Ser Residues Research Articles

In silico structural and mechanistic sights into the N-glycosidase mechanism of Shiga toxin.

Shiga toxin is the leading cause of food poisoning in the world. It is structurally similar to the plant type II ribosome-inactivating proteins (RIPs) and retains N-glycosidase activity. It acts specifically by depurinating the specific adenine A4605 of human 28S rRNA, ultimately inhibiting translation. Recent outbreaks and increasing demands for lab-scale meat assert the necessity for producing toxoids. In the current study, we have carried out the comparative structural and functional analysis of Shiga with ricin for N-glycosidase activity. Primary structural analysis indicates that Shiga is more flexible than ricin and one active site residue Gly121 (ricin), has been mutated to Ser (Shiga). Tertiary structure analysis confirms the conserved active site residue confirmation. Further, molecular dynamic studies indicate that the mutated Ser residue of Shiga imparts flexibility besides interacting with the conserved GAGA loop of 28s rRNA and contributes free energy of -5.39kcal/mol. We have observed a decreasing trend line of average free binding energy with an average of -23kcal/mol. The residue interaction network indicates that Arg is the key residue that protonates and initiates the N-glycosidase activity. Overall, these structural studies provide molecular insights into the N-glycosidase mechanism and serve as a prospect for the development of toxoids.

Self-contacting Cys, Ser, and Thr residues in high-resolution protein crystal structures: Tertiary constraints or hydrogen bonds?

Functional groups in the side-chains of at least 10 amino acids are mainly involved in tertiary interactions. However, structural and functional significance of intra-residue interactions has not been fully recognized. In this study, we have analyzed ~5800 non-redundant high-resolution protein structures and identified 1166 self-contacts between the side-chain S-H/O-H and backbone C=O groups in Cys, Ser, and Thr residues that satisfied the geometric criteria to form hydrogen bonds. Quantum chemical calculations using model compounds were used to evaluate single point energy for 45 representative examples from different allowed regions of Ramachandran map. Relative energy profiles obtained by varying the side-chain dihedral angle χ1 revealed that the energy difference between the crystal structure and the minimum energy conformations is between 0 and 3 kcal/mol. Natural bond orbital analysis (NBO) of self-contacting Cys residues revealed no charge transfer between Cys side-chain S-H and the backbone C=O groups. However, side-chain hydroxyl and the backbone C=O groups of 90%-95% of all self-contacting Ser and Thr residues are involved in charge transfer and the second order perturbation energy of majority of them is above 1 kcal/mol. Interaction energies calculated for model compounds along with NBO and NCIPLOT analyses demonstrate that the self-contacts observed in Ser and Thr residues can be described as hydrogen bonds. These interactions may provide stability to the loop/coil conformations. Self-contacting Cys residues are buried and the self-contacts appear to be mostly due to tertiary constraints. Dispersion between the self-contacting groups is one way to explain the close approach in Cys residues. Mutation studies will further validate and reveal the structural and functional significance of these self-contacting residues.

Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-InsP7.

Inositol pyrophosphates (PP-InsPs) are a sub-family of water soluble inositol phosphates that possess one or more diphosphate groups. PP-InsPs can transfer their β-phosphate group to a phosphorylated Ser residue to generate pyrophosphorylated Ser. This unique post-translational modification occurs on Ser residues that lie in acidic stretches within an intrinsically disordered protein sequence. Serine pyrophosphorylation is dependent on the presence of Mg2+ ions, but does not require an enzyme for catalysis. The mechanisms by which cells regulate PP-InsP-mediated pyrophosphorylation are still unknown. We performed mass spectrometry to identify interactors of IP6K1, an enzyme responsible for the synthesis of the PP-InsP 5-InsP7. Interestingly, IP6K1 interacted with several proteins that are known to undergo 5-InsP7-mediated pyrophosphorylation, including the nucleolar proteins NOLC1, TCOF and UBF1, and AP3B1, the β subunit of the AP3 adaptor protein complex. The IP6K1 interactome also included CK2, a protein kinase that phosphorylates Ser residues prior to pyrophosphorylation. We observe the formation of a protein complex between IP6K1, AP3B1, and the catalytic α-subunit of CK2, and show that disrupting IP6K1 binding to AP3B1 lowers its in vivo pyrophosphorylation. We propose that assembly of a substrate-CK2-IP6K complex would allow for coordinated pre-phosphorylation and pyrophosphorylation of the target serine residue, and provide a mechanism to regulate this enzyme-independent modification.

