-redox protein (ARP) represents the major AP site of cleavage activity. Despite the well-established understanding that the nucleosomal structure acts as a barrier to various DNA-templated processes, the regulatory mechanisms underlying BER at the chromatin level remain elusive, especially in plants. In this study, we identified plant chromatin remodeler Excision Repair Cross-Complementing protein group 6 (ERCC6) and histone chaperone Nucleosome Assembly Protein 1 (NAP1) as interacting proteins with ARP. The catalytic ATPase domain of ERCC6 facilitates its interaction with both ARP and NAP1. Additionally, ERCC6 and NAP1 synergistically contribute to nucleosome sliding and exposure of hindered endonuclease cleavage sites. Loss-of-function mutations in Arabidopsis (Arabidopsis thaliana) ERCC6 or NAP1 resulted in arp-dependent plant hypersensitivity to 5-fluorouracil, a toxic agent inducing BER, and the accumulation of AP sites. Furthermore, similar protein interactions are also found in yeast cells, suggesting a conserved recruitment mechanism employed by the AP endonuclease to overcome chromatin barriers during BER progression. APURINIC/APYRIMIDINIC ENDONUCLEASE REDOX PROTEIN recruits a chromatin remodeler and a histone chaperone to facilitate uracil N-glycosylase-triggered base excision repair. [ABSTRACT FROM AUTHOR]

Psoriasis is an inflammatory skin disease with a chronic relapsing course and an often-detrimental impact on patients' quality of life. Thanks to incredible advances in research over the past few decades, the therapeutic armamentarium of psoriasis is now reasonably broad and structured, with several therapeutic agents that have demonstrated successful long-term control of this condition. However, there are still unfulfilled gaps resulting from the inherent limitations of existing therapies, which have paved the way for the identification of new therapeutic strategies or the improvement of existing ones. A great deal of attention has recently been paid to the JAK/STAT pathway, playing a crucial role in chronic inflammatory skin diseases, including psoriasis. Indeed, in a disease with such a complex pathogenesis, the possibility to antagonize multiple molecular pathways via JAK/STAT inhibition offers an undeniable therapeutic advantage. However, data from clinical trials evaluating the use of oral JAK inhibitors in immune-mediated disorders, such as RA, have arisen safety concerns, suggesting a potentially increased risk of class-specific AEs such as infections, venous thromboembolism, and malignancies. New molecules are currently under investigation for the treatment of psoriasis, such as deucravacitinib, an oral selective inhibitor that binds to the regulatory domain of TYK2, brepocitinib (PF-06700841) and PF-06826647 that bind to the active site in the catalytic domain. Due to the selective TYK2 blockade allowing the inhibition of key cytokine-mediated signals, such as those induced by IL-12 and IL-23, anti-TYK2 agents appear to be very promising as the safety profile seems to be superior compared with pan-JAK inhibitors. The aim of our review is to thoroughly explore the rationale behind the usage of JAK inhibitors in PsO, their efficacy and safety profiles, with a special focus on oral TYK2 inhibitors, as well as to provide a forward-looking update on novel therapeutic strategies targeting the TYK2 pathway in psoriasis. [ABSTRACT FROM AUTHOR]

Amidst the rapid advancements in the fields of photovoltaics and optoelectronic devices, CsPbBr3 nanosheets (NSs) have emerged as a focal point of research due to their exceptional optical and electronic properties. This work explores the application potential of CsPbBr3 NSs in photonic and catalytic domains. Utilizing an optimized hot-injection method and a ZnBr2-assisted in situ passivation strategy, we successfully synthesized CsPbBr3 NSs with controlled dimensions and optical characteristics. Comprehensive characterization revealed that the nucleation environment and thickness significantly influenced the structure and optical performance of the materials. The results indicate that the optimized synthesis method enables control over the lateral dimensions of the nanoparticles, ranging from 9.1 ± 0.06 nm to 334.5 ± 4.40 nm, facilitating the tuning of the excitation wavelength from 460 nm (blue) to 510 nm (green). Further analyses involving photoresponse and electrochemical impedance spectroscopy demonstrated the substantial potential of these NSs in applications such as photocatalysis and energy conversion. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) of eukaryotes—retrotransposons and DNA transposons—are nucleotide sequences that can move from locus to locus of the genome, as well as between the genomes of different organisms. L31 DNA transposons are an ancient and diverse group belonging to the large IS630/Tc1/mariner group. L31 transposons are not widespread and are present in a limited number of taxa. In addition to the sequence encoding the DDE/D transposase, L31 transposons carry another ORF (ORF2). Detailed analysis of L31 elements in the genomes of six-rayed corals has provided detailed information on the distribution, diversity, and structure of the elements. Two large groups, L31-duo and L31-uno, were identified, differing in both catalytic domain pattern and structure. As a result of reconstruction of the evolution of L31 transposons, it was suggested that six-rayed corals received L31 transposons from bivalves. The split-off group L31-uno may have been obtained by mollusks as a result of horizontal transfer from corals. Studies of TE distribution and diversity in marine invertebrates will contribute to a better understanding of the evolutionary processes of TEs and their role in the evolutionary history of species. [ABSTRACT FROM AUTHOR]

Alginate lyases cleave the 1,4-glycosidic bond of alginate by eliminating sugar molecules from its bond. While earlier reported alginate lyases were primarily single catalytic domains, research on multi-module alginate lyases has been lfiguimited. This study identified VsAly7A, a multi-module alginate lyase present in Vibrio sp. QY108, comprising a "Pro-Asp-Thr(PDT)" fragment and two PL-7 catalytic domains (CD I and CD II). The "PDT" fragment enhances the soluble expression level and increases the thermostability and binding affinity to the substrate. Moreover, CD I exhibited greater catalytic efficiency than CD II. The incorporation of PDT-CD I resulted in an increase in the optimal temperature of VsAly7A, whereas CD II displayed a preference for polyG degradation. The multi-domain structure of VsAly7A provides a new idea for the rational design of alginate lyase, whilst the "PDT" fragment may serve as a fusion tag in the soluble expression of recombinant proteins. [ABSTRACT FROM AUTHOR]

Ubiquitin-specific protease 14 (USP14), one of the three major proteasome-associated deubiquitinating enzymes (DUBs), is known to be activated by the AKT-mediated phosphorylation at Ser432. Thereby, AKT can regulate global protein degradation by controlling the ubiquitin-proteasome system (UPS). However, the exact molecular mechanism of USP14 activation by AKT phosphorylation at the atomic level remains unknown. By performing the molecular dynamics (MD) simulation of the USP14 catalytic domain at three different states (inactive, active, and USP14-ubiquitin complex), we characterized the change in structural dynamics by phosphorylation. We observed that the Ser432 phosphorylation induced substantial conformational changes of USP14 in the blocking loop (BL) region to fold it from an open loop into a β-sheet, which is critical for USP14 activation. Furthermore, phosphorylation also increased the frequency of critical hydrogen bonding and salt bridge interactions between USP14 and ubiquitin, which is essential for DUB activity. Structural dynamics insights from this study pinpoint the important local conformational landscape of USP14 by the phosphorylation event, which would be critical for understanding USP14-mediated proteasome regulation and designing future therapeutics. [ABSTRACT FROM AUTHOR]

