A snapshot of the Physcomitrella N-terminome reveals N-terminal methylation of organellar proteins.

KEY MESSAGE: Analysis of the N-terminome of Physcomitrella reveals N-terminal monomethylation of nuclear-encoded, mitochondria-localized proteins. Post- or co-translational N-terminal modifications of proteins influence their half-life as well as mediating protein sorting to organelles via cleavable N-terminal sequences that are recognized by the respective translocation machinery. Here, we provide an overview on the current modification state of the N-termini of over 4500 proteins from the model moss Physcomitrella (Physcomitrium patens) using a compilation of 24 N-terminomics datasets. Our data reveal distinct proteoforms and modification states and confirm predicted targeting peptide cleavage sites of 1,144 proteins localized to plastids and the thylakoid lumen, to mitochondria, and to the secretory pathway. In addition, we uncover extended N-terminal methylation of mitochondrial proteins. Moreover, we identified PpNTM1 (P. patens alpha N-terminal protein methyltransferase 1) as a candidate for protein methylation in plastids, mitochondria, and the cytosol. These data can now be used to optimize computational targeting predictors, for customized protein fusions and their targeted localization in biotechnology, and offer novel insights into potential dual targeting of proteins.

Role of various virulence factors involved in the pathogenesis of Entamoeba histolytica.

Developing countries continuously face challenges to get rid of amoebiasis, a protozoan disease caused by Entamoeba histolytica. Every year around 900 million people get affected by amoebiasis, among them only 10 % of people show the symptoms of the disease while 90 % of people do not show any symptoms but still, serve as carriers of the disease. Asymptomatic persons carry cysts of Entamoeba in their fecal matter, which is carried by house flies to contaminate the food and water. Entamoeba histolytica is a very successful pathogen because it has very well-developed virulence factors that function in infection to host as well as in overcoming the host's immune response. However, researchers have very little information about the clear relationship between virulence factors and the virulence of Entamoeba histolytica, through various research, researchers have been able to identify key pathogenic factors that are crucial to the pathogenesis of amebiasis and have provided valuable insights into the development of the disease. The objective of this review is to underscore various virulence factors (Monosaccharides, Gal/GalNAc lectin, extracellular vesicles, cysteine proteases, amoeba-pores, and actin microfilament) involved in pathogenesis which may be helpful for designing of future drug or therapy.

Perspectives on Synthetic Protein Circuits in Mammalian Cells.

Mammalian synthetic biology aims to engineer cellular behaviors for therapeutic applications, such as enhancing immune cell efficacy against cancers or improving cell transplantation outcomes. Programming complex biological functions necessitates an understanding of molecular mechanisms governing cellular responses to stimuli. Traditionally, synthetic biology has focused on transcriptional circuits, but recent advances have led to the development of synthetic protein circuits, leveraging programmable binding, proteolysis, or phosphorylation to modulate protein interactions and cellular functions. These circuits offer advantages including robust performance, rapid functionality, and compact design, making them suitable for cellular engineering or gene therapies. This review outlines the post-translational toolkit, emphasizing synthetic protein components utilizing proteolysis or phosphorylation to program mammalian cell behaviors. Finally, we focus on key differences between rewiring native signaling pathways and creating orthogonal behaviors, alongside a proposed framework for translating synthetic protein circuits from tool development to pre-clinical applications in biomedicine.

Extracellular Release of a Disintegrin and Metalloproteinase Correlates With Periodontal Disease Severity.

AIM: Periodontal disease is driven by oral pathogens, including Porphyromonas gingivalis, and the release of inflammatory cytokines. These cytokines (e.g., TNF) or their receptors (e.g., IL-1R) are substrates of a disintegrin and metalloproteinases (ADAMs). In this study, we aimed to determine the effects of ADAMs on periodontal disease phenotypes. MATERIALS AND METHODS: Western blot and FRET-based activity measurements of the gingival crevicular fluid (GCF) of patients were compared with those of infected (P. gingivalis) or cytokine-stimulated oral keratinocytes and primary human neutrophils, respectively. This was accompanied by an analysis of the released extracellular vesicles and MMP9 activity. RESULTS: In the GCF of patients, ADAM8 protein expression and activity were correlated with disease stage, whereas ADAM10 protein expression was inversely correlated with disease stage. Infection and the resulting cytokine release orchestrated the release of soluble ADAM8 by oral keratinocytes and primary neutrophils as soluble ectodomain and on exosomes, respectively. Furthermore, ADAM8 regulated the release of ADAM10 and MMP9. CONCLUSION: Dysregulation of cell-associated and extracellular ADAM proteolytic activity may be an essential regulatory element in the progression of periodontal disease driven by ADAM8. The influence of ADAM8 on disease onset and the evaluation of targeting ADAM8 as a potential and novel local treatment option should be addressed in future translational in vivo studies.