GpsB Coordinates StkP Signaling as a PASTA Kinase Adaptor in Streptococcus pneumoniae Cell Division

StkP, the Ser/Thr protein kinase of the major human pathogen Streptococcus pneumoniae, monitors cell wall signals and regulates growth and division in response. In vivo, StkP interacts with GpsB, a cell division protein required for septal ring formation and closure, that affects StkP-dependent phosphorylation. Here, we report that although StkP has basal intrinsic kinase activity, GpsB promotes efficient autophosphorylation of StkP and phosphorylation of StkP substrates. Phosphoproteomic analyzes showed that GpsB is phosphorylated at several Ser and Thr residues. We confirmed that StkP directly phosphorylates GpsB in vitro and in vivo, with T79 and T83 being the major phosphorylation sites. In vitro, phosphoablative GpsB substitutions had a lower potential to stimulate StkP activity, whereas phosphomimetic substitutions were functional in terms of StkP activation. In vivo, substitutions of GpsB phosphoacceptor residues, either phosphoablative or mimetic, had a negative effect on GpsB function, resulting in reduced StkP-dependent phosphorylation and impaired cell division. The bacterial two-hybrid assay and co-immunoprecipitation of GpsB from cells with differentially active StkP indicated that increased phosphorylation of GpsB resulted in a more efficient interaction of GpsB with StkP. Our data suggest that GpsB acts as an adaptor that directly promotes StkP activity by mediating interactions within the StkP signaling hub, ensuring StkP recruitment into the complex and substrate specificity. We present a model that interaction of StkP with GpsB and its phosphorylation and dephosphorylation dynamically modulate kinase activity during exponential growth and under cell wall stress of S. pneumoniae, ensuring the proper functioning of the StkP signaling pathway.

Chemoselective Reagents for the Traceless Bioreversible Modification of Native Proteins.

Nature utilizes bioreversible post-translational modifications (PTMs) to spatiotemporally diversify protein function. Mimicking Nature's approach, chemists have developed a variety of chemoselective regents for traceless, bioreversible modification of native proteins. These strategies have found utility in the development of reversible covalent inhibitors and degraders as well as the synthesis of functional protein conjugates for delivery into cells. This Viewpoint provides a snapshot of such tools, which currently cover Cys, Ser, Thr, Lys, Asp, and Glu residues and the N terminus. Additionally, we explore how bioreversible reagents, originally developed by research communities with differing objectives, can be utilized synergistically. Looking forward, we discuss the need for developing bioreversible reagents for labeling His, Tyr, Arg, Trp, Asn, Gln, and Met residues and the C-terminus as well as the installation of dynamic PTMs. Finally, to broaden the applicability of these tools, we point out the importance of developing modular release scaffolds with tunable release times and responsiveness to multiple endogenous triggers. We anticipate that this Viewpoint will catalyze further research and technological breakthroughs in this rapidly evolving field.

SARS-CoV-2 E protein interacts with BRD2 and BRD4 SEED domains and alters transcription in a different way than BET inhibition

Bromodomain and extra-terminal (BET) proteins are relevant chromatin adaptors involved in the transcriptional control of thousands of genes. Two tandem N-terminal bromodomains are essential for chromatin attachment through acetyl-histone recognition. Recently, the BET proteins members BRD2 and BRD4 were found to interact with the SARS-CoV-2 envelope (E) protein, raising the question of whether the interaction constitutes a virus hijacking mechanism for transcription alteration in the host cell. To shed light on this question, we have compared the transcriptome of cells overexpressing E with that of cells treated with the BET inhibitor JQ1. Notably, E overexpression leads to a strong upregulation of natural immunity- and interferon response-related genes. However, BET inhibition results in the downregulation of most of these genes, indicating that these two conditions, far from causing a significant overlap of the altered transcriptomes, course with quite different outputs. Concerning the interaction of E protein with BET members, and differing from previous reports indicating that it occurs through BET bromodomains, we find that it relies on SEED and SEED-like domains, BET regions rich in Ser, Asp, and Glu residues. By taking advantage of this specific interaction, we have been able to direct selective degradation of E protein through a PROTAC system involving a dTAG-SEED fusion, highlighting the possible therapeutic use of this peptide for targeted degradation of a viral essential protein.