The post-translational modification of proteins by ubiquitin-like modifiers (UbLs), such as SUMO, ubiquitin, and Nedd8, regulates a vast array of cellular processes. Dedicated UbL deconjugating proteases families reverse these modifications. During bacterial infection, effector proteins, including deconjugating proteases, are released to disrupt host cell defenses and promote bacterial survival. NopD, an effector protein from rhizobia involved in legume nodule symbiosis, exhibits deSUMOylation activity and, unexpectedly, also deubiquitination and deNeddylation activities. Here, we present two crystal structures of Bradyrhizobium (sp. XS1150) NopD complexed with either Arabidopsis SUMO2 or ubiquitin at 1.50 Å and 1.94 Å resolution, respectively. Despite their low sequence similarity, SUMO and ubiquitin bind to a similar NopD interface, employing a unique loop insertion in the NopD sequence. In vitro binding and activity assays reveal specific residues that distinguish between deubiquitination and deSUMOylation. These unique multifaceted deconjugating activities against SUMO, ubiquitin, and Nedd8 exemplify an optimized bacterial protease that disrupts distinct UbL post-translational modifications during host cell infection. The unique structural features of the NopD catalytic domain, an effector protease from symbiotic bacteria rhizobia, grant a surprising versatility to remove various regulatory tags (SUMO, ubiquitin, Nedd8) from host plant proteins. [ABSTRACT FROM AUTHOR]

Serratiopeptidase, a proteolytic enzyme serves as an important anti-inflammatory and analgesic medication. Present study reports the production and purification of extracellular serratiopeptidase from an endophyte, Serratia marcescens MES-4, isolated from Morus rubra. Purification of the enzyme by Ion exchange chromatography led to the specific activity of 13,030 U/mg protein of serratiopeptidase, showcasing about 3.1 fold enhanced activity. The catalytic domain of the purified serratiopeptidase, composed of Zn coordinated with three histidine residues (His 209, His 213, and His 219), along with glutamate (Glu 210) and tyrosine (Tyr 249). The molecular mass, as determined by SDS-PAGE was ∼51 kDa. The purified serratiopeptidase displayed optimal activity at pH 9.0, temperature 50°C. Kinetic studies revealed V max and K m values of 33,333 U/mL and 1.66 mg/mL, respectively. Further, optimized conditions for the production of serratiopeptidase by Taguchi design led to the productivity of 87 U/mL/h with 87.9 fold enhanced production as compared to the previous conditions. • Purification and characterization of serratiopeptidase from the endophyte Serratia marcescens MES-4, isolated from Morus rubra. • Enhancement of serratiopeptidase activity by Taguchi L16 orthogonal array design. • Productivity of 87U/mL/h with 87.9 fold enhanced production was achieved as compared to the previous conditions. [ABSTRACT FROM AUTHOR]

The DNA methyltransferase DNMT3C appeared as a duplication of the DNMT3B gene in muroids and is required for silencing of young retrotransposons in the male germline. Using specialized assay systems, we investigate the flanking sequence preferences of DNMT3C and observe characteristic preferences for cytosine at the -2 and -1 flank that are unique among DNMT3 enzymes. We identify two amino acids in the catalytic domain of DNMT3C (C543 and V547) that are responsible for the DNMT3C-specific flanking sequence preferences and evolutionary conserved in muroids. Reanalysis of published data shows that DNMT3C flanking preferences are consistent with genome-wide methylation patterns in mouse ES cells only expressing DNMT3C. Strikingly, we show that CpG sites with the preferred flanking sequences of DNMT3C are enriched in murine retrotransposons that were previously identified as DNMT3C targets. Finally, we demonstrate experimentally that DNMT3C has elevated methylation activity on substrates derived from these biological targets. Our data show that DNMT3C flanking sequence preferences match the sequences of young murine retrotransposons which facilitates their methylation. By this, our data provide mechanistic insights into the molecular co-evolution of repeat elements and (epi)genetic defense systems dedicated to maintain genomic stability in mammals. A biochemical study discovers that the target site preferences of the DNA methyltransferase DNMT3C differ from its relatives DNMT3A and DNMT3B and they specifically match the biological repeat elements methylated by DNMT3C. [ABSTRACT FROM AUTHOR]

The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C–H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. Key points: • The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. • After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. • The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid. [ABSTRACT FROM AUTHOR]

Hao‐Fountain syndrome (HAFOUS, OMIM: #616863) is a neurodevelopmental disorder caused by pathogenic variants in the gene USP7 coding for USP7, a protein involved in several crucial cellular homeostatic mechanisms and the recently described MUST complex. The phenotype of HAFOUS is insufficiently understood, yet there is a great need to better understand the spectrum of disease, genotype–phenotype correlations, and disease trajectories. We now present a larger cohort of 32 additional individuals and provide further clinical information about six previously reported individuals. A questionnaire‐based study was performed to characterize the phenotype of Hao‐Fountain syndrome more clearly, to highlight new traits, and to better distinguish the disease from related neurodevelopmental disorders. In addition to confirming previously described features, we report hyperphagia and increased body weight in a subset of individuals. HAFOUS patients present an increased rate of birth complications, congenital anomalies, and abnormal pain thresholds. Speech impairment emerges as a potential hallmark of Hao‐Fountain syndrome. Cognitive testing reports reveal borderline intellectual functioning on average, although some individuals score in the range of intellectual disability. Finally, we created a syndrome‐specific severity score. This score neither indicates a sex‐ nor age‐specific difference of clinical severity, yet highlights a more severe outcome when amino acid changes colocalize to the catalytic domain of the USP7 protein. [ABSTRACT FROM AUTHOR]

Cholesterol is essential for both normal cell viability and cancer cell proliferation. Aberrant activity of squalene monooxygenase (SM, also known as squalene epoxidase), the ratelimiting enzyme of the committed cholesterol synthesis pathway, is accordingly implicated in a growing list of cancers. We previously reported that hypoxia triggers the truncation of SM to a constitutively active form, thus preserving sterol synthesis during oxygen shortfalls. Here, we show SM truncation is upregulated and correlates with the magnitude of hypoxia in endometrial cancer tissues, supporting the in vivo relevance of our earlier work. To further investigate the pathophysiological consequences of SM truncation, we examined its lipid dropletlocalized pool using complementary immunofluorescence and cell fractionation approaches and found that it exclusively comprises the truncated enzyme. This partitioning is facilitated by the loss of an endoplasmic reticulum-embedded region at the SM N terminus, whereas the catalytic domain containing membrane-associated C-terminal helices is spared. Moreover, we determined multiple amphipathic helices contribute to the lipid droplet localization of truncated SM. Taken together, our results expand on the striking differences between the two forms of SM and suggest upregulated truncation may contribute to SM-related oncogenesis. [ABSTRACT FROM AUTHOR]

