MoYvh1 subverts rice defense through functions of ribosomal protein MoMrt4 in Magnaporthe oryzae.

The accumulation of the reactive oxygen species (ROS) in rice is important in its interaction with the rice blast fungus Magnaporthe oryzae during which the pathogen scavenges ROS through the production of extracellular enzymes that promote blast. We previously characterized the MoYvh1 protein phosphatase from M. oryzae that plays a role in scavenging of ROS. To understand the underlying mechanism, we found that MoYvh1 is translocated into the nucleus following oxidative stress and that this translocation is dependent on MoSsb1 and MoSsz1 that are homologous to heat-shock protein 70 (Hsp70) proteins. In addition, we established a link between MoYvh1 and MoMrt4, a ribosome maturation factor homolog whose function also involves shuttling between the cytoplasm and the nucleus. Moreover, we found that MoYvh1 regulates the production of extracellular proteins that modulate rice-immunity. Taking together, our evidence suggests that functions of MoYvh1 in regulating ROS scavenging require its nucleocytoplasmic shuttling and the partner proteins MoSsb1 and MoSsz1, as well as MoMrt4. Our findings provide novel insights into the mechanism by which M. oryzae responds to and subverts host immunity through the regulation of ribosome biogenesis and protein biosynthesis.

MoPpe1 partners with MoSap1 to mediate TOR and cell wall integrity signalling in growth and pathogenicity of the rice blast fungus Magnaporthe oryzae.

In the rice blast fungus Magnaporthe oryzae, the cell wall integrity (CWI) signalling pathway governs cell wall changes in response to external cues and normal CWI signalling is critical for appressorium function and pathogenicity. We previously characterized the mitogen-activated protein kinase (MAPK) kinase MoMkk1 as an integral component of the CWI pathway. Using the affinity purification approach, we have identified MoMkk1-interacting MoPpe1 as a homologue of Saccharomyces cerevisiae serine/threonine protein phosphatase Sit4/Ppe1. We found that MoPpe1 is required for vegetative growth, conidiation and full virulence. In addition, we found that MoPpe1 interacts with MoSap1, a protein with functions similar to MoPpe1. Intriguingly, we found that MoPpe1-MoSap1 interaction is related to CWI and target of rapamycin (TOR) pathways. We presented evidence suggesting that MoPpe1 and MoSap1 function as an adaptor complex linking CWI and TOR signalling and that the activation of the TOR pathway leads to suppression of CWI signalling, resulting in defects in appressorium function and pathogenicity. Taken together, our studies not only reveal important functions of MoMkk1-MoPpe1-MoSap1 interactions in growth and pathogenicity of the blast fungus, but also highlight the complexity of regulatory networks involving conserved yet novel regulatory mechanisms of CWI and TOR signalling.

The Putative Protein Phosphatase MoYvh1 Functions Upstream of MoPdeH to Regulate the Development and Pathogenicity in Magnaporthe oryzae.

Protein phosphatases are critical regulators in eukaryotic cells. For example, the budding yeast Saccharomyces cerevisiae dual specificity protein phosphatase (DSP) ScYvh1 regulates growth, sporulation, and glycogen accumulation. Despite such importance, functions of Yvh1 proteins in filamentous fungi are not well understood. In this study, we characterized putative protein phosphatase MoYvh1, an Yvh1 homolog in the rice blast fungus Magnaporthe oryzae. Deletion of the MoYVH1 gene resulted in significant reductions in vegetative growth, conidial production, and virulence. The ΔMoyvh1 mutant also displayed defects in cell-wall integrity and was hyposensitive to the exogenous osmotic stress. Further examination revealed that the ΔMoyvh1 mutant had defects in appressorium function and invasive hyphae growth, resulting attenuated pathogenicity. Interestingly, we found that MoYvh1 affects the scavenging of host-derived reactive oxygen species that promotes M. oryzae infection. Finally, overexpression of the phosphodiesterase MoPDEH suppressed the defects in conidia formation and pathogenicity of the ΔMoyvh1 mutant, suggesting MoYvh1 could regulate MoPDEH for its function. Our study reveals not only the importance of MoYvh1 proteins in growth, differentiation, and virulence of the rice blast fungus but, also, a genetic link between MoYvh1 and MoPDEH-cAMP signaling in this fungus.

Diverse models of cavity engineering in enzyme modification: Creation, filling, and reshaping.