Natural zinc-rich oyster peptides ameliorate DSS-induced colitis via antioxidation, anti-inflammation, intestinal barrier repair, microbiota modulation, and SCFA accumulation

Natural oyster zinc-rich peptide (OZP) was extracted and purified. The higher zinc concentration in natural OZP may be closely related to the high zinc affinity of amino acids containing Lys, His, Cys, Val, Arg and Ser residues. FTIR analysis indicated that amino, amide, carboxyl and carbonyl groups may contribute to the binding sites for zinc and peptides. Administration of OZP ameliorated the symptoms of DSS-induced ulcerative colitis (UC) in mice, including weight loss, colonic damage, mucosal edema, goblet cell exhaustion and crypt disappearance. Natural OZP suppressed oxidative stress by significantly reducing the level of NO, MDA and MPO as well as increasing SOD activity. The anti-inflammatory effects of OZP were associated with the inhibition of TLR4/NF-κB pathways by diminishing NF-κB, TLR4, COX-2, iNOS, TNF-α, IL-6 and IFN-γ and elevating IL-10. By promoting mucus secretion, repairing goblet cells and enhancing the expression of tight junction proteins including occludin, claudin-1 and ZO-1, natural OZP maintained the intestinal barrier function. Furthermore, natural OZP regulated the microbial composition by remarkedly enhancing the beneficial microbial abundance like Faecalibaculum, Odoribacter and Anaerostipes, which in turn led to a significant increase in SCFA production. Collectively, natural OZP exerted the synergistic capacity to protect against DSS-induced UC by improving antioxidation and anti-inflammation, repairing intestinal barrier and modulating intestinal microbiota and SCFA.

Structural insight into the functional regulation of Elongation factor Tu by reactive oxygen species in Synechococcus elongatus PCC 7942

In cyanobacteria, Elongation factor Tu (EF-Tu) plays a crucial role in the repair of photosystem II (PSII), which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species (ROS). However, the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear. Previous research has shown that a mutated EF-Tu, where C82 is substituted with a Ser residue, can alleviate photoinhibition, highlighting the important role of C82 in EF-Tu photosensitivity. In this study, we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form (C82S) individually at resolutions of 1.7 Å and 2.0 Å in Synechococcus elongatus PCC 7942 complexed with GDP. Specifically, the GDP-bound form of EF-Tu adopts an open conformation with C82 located internally, making it resistant to oxidation. Coordinated conformational changes in switches I and II create a tunnel that positions C82 for ROS interaction, revealing the vulnerability of the closed conformation of EF-Tu to oxidation. An analysis of these two structures reveals that the precise spatial arrangement of C82 plays a crucial role in modulating EF-Tu's response to ROS, serving as a regulatory element that governs photosynthetic biosynthesis.

Covalent adduct formation of histone with organophosphorus pesticides in vitro

It is established that organophosphorus pesticide (OPP) toxicity results from modification of amino acids in active sites of target proteins. OPPs can also modify unrelated target proteins such as histones and such covalent histone modifications can alter DNA-binding properties and lead to aberrant gene expression. In the present study, we report on non-enzymatic covalent modifications of calf thymus histones adducted to selected OPPs and organophosphate flame retardants (OPFRs) in vitro using a bottom-up proteomics method approach. Histones were not found to form detectable adducts with the two tested OPFRs but were avidly modified by a few of the seven OPPs that were tested in vitro. Dimethyl phosphate (or diethyl phosphate) adducts were identified on Tyr, Lys and Ser residues. Most of the dialkyl phosphate adducts were identified on Tyr residues. Methyl and ethyl modified histones were also detected. Eleven amino residues in histones showed non-enzymatic covalent methylation by exposure of dichlorvos and malathion. Our bottom-up proteomics approach showing histone-OPP adduct formation warrants future studies on the underlying mechanism of chronic illness from exposure to OPPs.