Proteases: Role in Various Human Diseases.

Proteases, a group of hydrolytic enzymes catalyzing the hydrolysis of peptide bonds, play pivotal roles in various physiological processes and have emerged as key contributors to the pathogenesis of diverse diseases. This work provides an insight into the impact of protease activity on different disease contexts, highlighting their involvement in cancer, inflammatory disorders, cardiovascular diseases, infectious diseases, and neurodegenerative conditions. In cancer, proteases facilitate tumor growth, invasion, and metastasis, while in inflammatory diseases, dysregulated protease activity exacerbates tissue damage and inflammation. Cardiovascular diseases involve proteases in extracellular matrix remodeling, affecting arterial structure. In infectious diseases, proteases play crucial roles in pathogen invasion and immune evasion. Neurodegenerative diseases are characterized by protease dysregulation, contributing to protein misfolding and aggregation. As research progresses, understanding the intricate relationships between proteases and diseases becomes essential for developing targeted therapeutic strategies. This review aims to provide a comprehensive glimpse into the diverse impact of protease activities on various diseases, emphasizing their potential as crucial players in the landscape of disease pathology and potential therapeutic interventions.

Limited proteolysis of neutrophil granule proteins by the bacterial protease RgpB depletes neutrophil antimicrobial capacity.

Neutrophils are highly abundant in the gingival tissues where they play an essential role in immune homeostasis by preventing microbial invasion. Here, we show that the oral periodontal pathogen Porphyromonas gingivalis utilizes its cysteine proteases (gingipains) to disengage phagosomal antimicrobial capacity. Arginine gingipains are a sub-family of trypsin-like proteases produced by P. gingivalis that cleave several host proteins at arginine residues. We find that RgpB-mediated proteolysis of host proteins is not limited to the extracellular or plasma membrane-associated host proteins, but it can also degrade several intracellular proteins in neutrophils. Using 2D-DIGE coupled with mass spectrometry, we identified several cytoskeletal and cytoplasmic proteins, including metabolic enzymes and antimicrobial proteins such as neutrophil elastase, myeloperoxidase, and proteinase 3 within neutrophil granules that were cleaved by RgpB. Strikingly, despite the breakdown of multiple proteins, RgpB-treated neutrophils did not undergo apoptosis but instead increased integrin expression and underwent broad transcriptional changes consistent with proinflammatory programming. However, despite their primed status and augmented inflammatory capacity, RgpB-treated neutrophils were conducive to intracellular bacterial survival due to the reduced activity of granule proteins and oxidative burst. Thus, our data show a previously unknown role for P. gingivalis proteases in the attenuation of neutrophil microbicidal capacity via proteolysis of intracellular proteins.

In vivo and in vitro analyses of the role of the Prc protease in inducing mucoidy in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, alginate biosynthesis gene expression is inhibited by the transmembrane anti-sigma factor MucA, which sequesters the AlgU sigma factor. Cell envelope stress initiates cleavage of the MucA periplasmic domain by site-1 protease AlgW, followed by further MucA degradation to release AlgU. However, after colonizing the lungs of people with cystic fibrosis, P. aeruginosa converts to a mucoid form that produces alginate constitutively. Mucoid isolates often have mucA mutations, with the most common being mucA22, which truncates the periplasmic domain. MucA22 is degraded constitutively, and genetic studies suggested that the Prc protease is responsible. Some studies also suggested that Prc contributes to induction in strains with wild-type MucA, whereas others suggested the opposite. However, missing from all previous studies is a demonstration that Prc cleaves any protein directly, which leaves open the possibility that the effect of a prc null mutation is indirect. To address the ambiguities and shortfalls, we reevaluated the roles of AlgW and Prc as MucA and MucA22 site-1 proteases. In vivo analyses using three different assays and two different inducing conditions all suggested that AlgW is the only site-1 protease for wild-type MucA in any condition. In contrast, genetics suggested that AlgW or Prc act as MucA22 site-1 proteases in inducing conditions, whereas Prc is the only MucA22 site-1 protease in non-inducing conditions. For the first time, we also show that Prc is unable to degrade the periplasmic domain of wild-type MucA but does degrade the mutated periplasmic domain of MucA22 directly. IMPORTANCE: After colonizing the lungs of individuals with cystic fibrosis, Pseudomonas aeruginosa undergoes mutagenic conversion to a mucoid form, worsening the prognosis. Most mucoid isolates have a truncated negative regulatory protein MucA, which leads to constitutive production of the extracellular polysaccharide alginate. The protease Prc has been implicated, but not shown, to degrade the most common MucA variant, MucA22, to trigger alginate production. This work provides the first demonstration that the molecular mechanism of Prc involvement is direct degradation of the MucA22 periplasmic domain and perhaps other truncated MucA variants as well. MucA truncation and degradation by Prc might be the predominant mechanism of mucoid conversion in cystic fibrosis infections, suggesting that Prc activity could be a useful therapeutic target.