Protein kinases are essential regulators of cell function and represent one of the largest and most diverse protein families. They are particularly influential in signal transduction and coordinating complex processes like the cell cycle. Out of the 518 human protein kinases identified, 478 are part of a single superfamily sharing catalytic domains that are related in sequence. The dysregulation of protein kinases due to certain mutations has been associated with various diseases, including cancer. Although most of the protein kinase inhibitors identified as type I or type II primarily target the ATP-binding pockets of kinases, the structural and sequential resemblances among these pockets pose a significant challenge for selective inhibition. Therefore, targeting allosteric pockets that are beside highly conserved ATP pockets has emerged as a promising strategy to prevail current limitations, such as poor selectivity and drug resistance. In this article, we compared the binding pockets of various protein kinases for which allosteric (type III) inhibitors have already been developed. Additionally, understanding the structure and shape of existing ligands could aid in identifying key interaction sites within the allosteric pockets of kinases. This comprehensive review aims to facilitate the design of more effective and selective allosteric inhibitors. [ABSTRACT FROM AUTHOR]

Bacterial α-1,3-glucanase, classified as glycoside hydrolase (GH) family 87, has been divided into 3 subgroups based on differences in gene sequences in the catalytic domain. The enzymatic properties of subgroups 1 and 3 of several bacteria have been previously investigated and reported; however, the chemical characterization of subgroup 2 enzymes has not been previously conducted. The α-1,3-glucanase gene from Paenibacillus alginolyticus NBRC15375 (PaAgl) belonging to subgroup 2 of GH family 87 was cloned and expressed in Escherichia coli. PgAgl-N1 (subgroup 3) and PgAgl-N2 (subgroup 1) from P. glycanilyticus NBRC16188 were expressed in E. coli , and their enzymatic characteristics were compared. The amino acid sequence of PaAgl demonstrated that the homology was significantly lower in other subgroups when only the catalytic domain was compared. The oligosaccharide products of the mutan-degrading reaction seemed to have different characteristics among subgroups 1, 2, and 3 in GH family 87. [ABSTRACT FROM AUTHOR]

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single- molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well- characterized exo- cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate- binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate- binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy. [ABSTRACT FROM AUTHOR]

We report here the identification, characterization and three‐dimensional (3D) structure determination of NaNga, a newly identified β‐N‐acetylgalactosaminidase from the Gram‐negative soil bacterium Niabella aurantiaca DSM 17617. When recombinantly expressed in Escherichia coli, the enzyme selectively cleaved 4‐nitrophenyl‐N‐acetyl‐β‐d‐galactosamine (pNP‐β‐d‐GalpNAc). The X‐ray crystal structure of the protein was refined to 2.5 Å and consists of an N‐terminal β‐sandwich domain and a (β/α)8 barrel catalytic domain. Despite a mere 22% sequence identity, the 3D structure of NaNga is similar to those previously determined for family GH123 members, suggesting it also employs the same substrate‐assisted catalytic mechanism. Inhibition by N‐acetyl‐galactosamine thiazoline (GalNAc‐thiazoline) supports the suggested mechanism. A phylogenetic analysis of its proximal sequence space shows significant clustering of unknown sequences around NaNga with sufficient divergence with previously identified GH123 members to subdivide this family into distinct subfamilies. Although the actual biological substrate of our enzyme remains unknown, examination of the active site pocket suggests that it may be a β‐N‐acetylgalactosaminide substituted by a monosaccharide at O‐3. Analysis of the genomic context suggests, in turn, that this substituted β‐N‐acetylgalactosaminide may be appended to a d‐arabinan from an environmental Actinomycete. [ABSTRACT FROM AUTHOR]

Lysyl oxidase‐like 2 (LOXL2) is a Cu2+‐dependent amine oxidase that catalyzes the oxidative deamination of lysine residues of elastin and collagen to generate aldehyde groups. In the active state of LOXL2, its conserved catalytic domain has a lysine tyrosylquinone (LTQ) cofactor and a copper ion. LTQ is post‐translationally derived from Lys653 and Tyr689 of LOXL2. In this study, well‐tempered metadynamics simulations are used to investigate the conformation modulation from the inactive state to active state and obtain the free energy landscape. Interestingly, the simulations show that LTQ precursor residues cannot get close in Zn2+−bound LOXL2, whereas the copper ion binding triggers a conformation change to form an optional structure for LTQ biosynthesis. It is found that a prominent loop, consisting of residues 654–658, hinders the formation of LTQ in Zn2+−LOXL2 by preventing Lys653 from moving into active site of LOXL2, whereas this loop is relatively flexible in Cu2+−LOXL2. After the formation of LTQ, it needs to be activated by adjusting its position to be exposed to the solvent, thus allowing it to react with its substrate proteins. The results indicate that this process involves an intermediate state and that the activation of LTQ overcomes an energy barrier of 5.9 kcal mol−1. [ABSTRACT FROM AUTHOR]

An uncharacterized gene encoding a glycoside hydrolase family 43-like enzyme from Clostridium boliviensis strain E-1 was identified from genomic sequence data, and the encoded enzyme, CbE1Xyn43-l, was produced in Escherichia coli. CbE1Xyn43-l (52.9 kDa) is a two-domain endo-ß-xylanase consisting of a C-terminal CBM6 and a GH43-like catalytic domain. The positions of the catalytic dyad conserved in GH43, the catalytic base (Asp74), and proton donor (Glu240) were identified in alignments including GH43-enzymes of known 3D-structure from different subfamilies. CbE1Xyn43-l is active at pH 7.0-9.0, with optimum temperature at 65°C, and a more than 7 days' half-life in irreversible deactivation studies at this temperature. The enzyme hydrolyzed birchwood xylan, quinoa stalks glucuronoarabinoxylan, and wheat arabinoxylan with xylotriose and xylotetraose as major hydrolysis products. CbE1Xyn43-l also released xylobiose from pNPX2 with low turnover (kcat of 0.044 s-1) but was inactive on pNPX, showing that a degree of polymerization of three (DP3) was the smallest hydrolyzable substrate. Divalent ions affected the specific activity on xylan substrates, which dependent on the ion could be increased or decreased. In conclusion, CbE1Xyn43-l from C. boliviensis strain E-1 is the first characterized member of a large group of homologous hypothetical proteins annotated as GH43-like and is a thermostable endo-xylanase, producing xylooligosaccharides of high DP (xylotriose and xylotetraose) producer. IMPORTANCE The genome of Clostridium boliviensis strain E-1 encodes a number of hypothetical enzymes, annotated as glycoside hydrolase-like but not classified in the Carbohydrate Active Enzyme Database (CAZy). A novel thermostable GH43-like enzyme is here characterized as an endo-ß-xylanase of interest in the production of prebiotic xylooligosaccharides (XOs) from different xylan sources. CbE1Xyn43-l is a two-domain enzyme composed of a catalytic GH43-l domain and a CBM6 domain, producing xylotriose as main XO product. The enzyme has homologs in many related Clostridium strains which may indicate a similar function and be a previously unknown type of endo-xylanase in this evolutionary lineage of microorganisms. [ABSTRACT FROM AUTHOR]