An alternative 3′ splice site of PeuHKT1;3 improves the response to salt stress through enhancing affinity to K+ in Populus

Alternative splicing (AS) serves as a crucial post-transcriptional regulator in plants that contributes to the resistance to salt stress. However, the underlying mechanism is largely unknown. In this research, we identified an important AS transcript in Populus euphratica, PeuHKT1:3a, generated by alternative 3′ splice site splicing mode that resulted in the removal of 252 bases at the 5′ end of the first exon in PeuHKT1:3. Protein sequence comparison showed that the site of AS occurred in PeuHKT1:3 is located at a crucial Ser residue within the first pore-loop domain, which leads to inefficient K+ transport in HKT I-type transporters. Expressing PeuHKT1;3a in an axt3 mutant yeast strain can effectively compensate for the lack of intracellular K+, whereas the expression of PeuHKT1;3 cannot yield the effect. Furthermore, in transgenic Arabidopsis and poplar plants, it was observed that lines expressing PeuHKT1;3a exhibited greater salt tolerance compared to those expressing the PeuHKT1;3 strain. Analysis of ion content and flux demonstrated that the transgenic PeuHKT1;3a line exhibited significantly higher K+ content compared to the PeuHKT1;3 line, while there was no significant difference in Na+ content. In conclusion, our findings revealed that AS can give rise to novel variants of HKT I-type proteins in P. euphratica with modified K+ selectivity to keep a higher K+/Na+ ratio to enhanced salt tolerance.

Elevated calcineurin activity in primary astrocytes leads to the dephosphorylation of connexin 43 in conjunction with increased membrane permeability.

Hyperactivation of the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) is observed in reactive astrocytes associated with neuroinflammation and progressive degenerative diseases, like Alzheimer's disease. Apart from key transcription factors (e.g. nuclear factor of activated t cells and nuclear factor-κB) very few other CN-dependent pathways have been studied in astrocytes. The hemichannel protein, connexin 43 (Cx43) is found at high levels in astrocytes and contains a CN-sensitive Ser residue near its carboxy terminus. CN-dependent dephosphorylation of Cx43 has been reported in primary astrocytes treated with injurious stimuli, but much remains unknown about CN/Cx43 interactions in the context of neuroinflammation and disease. Western blots were used to assess total Cx43 and dephosphorylated Cx43 subtypes in rat embryonic primary astrocytes treated with a hyperactive CN fragment (ΔCN, via adenovirus), or with a proinflammatory cytokine cocktail. Under similar treatment conditions, an ethidium bromide (EtBr) uptake assay was used to assess membrane permeability. Effects of ΔCN and cytokines were tested in the presence or absence of the CN inhibitor, cyclosporin A. A connexin inhibitor, carbenoxolone was also used in EtBr assays to assess the involvement of connexins in membrane permeability. Treatment with ΔCN or cytokines increased dephosphorylated Cx43 levels in conjunction with increased membrane permeability (elevated EtBr uptake). Effects of ΔCN or cytokine treatment were blocked by cyclosporine A. Treatment-induced changes in EtBr uptake were also inhibited by carbenoxolone. The results suggest that Cx43 hemichannels could be an important mechanism through which astrocytic CN disrupts neurologic function associated with neurodegenerative disease.

SKGQA, a Peptide Derived from the ANA/BTG3 Protein, Cleaves Amyloid-β with Proteolytic Activity.

Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-β (Aβ) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aβ42 (a-Aβ42) and solid insoluble form s-Aβ42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aβ42 for AD treatment.

Serine/Threonine kinase 16 phosphorylates STAT3 and confers a JAK2-Inhibition resistance phenotype in triple-negative breast cancer

Although Janus kinase 2 (JAK2) plays a critical role in the progression of triple-negative breast cancer (TNBC), its inhibitors are incapable of eradicating these tumor cells, implicating drug resistance mechanisms exist. Our evidences show that TNBC cells express high level of Serine/Threonine Kinase 16 (STK16) when JAK2 signaling is blocked. Pharmacological inhibition or silencing of STK16 significantly enhances the sensitivity of TNBC cells to JAK2 inhibition, while over-expression of STK16 alleviates the anti-tumor effect of JAK2-inhibitor. Mechanistically, elevated STK16 expression rescues the phosphorylation status and transcriptional activity of STAT3, as STK16 is able to directly catalyze the phosphorylation of STAT3 at ser-727 residue. Our data indicate that upon JAK2 inhibition, TNBC cells express STK16 to maintain STAT3 transcriptional activity, dual-inhibition of JAK2/STK16 offers a potential way to treat TNBC patients.