Comparative Genomic Analyses of Colletotrichum lindemuthianum Pathotypes with Different Virulence Levels and Lifestyles.

Colletotrichum lindemuthianum is the most frequent pathogenic fungus of the common bean Phaseolus vulgaris. This filamentous fungus employs a hemibiotrophic nutrition/infection strategy, which is characteristic of many Colletotrichum species. Due to host-pathogen coevolution, C. lindemuthianum includes pathotypes with a diversity of virulence against differential common bean varieties. In this study, we performed comparative genomic analyses on three pathotypes with different virulence levels and a non-pathogenic pathotype, isolated from different geographical areas in Mexico. Our results revealed large genomes with high transposable element contents that have undergone expansions, generating intraspecific diversity. All the pathotypes exhibited a similar number of clusters of orthologous genes (COGs) and Gene Ontology (GO) terms. TFomes contain families that are typical in fungal genomes; however, they show different contents between pathotypes, mainly in transcription factors with the fungal-specific TF and Zn2Cys6 domains. Peptidase families mainly contain abundant serine peptidases, metallopeptidases, and cysteine peptidases. In the secretomes, the number of genes differed between the pathotypes, with a high percentage of candidate effectors. Both the virulence gene and CAZyme gene content for each pathotype was abundant and diverse, and the latter was enriched in hemicellulolytic enzymes. We provide new insights into the nature of intraspecific diversity among C. lindemuthianum pathotypes and the origin of their ability to rapidly adapt to genetic changes in its host and environmental conditions.

Allergic Bronchopulmonary Aspergillosis (ABPA) in the Era of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Modulators.

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity disease caused by Aspergillus fumigatus (Af), prevalent in persons with cystic fibrosis (CF) or asthma. In ABPA, Af proteases drive a T-helper cell-2 (Th2)-mediated allergic immune response leading to inflammation that contributes to permanent lung damage. Corticosteroids and antifungals are the mainstays of therapies for ABPA. However, their long-term use has negative sequelae. The treatment of patients with CF (pwCF) has been revolutionized by the efficacy of cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy. Pharmacological improvement in CFTR function with highly effective elexacaftor/tezacaftor/ivacaftor (ETI) provides unprecedented improvements in lung function and other clinical outcomes of pwCF. The mechanism behind the improvement in patient outcomes is a continued topic of investigation as our understanding of the role of CFTR function evolves. As ETI therapy gains traction in CF management, understanding its potential impact on ABPA, especially on the allergic immune response pathways and Af infection becomes increasingly crucial for optimizing patient outcomes. This literature review aims to examine the extent of these findings and expand our understanding of the already published research focusing on the intersection between ABPA therapeutic approaches in CF and the rapid impact of the evolving CFTR modulator landscape. While our literature search yielded limited reports specifically focusing on the role of CFTR modulator therapy on CF-ABPA, findings from epidemiologic and retrospective studies suggest the potential for CFTR modulator therapies to positively influence pulmonary outcomes by addressing the underlying pathophysiology of CF-ABPA, especially by decreasing inflammatory response and Af colonization. Thus, this review highlights the promising scope of CFTR modulator therapy in decreasing the overall prevalence and incidence of CF-ABPA.

New Bacillus paralicheniformis strain with high proteolytic and keratinolytic activity.