4-α-glucanotransferases (4αGTs, EC 2.4.1.25) from glycoside hydrolase family 77 (GH77) catalyze chain elongation of starch amylopectin chains and can be utilized to structurally modify starch to tailor its gelation properties. The potential relationship between the structural design of 4αGTs and functional starch modification is unknown. Here, family GH77 was mined in silico for enzyme candidates based on sub-grouping guided by Conserved Unique Peptide Patterns (CUPP) bioinformatics categorization. From + 12,000 protein sequences a representative set of 27 4αGTs, representing four different domain architectures, different bacterial origins and diverse CUPP groups, was selected for heterologous expression and further study. Most of the enzymes catalyzed starch modification, but their efficacies varied substantially. Five of the 4αGTs were characterized in detail, and their action was compared to that of the industrial benchmark enzyme, Tt4αGT (CUPP 77_1.2), from Thermus thermophilus. Reaction optima of the five 4αGTs ranged from ∼40–60 °C and pH 7.3–9.0. Several were stable for a minimum 4 h at 70 °C. Domain architecture type A proteins, consisting only of a catalytic domain, had high thermal stability and high starch modification ability. All five novel 4αGTs (and Tt4αGT) induced enhanced gelling of potato starch. One, At4αGT from Azospirillum thermophilum (CUPP 77_2.4), displayed distinct starch modifying abilities, whereas T24αGT from Thermus sp. 2.9 (CUPP 77_1.2) modified the starch similarly to Tt4αGT, but slightly more effectively. T24αGT and At4αGT are thus interesting candidates for industrial starch modification. A model is proposed to explain the link between the 4αGT induced molecular modifications and macroscopic starch gelation. [Display omitted] • Bioinformatics based categorization of + 2200 4αGTs using CUPP informatics. • In silico identification of 27 microbial, potentially thermostable 4αGTs. • Reaction optima, thermal stability, and starch gelation ability of five novel 4αGTs. • Identification of type A 4αGTs as the most efficient and thermostable type. • Model to explain how 4αGT catalyzed chain elongation causes starch gelation. [ABSTRACT FROM AUTHOR]

Flavodiiron proteins (FDPs) are a family of enzymes with a significant role in O2/H2O2 and/or NO detoxification through the reduction of these species to H2O or N2O, respectively. All FDPs contain a minimal catalytic unit of two identical subunits, each one having a metallo‐β‐lactamase‐like domain harboring the catalytic diiron site, and a flavodoxin‐like domain. However, more complex and diverse arrangements in terms of domains are found in this family, of which the class H enzymes are among the most complex. One of such FDPs is encoded in the genome of the anaerobic bacterium Syntrophomonas wolfei subsp. wolfei str. Goettingen G311. Besides the core domains, this protein is predicted to have three additional ones after the flavodoxin core domain: two short‐chain rubredoxins and a NAD(P)H:rubredoxin oxidoreductase‐like domain. This enzyme, FDP_H, was produced and characterized and the presence of the predicted cofactors was investigated by a set of biochemical and spectroscopic methodologies. Syntrophomonas wolfei FDP_H exhibited a remarkable O2 reduction activity with a kcat = 52.0 ± 1.2 s−1 and a negligible NO reduction activity (~ 100 times lower than with O2), with NADH as an electron donor, that is, it is an oxygen‐selective FDP. In addition, this enzyme showed the highest turnover value for H2O2 reduction (kcat = 19.1 ± 2.2 s−1) ever observed among FDPs. Kinetic studies of site‐directed mutants of iron‐binding cysteines at the two rubredoxin domains demonstrated the essential role of these centers since their absence leads to a significant decrease or even abolishment of O2 and H2O2 reduction activities. [ABSTRACT FROM AUTHOR]

Structural organization of HIV-1 integrase is based on a tetramer formed by two protein dimers. Within this tetramer, the catalytic domain of one subunit of the first dimer interacts with the N-terminal domain of the second dimer subunit. It is the tetrameric structure that allows both ends of the viral DNA to be correctly positioned relative to the cellular DNA and to realize catalytic functions of integrase, namely 3′-processing and strand transfer. However, during the HIV-1 replicative cycle, integrase is responsible not only for the integration stage, it is also involved in reverse transcription and is necessary at the stage of capsid formation of the newly formed virions. It has been suggested that HIV-1 integrase is a structurally dynamic protein and its biological functions depend on its structure. Accordingly, studying interactions between the domains of integrase that provide its tetrameric structure is important for understanding its multiple functions. In this work, we investigated the role of three amino acids of the catalytic domain, I182, R187, and K188, located in the contact region of two integrase dimers in the tetramer structure, in reverse transcription and integration. It has been shown that the R187 residue is extremely important for formation of the correct integrase structure, which is necessary at all stages of its functional activity. The I182 residue is necessary for successful integration and is not important for reverse transcription, while the K188 residue, on the contrary, is involved in formation of the integrase structure, which is important for the effective reverse transcription. [ABSTRACT FROM AUTHOR]

Enzymes of the carbohydrate esterase family 4 (CE4) deacetylate a broad range of substrates, including linear, branched and mesh-like polysaccharides. Although they are enzymes of variable amino acid sequence length, they all comprise the conserved catalytic domain NodB. NodB carries the metal binding and active site residues and is characterized by a set of conserved sequence motifs, which are linked to the deacetylation activity. Besides a non-structured, flexible peptide of variable length that precedes NodB, several members of the CE4 family contain additional domains whose function or contribution to substrate specificity are not efficiently characterized. Evidence suggests that CE4 family members comprising solely the NodB domain have developed features linked to a variety of substrate specificities. To understand the NodB-based substrate diversity within the CE4 family, we perform a comparative analysis of all NodB domains structurally characterized so far. We show that amino acid sequence variations, topology diversities and excursions away from the framework structure give rise to different NodB domain classes associated with different substrate specificities and particular functions within and beyond the CE4 family. Our work reveals a link between specific NodB domain characteristics and substrate recognition. Thus, the details of the fold are clarified, and the structural basis of its variations is deciphered and associated with function. The conclusions of this work are also used to make predictions and propose specific functions for biochemically/enzymatically uncharacterized NodB-containing proteins, which have generally been considered as putative CE4 deacetylases. We show that some of them probably belong to different enzymatic families. [ABSTRACT FROM AUTHOR]