Most enzyme modification strategies focus on designing the active sites or their surrounding structures. Interestingly, a large portion of the enzymes (60%) feature active sites located within spacious cavities. Despite recent discoveries, cavity-mediated enzyme engineering remains crucial for enhancing enzyme properties and unraveling folding-unfolding mechanisms. Cavity engineering influences enzyme stability, catalytic activity, specificity, substrate recognition, and docking. This article provides a comprehensive review of various cavity engineering models for enzyme modification, including cavity creation, filling, and reshaping. Additionally, it also discusses feasible tools for geometric analysis, functional assessment, and modification of cavities, and explores potential future research directions in this field. Furthermore, a promising universal modification strategy for cavity engineering that leverages state-of-the-art technologies and methodologies to tailor cavities according to the specific requirements of industrial production conditions is proposed.

Computer-Aided Reconstruction and Application of Bacillus halodurans S7 Xylanase with Heat and Alkali Resistance.

ß-1,4-Endoxylanase is the most critical hydrolase for xylan degradation during lignocellulosic biomass utilization. However, its poor stability and activity in hot and alkaline environments hinder its widespread application. In this study, BhS7Xyl from Bacillus halodurans S7 was improved using a computer-aided design through isothermal compressibility (ßT) perturbation engineering and by combining three thermostability prediction algorithms (ICPE-TPA). The best variant with remarkable improvement in specific activity, heat resistance (70 °C), and alkaline resistance (both pH 9.0 and 70 °C), R69F/E137M/E145L, exhibited a 4.9-fold increase by wild-type in specific activity (1368.6 U/mg), a 39.4-fold increase in temperature half-life (458.1 min), and a 57.6-fold increase in pH half-life (383.1 min). Furthermore, R69F/E137M/E145L was applied to the hydrolysis of agricultural waste (corncob and hardwood pulp) to efficiently obtain a higher yield of high-value xylooligosaccharides. Overall, the ICPE-TPA strategy has the potential to improve the functional performance of enzymes under extreme conditions for the high-value utilization of lignocellulosic biomass.

Directed Evolution of the UDP-Glycosyltransferase UGTBL1 for Highly Regioselective and Efficient Biosynthesis of Natural Phenolic Glycosides.

The O-glycosylation of polyphenols for the synthesis of glycosides has garnered substantial attention in food research applications. However, the practical utility of UDP-glycosyltransferases (UGTs) is significantly hindered by their low catalytic efficiency and suboptimal regioselectivity. The concurrent optimization of the regioselectivity and activity during the glycosylation of polyphenols presents a formidable challenge. Here, we addressed the long-standing activity-regioselectivity tradeoff in glycosyltransferase UGTBL1 through systematic enzyme engineering. The optimal combination of mutants, N61S/I62M/D63W/A208R/P218W/R282W (SMWRW1W2), yielded a 6.1-fold improvement in relative activity and a 17.3-fold increase in the ratio of gastrodin to para-hydroxybenzyl alcohol-4'-O-ß-glucoside (with 89.5% regioselectivity for gastrodin) compared to those of the wild-type enzyme and ultimately allowed gram-scale production of gastrodin (1,066.2 mg/L) using whole-cell biocatalysis. In addition, variant SMWRW1W2 exhibited a preference for producing phenolic glycosides from several substrates. This study lays the foundation for the engineering of additional UGTs and the practical applications of UGTs in regioselective retrofitting.

Homeostasis of cell wall integrity pathway phosphorylation is required for the growth and pathogenicity of Magnaporthe oryzae.

The cell wall provides a crucial barrier to stress imposed by the external environment. In the rice blast fungus Magnaporthe oryzae, this stress response is mediated by the cell wall integrity (CWI) pathway, consisting of a well-characterized protein phosphorylation cascade. However, other regulators that maintain CWI phosphorylation homeostasis, such as protein phosphatases (PPases), remain unclear. Here, we identified two PPases, MoPtc1 and MoPtc2, that function as negative regulators of the CWI pathway. MoPtc1 and MoPtc2 interact with MoMkk1, one of the key components of the CWI pathway, and are crucial for the vegetative growth, conidial formation, and virulence of M. oryzae. We also demonstrate that both MoPtc1 and MoPtc2 dephosphorylate MoMkk1 in vivo and in vitro, and that CWI stress leads to enhanced interaction between MoPtc1 and MoMkk1. CWI stress abolishes the interaction between MoPtc2 and MoMkk1, providing a means of deactivation for CWI signalling. Our studies reveal that CWI signalling in M. oryzae is a highly coordinated regulatory mechanism vital for stress response and pathogenicity.

Stathmin 1 is a biomarker for diagnosis of microvascular invasion to predict prognosis of early hepatocellular carcinoma.