In Vitro Identification of Phosphorylation Sites on TcPolβ by Protein Kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 and Effect of Phorbol Ester on Activation by TcPKC of TcPolβ in Trypanosoma cruzi Epimastigotes.

Chagas disease is caused by the single-flagellated protozoan Trypanosoma cruzi, which affects several million people worldwide. Understanding the signal transduction pathways involved in this parasite's growth, adaptation, and differentiation is crucial. Understanding the basic mechanisms of signal transduction in T. cruzi could help to develop new drugs to treat the disease caused by these protozoa. In the present work, we have demonstrated that Fetal Calf Serum (FCS) can quickly increase the levels of both phosphorylated and unphosphorylated forms of T. cruzi DNA polymerase beta (TcPolβ) in tissue-cultured trypomastigotes. The in vitro phosphorylation sites on TcPolβ by protein kinases TcCK1, TcCK2, TcAUK1, and TcPKC1 have been identified by Mass Spectrometry (MS) analysis and with antibodies against phosphor Ser-Thr-Tyr. MS analysis indicated that these protein kinases can phosphorylate Ser and Thr residues on several sites on TcPolβ. Unexpectedly, it was found that TcCK1 and TcPKC1 can phosphorylate a different Tyr residue on TcPolβ. By using a specific anti-phosphor Tyr monoclonal antibody, it was determined that TcCK1 can be in vitro autophosphorylated on Tyr residues. In vitro and in vivo studies showed that phorbol 12-myristate 13-acetate (PMA) can activate the PKC to stimulate the TcPolβ phosphorylation and enzymatic activity in T. cruzi epimastigotes.

Acquiring insights through a sequence-based approach to the critical Zika virus MTase domain

Background: ZIKV is one of the re-emerging arboviruses (viruses carried by arthropods), which is spread through the Aedes mosquito. It is an RNA virus with only one strand that is appropriate to the family Flaviviridae's Flavivirus (genus) & has been linked to other Flaviviruses such as the West Nile virus, chikungunya virus, & dengue (DENV) virus. The envelope, precursor membrane, and capsid are three structural proteins, and seven nonstructural proteins are also encoded by the Zika virus genome.Methods: We conducted an in-silico analysis of the Zika virus' MTase domain protein for this publication. We predicted that methylation would play a significant role in the available Prosite, Pfam, and InterProScan tools to aid in locating the MTase domain. Along with alignment, amino acid composition, charged amino acids, atomic level studies, & molecular weight, we also make predictions for these variables, including theoretical Pi.Results: We also examine the MTase domain's simulated structure (alpha helix, beta sheet, turn) and its specifics, including secondary structure. We also pinpoint the locations where proteins, DNA, and RNA bind. Potential phosphorylation sites can be found on the Ser, Thr, and Tyr residues in the MTase domain.Conclusion: These outcomes imply a complicated interaction between different phosphorylation modifications that modulates the activity of the MTase domain. To fully appreciate the auxiliary and practical perspectives and to clarify the varied roles of PTM in the MTase domain will be a primary goal of future study.Keywords: I-TASSER; Secondary structure; Prosite; α-helix; Pfam; InterProScan; Binding sites; Posttranslational modification; SOPMA; Phyre2

Charge Relay Without Proton Transfer: Coupling of Two Short Hydrogen Bonds via Imidazole in Models of Catalytic Triad of Serine Protease Active Site.

A homologous series of 20 substituted alcohol-imidazole-acetate model complexes imitating the charge relay system in Ser-His-Asp catalytic triad of serine proteases is considered quantum-chemically. We show qualitatively that the geometries of alcohol-imidazole and imidazole-acetate short hydrogen bonds are strongly coupled via the central imidazole and such complexes are capable of effectively relaying the charge from acetate to alcohol moiety upon relatively small concerted proton displacements. We hypothesize an alternative catalytic mechanism of serine proteases that does not require two complete proton transfers or hydrogen bond breakage between Ser and His residues.