Selenocysteine, the 21st amino acid specified by the genetic code, is a rare selenium-containing residue found in the catalytic site of selenoprotein oxidoreductases. Selenocysteine is analogous to the common cysteine amino acid, but its selenium atom offers physical–chemical properties not provided by the corresponding sulfur atom in cysteine. Catalytic sites with selenocysteine in selenoproteins of vertebrates are under strong purifying selection, but one enzyme, glutathione peroxidase 6 (GPX6), independently exchanged selenocysteine for cysteine <100 million years ago in several mammalian lineages. We reconstructed and assayed these ancient enzymes before and after selenocysteine was lost and up to today and found them to have lost their classic ability to reduce hydroperoxides using glutathione. This loss of function, however, was accompanied by additional amino acid changes in the catalytic domain, with protein sites concertedly changing under positive selection across distant lineages abandoning selenocysteine in glutathione peroxidase 6. This demonstrates a narrow evolutionary range in maintaining fitness when sulfur in cysteine impairs the catalytic activity of this protein, with pleiotropy and epistasis likely driving the observed convergent evolution. We propose that the mutations shared across distinct lineages may trigger enzymatic properties beyond those in classic glutathione peroxidases, rather than simply recovering catalytic rate. These findings are an unusual example of adaptive convergence across mammalian selenoproteins, with the evolutionary signatures possibly representing the evolution of novel oxidoreductase functions. [ABSTRACT FROM AUTHOR]

Interferon-gamma-inducible large GTPases, hGBPs, possess antipathogenic and antitumor activities in human cells. Like hGBP1, its closest homolog, hGBP3 has two domains; an Nterminal catalytic domain and a C-terminal helical domain, connected by an intermediate region. The biochemical function of this protein and the role of its domains in substrate hydrolysis have not yet been investigated. Here, we report that while hGBP3 can produce both GDP and GMP, GMP is the minor product, 30% (unlike 85% in hGBP1), indicating that hGBP3 is unable to produce enhanced GMP. To understand which domain(s) are responsible for this deficiency, we created hGBP3 truncated variants. Surprisingly, GMP production was similar upon deletion of the helical domain, suggesting that in contrast to hGBP1, the helical domain of hGBP3 cannot stimulate the second phosphate cleavage of GTP. We conducted computational and solution studies to understand the underlying basis. We found that the regulatory residue W79, present in the catalytic domain, forms an H-bond with the backbone carbonyl of K76 (located in the catalytic loop) of the substrate-bound hGBP3. However, after gamma-phosphate cleavage of GTP, the W79-containing region does not undergo a conformational change, failing to redirect the catalytic loop toward the beta-phosphate. This is necessary for efficient GMP formation because hGBP homologs utilize the same catalytic residue for both phosphate cleavages. We suggest that the lack of specific interdomain contacts mediated by the helical domain prevents the catalytic loop movement, resulting in reduced GMP formation. These findings may provide insight into how hGBP3 contributes to immunity. [ABSTRACT FROM AUTHOR]

The toxicity of botulinum multi-domain neurotoxins (BoNTs) arises from a sequence of molecular events, in which the translocation of the catalytic domain through the membrane of a neurotransmitter vesicle plays a key role. A recent structural study of the translocation domain of BoNTs suggests that the interaction with the membrane is driven by the transition of an α helical switch towards a β hairpin. Atomistic simulations in conjunction with the mesoscopic Twister model are used to investigate the consequences of this proposition for the toxin–membrane interaction. The conformational mobilities of the domain, as well as the effect of the membrane, implicitly examined by comparing water and water–ethanol solvents, lead to the conclusion that the transition of the switch modifies the internal dynamics and the effect of membrane hydrophobicity on the whole protein. The central two α helices, helix 1 and helix 2, forming two coiled-coil motifs, are analyzed using the Twister model, in which the initial deformation of the membrane by the protein is caused by the presence of local torques arising from asymmetric positions of hydrophobic residues. Different torque distributions are observed depending on the switch conformations and permit an origin for the mechanism opening the membrane to be proposed. [ABSTRACT FROM AUTHOR]

The prevalence and clinical significance of heterozygous factor XIII (FXIII) deficiency has long been debated, with controversial reports emerging since 1988. In the absence of large epidemiologic studies, but based on a few studies, a prevalence of 1 per 1,000 to 5,000 is estimated. In southeastern Iran, a hotspot area for the disorder, a study of more than 3,500 individuals found an incidence of 3.5%. Between 1988 and 2023, a total of 308 individuals were found with heterozygous FXIII deficiency, of which molecular, laboratory, and clinical presentations were available for 207 individuals. A total of 49 variants were found in the F13A gene, most of which were missense (61.2%), followed by nonsense (12.2%) and small deletions (12.2%), most occurring in the catalytic domain (52.1%) of the FXIII-A protein and most frequently in exon 4 (17%) of the F13A gene. This pattern is relatively similar to homozygous (severe) FXIII deficiency. In general, heterozygous FXIII deficiency is an asymptomatic condition without spontaneous bleeding tendency, but it can lead to hemorrhagic complications in hemostatic challenges such as trauma, surgery, childbirth, and pregnancy. Postoperative bleeding, postpartum hemorrhage, and miscarriage are the most common clinical manifestations, while impaired wound healing has been rarely reported. Although some of these clinical manifestations can also be observed in the general population, they are more common in heterozygous FXIII deficiency. While studies of heterozygous FXIII deficiency conducted over the past 35 years have shed light on some of the ambiguities of this condition, further studies on a large number of heterozygotes are needed to answer the major questions related to heterozygous FXIII deficiency. [ABSTRACT FROM AUTHOR]