Microvascular invasion (MVI) is presently evaluated as a high-risk factor to be directly relative to postoperative prognosis of hepatocellular carcinoma (HCC). Up to now, diagnosis of MVI mainly depends on the postoperative pathological analyses with H&E staining assay, based on numbers and distribution characteristics of MVI to classify the risk levels of MVI. However, such pathological analyses lack the specificity to discriminate MVI in HCC specimens, especially in complicated pathological tissues. In addition, the efficiency to precisely define stages of MVI is not satisfied. Thus, any biomarker for both conforming diagnosis of MVI and staging its levels will efficiently and effectively promote the prediction of early postoperative recurrence and metastasis for HCC. Through bioinformatics analysis and clinical sample verification, we discovered that Stathmin 1 (STMN1) gene was significantly up-regulated at the locations of MVI. Combining STMN1 immunostaining with classic H&E staining assays, we established a new protocol for MVI pathological diagnosis. Next, we found that the degrees of MVI risk could be graded according to expression levels of STMN1 for prognosis prediction on recurrence rates and overall survival in early HCC patients. STMN1 affected epithelial-mesenchymal transformation (EMT) of HCC cells by regulating the dynamic balance of microtubules through signaling of "STMN1-Microtubule-EMT" axis. Inhibition of STMN1 expression in HCC cells reduced their lung metastatic ability in recipients of mouse model, suggesting that STMN1 also could be a potential therapeutic target for inhibiting HCC metastasis. Therefore, we conclude that STMN1 has potentials for clinical applications as a biomarker for both pathological diagnosis and prognostic prediction, as well as a therapeutic target for HCC.

A self-balancing circuit centered on MoOsm1 kinase governs adaptive responses to host-derived ROS in Magnaporthe oryzae.

The production of reactive oxygen species (ROS) is a ubiquitous defense response in plants. Adapted pathogens evolved mechanisms to counteract the deleterious effects of host-derived ROS and promote infection. How plant pathogens regulate this elaborate response against ROS burst remains unclear. Using the rice blast fungus Magnaporthe oryzae, we uncovered a self-balancing circuit controlling response to ROS in planta and virulence. During infection, ROS induces phosphorylation of the high osmolarity glycerol pathway kinase MoOsm1 and its nuclear translocation. There, MoOsm1 phosphorylates transcription factor MoAtf1 and dissociates MoAtf1-MoTup1 complex. This releases MoTup1-mediated transcriptional repression on oxidoreduction-pathway genes and activates the transcription of MoPtp1/2 protein phosphatases. In turn, MoPtp1/2 dephosphorylate MoOsm1, restoring the circuit to its initial state. Balanced interactions among proteins centered on MoOsm1 provide a means to counter host-derived ROS. Our findings thereby reveal new insights into how M. oryzae utilizes a phosphor-regulatory circuitry to face plant immunity during infection.

Conversion of hepatoma cells to hepatocyte-like cells by defined hepatocyte nuclear factors.

Normal cells become cancer cells after a malignant transformation, but whether cancer cells can be reversed to normal status remains elusive. Here, we report that the combination of hepatocyte nuclear factor 1A (HNF1A), HNF4A and forkhead box protein A3 (FOXA3) synergistically reprograms hepatocellular carcinoma (HCC) cells to hepatocyte-like cells (reprogrammed hepatocytes, rHeps). Our results show that rHeps lose the malignant phenotypes of cancer cells and retrieve hepatocyte-specific characteristics including hepatocyte-like morphology; global expression pattern of genes and specific biomarkers of hepatocytes; and the unique hepatic functions of albumin (ALB) secretion, glycogen synthesis, low-density lipoprotein (LDL) uptake, urea production, cytochrome P450 enzymes induction and drug metabolism. Intratumoral injection of these three factors efficiently shrank patient-derived tumor xenografts and reprogrammed HCC cells in vivo. Most importantly, transplantation of rHeps in the liver of fumarylacetoacetate hydrolase-deficient (Fah-/-) mice led to the reconstruction of hepatic lobules and the restoration of hepatic function. Mechanistically, exogenous expression of HNF1A, HNF4A and FOXA3 in HCC cells initiated the endogenous expression of numerous hepatocyte nuclear factors, which promoted the conversion of HCC cells to hepatocyte-like cells. Collectively, our results indicate the successful conversion of hepatoma cells to hepatocyte-like cells, not only extending our current knowledge of cell reprogramming but also providing a route towards a novel therapeutic strategy for cancer.

Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens.