Diverse roles of the metal binding domains and transport mechanism of copper transporting P-type ATPases

Copper transporting P-type (P1B-1-) ATPases are essential for cellular homeostasis. Nonetheless, the E1-E1P-E2P-E2 states mechanism of P1B-1-ATPases remains poorly understood. In particular, the role of the intrinsic metal binding domains (MBDs) is enigmatic. Here, four cryo-EM structures and molecular dynamics simulations of a P1B-1-ATPase are combined to reveal that in many eukaryotes the MBD immediately prior to the ATPase core, MBD−1, serves a structural role, remodeling the ion-uptake region. In contrast, the MBD prior to MBD−1, MBD−2, likely assists in copper delivery to the ATPase core. Invariant Tyr, Asn and Ser residues in the transmembrane domain assist in positioning sulfur-providing copper-binding amino acids, allowing for copper uptake, binding and release. As such, our findings unify previously conflicting data on the transport and regulation of P1B-1-ATPases. The results are critical for a fundamental understanding of cellular copper homeostasis and for comprehension of the molecular bases of P1B-1-disorders and ongoing clinical trials.

Quantification of serine residue stereoinversion in a short peptide by reversed-phase high-performance liquid chromatography: analysis of mechanisms promoting serine stereoinversion.

Stereoinversion of Ser residues within proteins, which has been identified in long-lived proteins, influences protein function. To quantify the stereoinversion of Ser residues, we investigated the potential adaptation of our direct peptide analytical method originally established for analyzing the isomerization of asparaginyl/aspartyl residues. Peptide pairs containing L-Ser or D-Ser residues with lengths of four or five residues were synthesized. Separation conditions for these peptide pairs were systematically examined by precisely adjusting the pH of the elution solvent using reverse-phase high-performance liquid chromatography (HPLC). Optimal separation conditions were successfully developed for all peptide pairs, enabling the direct quantification of Ser residue stereoinversion through a single HPLC run. Subsequently, the degree of Ser stereoinversion within the model peptide, Gly-Ser-Gly-Tyr, was determined using the method established in this study. Surprisingly, the stereoinversion of Ser residues occurred only when the absolute configurations of Ser and Tyr residues of the peptide differed from each other, whereas no stereoinversion was observed when their absolute configurations were identical. The experiments using peptides similar to the model peptide reveal that both the N-terminal amino group and the hydroxyl group of the C-terminal Tyr residue are involved in the stereoinversion of the Ser residue. By applying a simple method to quantify the stereoinversion of Ser residues, valuable insights into the mechanisms governing these stereoinversions were obtained.

Two DOT1 enzymes cooperatively mediate efficient ubiquitin-independent histone H3 lysine 76 tri-methylation in kinetoplastids

In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx6K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site loop flexible, implying a distinct mechanism of substrate recognition. We further reveal distinct methylation kinetics and substrate preferences of DOT1A (H3K76me0) and DOT1B (DOT1A products H3K76me1/me2) in vitro, determined by a Ser and Ala residue within motif IV, respectively, enabling DOT1A and DOT1B to mediate efficient H3K76 tri-methylation non-processively but cooperatively, and suggesting why kinetoplastids have evolved two DOT1 enzymes.

Characterization of two SGNH family cell death-inducing proteins from the horticulturally important fungal pathogen Botrytis cinerea based on the optimized prokaryotic expression system

Botrytis cinerea is one of the most destructive phytopathogenic fungi, causing significant losses to horticultural crops. As a necrotrophic fungus, B. cinerea obtains nutrients by killing host cells. Secreted cell death-inducing proteins (CDIPs) play a crucial role in necrotrophic infection; however, only a limited number have been reported. For high-throughput CDIP screening, we optimized the prokaryotic expression system and compared its efficiency with other commonly used protein expression systems. The optimized prokaryotic expression system showed superior effectiveness and efficiency and was selected for subsequent CDIP screening. The screening system verified fifty-five candidate proteins and identified two novel SGNH family CDIPs: BcRAE and BcFAT. BcRAE and BcFAT exhibited high expression levels throughout the infection process. Site-directed mutagenesis targeting conserved Ser residues abolished the cell death-inducing activity of both BcRAE and BcFAT. Moreover, the transient expression of BcRAE and BcFAT in plants enhanced plant resistance against B. cinerea without inducing cell death, independent of their enzymatic activities. Our results suggest a high-efficiency screening system for high-throughput CDIP screening and provide new targets for further study of B. cinerea-plant interactions.