Modulation of cell death is a powerful strategy employed by pathogenic bacteria to evade host immune clearance and occupy profitable replication niches during infection. Intracellular pathogens employ the type III secretion system (T3SS) to deliver effectors, which interfere with regulated cell death pathways to evade immune defenses. Here, we reveal that poly(ADP-ribose) polymerase-1 (PARP1)-dependent cell death restrains Edwardsiella piscicida's proliferation in mouse monocyte macrophages J774A.1, of which PARP1 activation results in the accumulation of poly(ADP-ribose) (PAR) and enhanced inflammatory response. Moreover, E. piscicida, an important intracellular pathogen, leverages a T3SS effector YfiD to impair PARP1's activity and inhibit PAR accumulation. Once translocated into the host nucleus, YfiD binds to the ADP-ribosyl transferase (ART) domain of PARP1 to suppress its PARylation ability as the pharmacological inhibitor of PARP1 behaves. Furthermore, the interaction between YfiD and ART mainly relies on the complete unfolding of the helical domain, which releases the inhibitory effect on ART. In addition, YfiD impairs the inflammatory response and cell death in macrophages and promotes in vivo colonization and virulence of E. piscicida. Collectively, our results establish the functional mechanism of YfiD as a potential PARP1 inhibitor and provide more insights into host defense against bacterial infection. The bacterial T3SS effector YfiD interacts with the catalytic domain of host PARP1 during infection. Inhibition of PARP1 activity impairs the pro-inflammatory response against bacterial infection. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) is a type of lung cancer associated with translocation of the EML4 and ALK genes on the short arm of chromosome 2. This leads to the development of an aberrant protein kinase with a deregulated catalytic domain, the cdALK+. Currently, different ALK inhibitors (iALKs) have been proposed to treat ALK+ NSCLC patients. However, the recent resistance to iALKs stimulates the exploration of new iALKs for NSCLC. Here, we describe an in silico approach to finding FDA-approved drugs that can be used by pharmacological repositioning as iALK. We used homology modelling to obtain a structural model of cdALK+ protein and then performed molecular docking and molecular dynamics of the complex cdALK+-iALKs to generate the pharmacophore model. The pharmacophore was used to identify potential iALKs from FDA-approved drugs library by ligand-based virtual screening. Four pharmacophores with different atomistic characteristics were generated, resulting in six drugs that satisfied the proposed atomistic positions and coupled at the ATP-binding site. Mitoxantrone, riboflavin and abacavir exhibit the best interaction energies with 228.29, 165.40 and 133.48 KJoul/mol respectively. In addition, the special literature proposed these drugs for other types of diseases due to pharmacological repositioning. This study proposes FDA-approved drugs with ALK inhibitory characteristics. Moreover, we identified pharmacophores sites that can be tested with other pharmacological libraries. [ABSTRACT FROM AUTHOR]

Estrogens play critical roles in embryonic development, gonadal sex differentiation, behavior, and reproduction in vertebrates and in several human cancers. Estrogens are synthesized from testosterone and androstenedione by the endoplasmic reticulum membrane-bound P450 aromatase/cytochrome P450 oxidoreductase complex (CYP19/CPR). Here, we report the characterization of novel mammalian CYP19 isoforms encoded by CYP19 gene copies. These CYP19 isoforms are all defined by a combination of mutations in the N-terminal transmembrane helix (E42K, D43N) and in helix C of the catalytic domain (P146T, F147Y). The mutant CYP19 isoforms show increased androgen conversion due to the KN transmembrane helix. In addition, the TY substitutions in helix C result in a substrate preference for androstenedione. Our structural models suggest that CYP19 mutants may interact differently with the membrane (affecting substrate uptake) and with CPR (affecting electron transfer), providing structural clues for the catalytic differences. [ABSTRACT FROM AUTHOR]

[Display omitted] • We for the first time designed a targeted ITGA6 multi-locus guide (g)RNA and established a bidirectional deactivated RfxCas13d (dCasRx)-based m6A-editing platform. • The dCasRx–m6A editors have a high on-target efficiency. • Multi-target demethylation using dCasRx–m6A editors reduces ITGA6 translation. • Multi-target demethylation of ITGA6 mRNA inhibits BCa progression. • Adeno-associated delivery of dCasRx–ALKBH5-CD and ITGA6-multi-gRNA inhibits BCa progression. N6-methyladenosine (m6A) modification contributes to the pathogenesis and development of various cancers, including bladder cancer (BCa). In particular, integrin α6 (ITGA6) promotes BCa progression by cooperatively regulating multisite m6A modification. However, the therapeutic effect of targeting ITGA6 multisite m6A modifications in BCa remains unknown. We aim to develop a multisite dCasRx– m6A editor for assessing the effects of the multisite dCasRx–m6A editor targeted m6A demethylation of ITGA6 mRNA in BC growth and progression. The multisite dCasRx– m6A editor was generated by cloning. m6A-methylated RNA immunoprecipitation (meRIP), luciferase reporter, a single-base T3 ligase-based qPCR-amplification, Polysome profiling and meRIP-seq experiments were performed to determine the targeting specificity of the multisite dCasRx–m6A editor. We performed cell phenotype analysis and used in vivo mouse xenograft models to assess the effects of the multisite dCasRx–m6A editor in BC growth and progression. We designed a targeted ITGA6 multi-locus guide (g)RNA and established a bidirectional deactivated RfxCas13d (dCasRx)-based m6A-editing platform, comprising a nucleus-localized dCasRx fused with the catalytic domains of methyltransferase-like 3 (METTL3-CD) or α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5-CD), to simultaneously manipulate the methylation of ITGA6 mRNA at four m6A sites. The results confirmed the dCasRx–m6A editor modified m6A at multiple sites in ITGA6 mRNA, with low off-target effects. Moreover, targeted m6A demethylation of ITGA6 mRNA by the multisite dCasRx–m6A editor significantly reduced BCa cell proliferation and migration in vitro and in vivo. Furthermore, the dCasRx–ALKBH5-CD and ITGA6 multi-site gRNA delivered to 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice via adeno-associated viral vectors significantly inhibited BCa cell growth. Our study proposes a novel therapeutic tool for the treatment of BC by applying the multisite dCasRx–m6A editor while highlighting its potential efficacy for treating other diseases associated with abnormal m6A modifications. [ABSTRACT FROM AUTHOR]

TFIIIC is a multi-subunit complex required for tRNA transcription by RNA polymerase III. Human TFIIIC holo-complex possesses lysine acetyltransferase activity that aids in relieving chromatin-mediated repression for RNA polymerase III-mediated transcription and chromatin assembly. Here we have characterized the acetyltransferase activity of the largest and DNA-binding subunit of TFIIIC complex, TFIIIC220. Purified recombinant human TFIIIC220 acetylated core histones H3, H4 and H2A in vitro. Moreover, we have identified the putative catalytic domain of TFIIIC220 that efficiently acetylates core histones in vitro. Mutating critical residues of the putative acetyl-CoA binding 'P loop' drastically reduced the catalytic activity of the acetyltransferase domain. Further analysis showed that the knockdown of TFIIIC220 in mammalian cell lines dramatically reduces global H3K18 acetylation level, which was rescued by overexpression of the putative acetyltransferase domain of human TFIIIC220. Our findings indicated a possibility of a crucial role for TFIIIC220 in maintaining acetylation homeostasis in the cell. [ABSTRACT FROM AUTHOR]