The study of pathophysiological mechanisms in human liver disease has been constrained by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. We and others have previously shown that mouse mature hepatocytes can be converted to liver progenitor-like cells in vitro with defined chemical factors. Here we describe a protocol achieving efficient conversion of human primary hepatocytes into liver progenitor-like cells (HepLPCs) through delivery of developmentally relevant cues, including NAD + -dependent deacetylase SIRT1 signaling. These HepLPCs could be expanded significantly during in vitro passage. The expanded cells can readily be converted back into metabolically functional hepatocytes in vitro and upon transplantation in vivo. Under three-dimensional culture conditions, differentiated cells generated from HepLPCs regained the ability to support infection or reactivation of hepatitis B virus (HBV). Our work demonstrates the utility of the conversion between hepatocyte and liver progenitor-like cells for studying HBV biology and antiviral therapies. These findings will facilitate the study of liver diseases and regenerative medicine.

Senescence and cell death in chronic liver injury: roles and mechanisms underlying hepatocarcinogenesis.

Chronic liver injury (CLI) is a complex pathological process typically characterized by progressive destruction and regeneration of liver parenchymal cells due to diverse risk factors such as alcohol abuse, drug toxicity, viral infection, and genetic metabolic disorders. When the damage to hepatocytes is mild, the liver can regenerate itself and restore to the normal state; when the damage is irreparable, hepatocytes would undergo senescence or various forms of death including apoptosis, necrosis and necroptosis. These pathological changes not only promote the progression of the existing hepatopathies via various underlying mechanisms but are closely associated with hepatocarcinogenesis. In this review, we discuss the pathological changes that hepatocytes undergo during CLI, and their roles and mechanisms in the progression of hepatopathies and hepatocarcinogenesis. We also give a brief introduction about some animal models currently used for the research of CLI and progress in the research of CLI.

The extent of liver injury determines hepatocyte fate toward senescence or cancer.

It is well known that induction of hepatocyte senescence could inhibit the development of hepatocellular carcinoma (HCC). Until now, it is still unclear how the degree of liver injury dictates hepatocyte senescence and carcinogenesis. In this study, we investigated whether the severity of injury determines cell fate decisions between hepatocyte senescence and carcinogenesis. After testing of different degrees of liver injury, we found that hepatocyte senescence is strongly induced in the setting of severe acute liver injury. Longer-term, moderate liver injury, on the contrary did not result into hepatocyte senescence, but led to a significant incidence of HCC instead. In addition, carcinogenesis was significantly reduced by the induction of severe acute injury after chronic moderate liver injury. Meanwhile, immune surveillance, especially the activations of macrophages, was activated after re-induction of senescence by severe acute liver injury. We conclude that severe acute liver injury leads to hepatocyte senescence along with activating immune surveillance and a low incidence of HCC, whereas chronic moderate injury allows hepatocytes to proliferate rather than to enter into senescence, and correlates with a high incidence of HCC. This study improves our understanding in hepatocyte cell fate decisions and suggests a potential clinical strategy to induce senescence to treat HCC.

Carbamoyl Phosphate Synthetase Subunit MoCpa2 Affects Development and Pathogenicity by Modulating Arginine Biosynthesis in Magnaporthe oryzae.

Arginine is a semi-essential amino acid that affects physiological and biochemical functions. The CPA2 gene in yeast encodes a large subunit of arginine-specific carbamoyl phosphate synthetase (CPS) and is involved in arginine biosynthesis. Here, an ortholog of yeast CPA2 was identified in the rice blast fungus Magnaporthe oryzae, and was named MoCPA2. MoCpa2 is an 1180-amino acid protein which contains an ATP grasp domain and two CPSase domains. Targeted deletion of MoCPA2 supported its role in de novo arginine biosynthesis in M. oryzae as mutant phenotypes were complemented by arginine but not ornithine. The ΔMocpa2 mutant exhibited defects in asexual development and pathogenicity but not appressorium formation. Further examination revealed that the invasive hyphae of the ΔMocpa2 mutant were restricted mainly to the primary infected cells. In addition, the ΔMocpa2 mutant was unable to induce a plant defense response and had the ability to scavenge ROS during pathogen-plant interactions. Structure analysis revealed that the ATP grasp domain and each CPS domain were indispensable for the proper localization and full function of MoCpa2. In summary, our results indicate that MoCpa2 plays an important role in arginine biosynthesis, and affects growth, conidiogenesis, and pathogenicity. These results suggest that research into metabolism and processes that mediate amino acid synthesis are valuable for understanding M. oryzae pathogenesis.