Cyclic β-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic β-1,2-glucans (CβGs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that β-glucosidase-resistant compounds are produced by TiCGSCy with linear β-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are CβGs. Next, action pattern analyses using β-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide being the minimum length of the substrate. These analyses also showed that longer substrate β-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce CβGs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of β-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear differences in the reaction pathways between these enzymes. Overall, we propose a novel reaction mechanism of TiCGSCy. Thus, the present group of CGSs defines a new glycoside hydrolase family, GH189. Key points: • It was clearly evidenced that cyclization domain alone produces cyclic β-1,2-glucans. • The domain exclusively catalyzes transglycosylation without hydrolysis. • The present catalytic domain defines as a new glycoside hydrolase family 189. [ABSTRACT FROM AUTHOR]

Exonuclease VII (ExoVII) is a ubiquitous bacterial nuclease. Encoded by the xseA and xseB genes, ExoVII participates in multiple nucleic acid-dependent pathways including the processing of multicopy single-stranded DNA and the repair of covalent DNA-protein crosslinks (DPCs). Although many biochemical properties of ExoVII have been defined, little is known about its structure/function relationships. Here, we use cryoelectron microscopy (cryoEM) to determine that Escherichia coli ExoVII comprises a highly elongated XseA4·XseB24 holo-complex. Each XseA subunit dimerizes through a central extended a-helical segment decorated by six XseB subunits and a C-terminal, domain-swapped ß-barrel element; two XseA2·XseB12 subcomplexes further associate using N-terminal OB (oligonucleotide/oligosaccharide-binding) folds and catalytic domains to form a spindle-shaped, catenated octaicosamer. The catalytic domains of XseA, which adopt a nuclease fold related to 3-dehydroquinate dehydratases, are sequestered in the center of the complex and accessible only through large pores formed between XseA tetramers. The architectural organization of ExoVII, combined with biochemical studies, indicate that substrate selectivity is controlled by steric access to its nuclease elements and that tetramer dissociation results from substrate DNA binding. Despite a lack of sequence and fold homology, the physical organization of ExoVII is reminiscent of Mre11·Rad50/SbcCD ATP (adenosine triphosphate)-dependent nucleases used in the repair of double-stranded DNA breaks, including those formed by DPCs through aberrant topoisomerase activity, suggesting that there may have been convergent evolutionary pressure to contend with such damage events. [ABSTRACT FROM AUTHOR]

HDAC11 is a class IV histone deacylase with no crystal structure reported so far. The catalytic domain of HDAC11 shares low sequence identity with other HDAC isoforms, which makes conventional homology modeling less reliable. AlphaFold is a machine learning approach that can predict the 3D structure of proteins with high accuracy even in absence of similar structures. However, the fact that AlphaFold models are predicted in the absence of small molecules and ions/cofactors complicates their utilization for drug design. Previously, we optimized an HDAC11 AlphaFold model by adding the catalytic zinc ion and minimization in the presence of reported HDAC11 inhibitors. In the current study, we implement a comparative structure-based virtual screening approach utilizing the previously optimized HDAC11 AlphaFold model to identify novel and selective HDAC11 inhibitors. The stepwise virtual screening approach was successful in identifying a hit that was subsequently tested using an in vitro enzymatic assay. The hit compound showed an IC50 value of 3.5 µM for HDAC11 and could selectively inhibit HDAC11 over other HDAC subtypes at 10 µM concentration. In addition, we carried out molecular dynamics simulations to further confirm the binding hypothesis obtained by the docking study. These results reinforce the previously presented AlphaFold optimization approach and confirm the applicability of AlphaFold models in the search for novel inhibitors for drug discovery. [ABSTRACT FROM AUTHOR]

NSD3 (nuclear receptor-binding SET domain protein 3) is a member of the NSD histone methyltransferase family of proteins. In recent years, it has been identified as a potential oncogene in certain types of cancer. The NSD3 gene encodes three isoforms, the long version (NSD3L), a short version (NSD3S) and the WHISTLE isoforms. Importantly, the NSD3S isoform corresponds to the N-terminal region of the full-length protein, lacking the methyltransferase domain. The chromosomal location of NSD3 is frequently amplified across cancer types, such as breast, lung, and colon, among others. Recently, this amplification has been correlated to a chromothripsis event, that could explain the different NSD3 alterations found in cancer. The fusion proteins containing NSD3 have also been reported in leukemia (NSD3-NUP98), and in NUT (nuclear protein of the testis) midline carcinoma (NSD3-NUT). Its role as an oncogene has been described by modulating different cancer pathways through its methyltransferase activity, or the short isoform of the protein, through protein interactions. Specifically, in this review we will focus on the functions that have been characterized as methyltransferase dependent, and those that have been correlated with the expression of the NSD3S isoform. There is evidence that both the NSD3L and NSD3S isoforms are relevant for cancer progression, establishing NSD3 as a therapeutic target. However, further functional studies are needed to differentiate NSD3 oncogenic activity as dependent or independent of the catalytic domain of the protein, as well as the contribution of each isoform and its clinical significance in cancer progression. [ABSTRACT FROM AUTHOR]

Lignin, an inherently heterogeneous arylpropanoid polymer, presents a unique resource for the sustainable manufacture of aromatic chemicals and fuels. Recently, numerous catalytic strategies for lignin valorization have been explored. Polyoxometalates (POMs), with their distinctive structures enabling control of redox processes in lignin conversion, have been of particular interest. However, a comprehensive assessment of this emerging field of study is currently missing. This paper aims to fill this gap with a detailed review of the use of POMs for catalytic lignin conversion into value‐added compounds. This review delves into various catalytic lignin conversion techniques, including thermal catalysis, electrocatalysis, and tandem processes for lignin separation, depolymerization, and upgrading. Moreover, we highlight innovative investigations utilizing polyoxometalate‐ionic liquids (POM‐ILs), POM‐deep eutectic solvents (POM‐DESs), and solidification or immobilization techniques for POMs as lignin valorization catalysts. Lastly, we discuss the future prospects and challenges in the domain of POM catalytic lignin conversion. [ABSTRACT FROM AUTHOR]

Mutation is the major cause of phenotypic innovations. Apart from DNA mutations, the alteration on RNA such as the ADAR-mediated A-to-I RNA editing could also shape the phenotype. These two layers of variations have not been systematically combined to study their collective roles in cancers. We collected the high-quality transcriptomes of ten hepatocellular carcinoma (HCC) and the matched control samples. We systematically identified HCC-specific mutations in the exonic regions and profiled the A-to-I RNA editome in each sample. All ten HCC samples had mutations in the CDS of ADAR2 gene (dsRNA-binding domain or catalytic domain). The consequence of these mutations converged to the elevation of ADAR2 efficiency as reflected by the global increase of RNA editing levels in HCC. The up-regulated editing sites (UES) were enriched in the CDS and UTR of oncogenes and tumor suppressor genes (TSG), indicating the possible roles of these target genes in HCC oncogenesis. We present the mutation-ADAR2-UES-oncogene/TSG-HCC axis that explains how mutations at different layers would finally lead to abnormal phenotype. In the light of central dogma, our work provides novel insights into how to fully take advantage of the transcriptome data to decipher the consequence of mutations. [ABSTRACT FROM AUTHOR]

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,Dtranspeptidases, which make bonds between the amino acid in position four of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software that can identify 1-3 crosslinks and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their Cterminus. Using a bioinformatics approach, we identified a G. oxydans mutant from a transposon library with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) oxidatively depolymerize recalcitrant polysaccharides, which is important for biomass conversion. The catalytic domains of many LPMOs are linked to carbohydrate-binding modules (CBMs) through flexible linkers, but the function of these CBMs in LPMO catalysis is not well understood. In this study, we utilized MtLPMO9L and MtLPMO9G derived from Myceliophthora thermophila to investigate the impact of CBMs on LPMO activity, with particular emphasis on their influence on H2O2 tolerance. Using truncated forms of MtLPMO9G generated by removing the CBM, we found reduced substrate binding affinity and enzymatic activity. Conversely, when the CBM was fused to the C terminus of the single-domain MtLPMO9L to create MtLPMO9L-CBM, we observed a substantial improvement in substrate binding affinity, enzymatic activity, and notably, H2O2 tolerance. Furthermore, molecular dynamics simulations confirmed that the CBM fusion enhances the proximity of the active site to the substrate, thereby promoting multilocal cleavage and impacting the exposure of the copper active site to H2O2. Importantly, the fusion of CBM resulted in more efficient consumption of H2O2 by LPMO, leading to improved enzymatic activity and reduced auto-oxidative damage of the copper active center. [ABSTRACT FROM AUTHOR]

Bacteriophage endolysins are bacteriolytic enzymes that have been explored as potential weapons to fight antibiotic-resistant bacteria. Despite several studies support the application of endolysins as enzybiotics, detailed knowledge on cellular and enzymatic factors affecting their lytic activity is still missing. The bacterial membrane proton motive force (PMF) and certain cell wall glycopolymers of Gram-positive bacteria have been implicated in some tolerance to endolysins. Here, we studied how the anti-staphylococcal endolysin Lys11, a modular enzyme with two catalytic domains (peptidase and amidase) and a cell binding domain (CBD11), responded to changes in the chemical and/or electric gradients of the PMF (ΔpH and Δψ, respectively). We show that simultaneous dissipation of both gradients enhances endolysin binding to cells and lytic activity. The collapse of ΔpH is preponderant in the stimulation of Lys11 lytic action, while the dissipation of Δψ is mainly associated with higher endolysin binding. Interestingly, this binding depends on the amidase domain. The peptidase domain is responsible for most of the Lys11 bacteriolytic activity. Wall teichoic acids (WTAs) are confirmed as major determinants of endolysin tolerance, in part by severely hindering CBD11 binding activity. In conclusion, the PMF and WTA interfere differently with the endolysin functional domains, affecting both the binding and catalytic efficiencies. [ABSTRACT FROM AUTHOR]

Abnormal activation of receptor tyrosine kinases (RTKs) contributes to tumorigenesis, while protein tyrosine phosphatases (PTPs) contribute to tumor control. One of the most representative PTPs is Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1), which is associated with either an increased or decreased survival rate depending on the cancer type. Hypermethylation in the promoter region of PTPN6, the gene for the SHP-1 protein, is a representative epigenetic regulation mechanism that suppresses the expression of SHP-1 in tumor cells. SHP-1 comprises two SH2 domains (N-SH2 and C-SH2) and a catalytic PTP domain. Intramolecular interactions between the N-SH2 and PTP domains inhibit SHP-1 activity. Opening of the PTP domain by a conformational change in SHP-1 increases enzymatic activity and contributes to a tumor control phenotype by inhibiting the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT3) pathway. Although various compounds that increase SHP-1 activation or expression have been proposed as tumor therapeutics, except sorafenib and its derivatives, few candidates have demonstrated clinical significance. In some cancers, SHP-1 expression and activation contribute to a tumorigenic phenotype by inducing a tumor-friendly microenvironment. Therefore, developing anticancer drugs targeting SHP-1 must consider the effect of SHP-1 on both cell biological mechanisms of SHP-1 in tumor cells and the tumor microenvironment according to the target cancer type. Furthermore, the use of combination therapies should be considered. [ABSTRACT FROM AUTHOR]

DNA methylation is critically involved in the regulation of chromatin states and cell-type-specific gene expression. The exclusive expression of imprinted genes from either the maternal or the paternal allele is regulated by allele-specific DNA methylation at imprinting control regions (ICRs). Aberrant DNA hyper- or hypomethylation at the ICR1 of the H19/IGF2 imprinting locus is characteristic for the imprinting disorders Beckwith–Wiedemann syndrome (BWS) and Silver–Russell syndrome (SRS), respectively. In this paper, we performed epigenome editing to induce targeted DNA demethylation at ICR1 in HEK293 cells using dCas9-SunTag and the catalytic domain of TET1. 5-methylcytosine (5mC) levels at the target locus were reduced up to 90% and, 27 days after transient transfection, >60% demethylation was still observed. Consistent with the stable demethylation of CTCF-binding sites within the ICR1, the occupancy of the DNA methylation-sensitive insulator CTCF protein increased by >2-fold throughout the 27 days. Additionally, the H19 expression was increased by 2-fold stably, while IGF2 was repressed though only transiently. Our data illustrate the ability of epigenome editing to implement long-term changes in DNA methylation at imprinting control regions after a single transient treatment, potentially paving the way for therapeutic epigenome editing approaches in the treatment of imprinting disorders. [ABSTRACT FROM AUTHOR]

SHP2 phosphatase plays an important role in regulating several intracellular signaling pathways. Pathogenic mutations of SHP2 cause developmental disorders and are linked to hematological malignancies and cancer. SHP2 comprises two tandemly-arranged SH2 domains, a catalytic PTP domain, and a disordered C-terminal tail. Under physiological, non-stimulating conditions, the catalytic site of PTP is occluded by the N-SH2 domain, so that the basal activity of SHP2 is low. Whereas the autoinhibited structure of SHP2 has been known for two decades, its active, open structure still represents a conundrum. Since the oncogenic mutant SHP2E76K almost completely populates the active, open state, this mutant has been extensively studied as a model for activated SHP2. By molecular dynamics simulations and accurate explicit-solvent SAXS curve predictions, we present the heterogeneous atomistic ensemble of constitutively active SHP2E76K in solution, encompassing a set of conformational arrangements and radii of gyration in agreement with experimental SAXS data. The heterogeneous structural ensemble of the constitutively active SHP2E76K mutant has been determined by means molecular dynamics simulations in agreement with experimental data from small-angle X-ray scattering. [ABSTRACT FROM AUTHOR]