Bacillus paralicheniformis T7, which exhibits high proteolytic and keratinolytic activities, was isolated from soil in Kazakhstan. Its secreted proteases were thermostable and alkaline, demonstrating maximum activity at 70 °C and pH 9.0. The proteases and keratinases of this strain were sensitive to Ni2+, Co2+, Mn2+, and Cd2+, with Cu2+, Co2+ and Cd2+ negatively affecting keratinolytic activity, and Fe3+ ions have a strong inhibitory effect on proteolytic and keratinolytic activity. Seven proteases were identified in the enzymatic extract of B. paralicheniformis T7: four from the serine peptidase family and three from the metallopeptidase family. The proteases hydrolyzed 1 mg of casein, hemoglobin, gelatin, ovalbumin, bovine serum albumin, or keratin within 15 s to 30 min. The high keratinolytic activity of this strain was confirmed through the degradation of chicken feathers, horns, hooves, wool, and cattle hide. Chicken feathers were hydrolyzed in 4 days, and the degrees of hydrolysis for cattle hide, wool, hoof, and horn after 7 days of cultivation were 97.2, 34.5, 29.6, and 3.6%, respectively. During submerged fermentation with feather medium in a laboratory bioreactor, the strain secreted enzymes with 249.20 ± 7.88 U/mL protease activity after 24 h. Thus, B. paralicheniformis T7 can be used to produce proteolytic and keratinolytic enzymes for application in processing proteinaceous raw materials and keratinous animal waste.

First crystal structure of the DUF2436 domain of virulence proteins from Porphyromonas gingivalis.

Porphyromonas gingivalis is a major pathogenic oral bacterium that is responsible for periodontal disease. It is linked to chronic periodontitis, gingivitis and aggressive periodontitis. P. gingivalis exerts its pathogenic effects through mechanisms such as immune evasion and tissue destruction, primarily by secreting various factors, including cysteine proteases such as gingipain K (Kgp), gingipain R (RgpA and RgpB) and PrtH (UniProtKB ID P46071). Virulence proteins comprise multiple domains, including the pro-peptide region, catalytic domain, K domain, R domain and DUF2436 domain. While there is a growing database of knowledge on virulence proteins and domains, there was no prior evidence or information regarding the structure and biological function of the well conserved DUF2436 domain. In this study, the DUF2436 domain of PrtH from P. gingivalis (PgDUF2436) was determined at 2.21 Šresolution, revealing a noncanonical ß-jelly-roll sandwich topology with two antiparallel ß-sheets and one short α-helix. Although the structure of PgDUF2436 was determined by the molecular-replacement method using an AlphaFold model structure as a template, there were significant differences in the positions of ß1 between the AlphaFold model and the experimentally determined PgDUF2436 structure. The Basic Local Alignment Search Tool sequence-similarity search program showed no sequentially similar proteins in the Protein Data Bank. However, DaliLite search results using structure-based alignment revealed that the PgDUF2436 structure has structural similarity Z-scores of 5.9-5.4 with the C-terminal domain of AlgF, the D4 domain of cytolysin, IglE and the extracellular domain structure of PepT2. This study has elucidated the structure of the DUF2436 domain for the first time and a comparative analysis with similar structures has been performed.

Production of milk-coagulating protease by fungus Pleurotus djamor through solid state fermentation using wheat bran as the low-cost substrate.

Proteases are enzymes that hydrolyze peptide bonds present in proteins and peptides. They are widely used for various industrial applications, such as in the detergent, food, and dairy industries. Cheese is one of the most important products of the dairy industry, and the coagulation stage is crucial during the cheese-making process. Enzymatic coagulation is the most common technique utilized for this purpose. Microbial enzymes are frequently used for coagulation due to their advantages in terms of availability, sustainability, quality control, product variety, and compliance with dietary and cultural/religious requirements. In the present study, we identified and subsequently characterized milk coagulant activity from the fungus Pleurotus djamor PLO13, obtained during a solid-state fermentation process, using the agro-industrial residue, wheat bran, as the fermentation medium. Maximum enzyme production and caseinolytic activity occurred 120 h after cultivation. When the enzyme activity against various protease-specific synthetic substrates and inhibitors was analyzed, the enzyme was found to be a serine protease, similar to elastase 2. This elastase-2-like serine protease was able to coagulate pasteurized whole and reconstituted skim milk highly efficiently in the presence and absence of calcium, even at room temperature. The coagulation process was influenced by factors such as temperature, time, and calcium concentration. We demonstrate here, for the first time, an elastase-2-like enzyme in a microorganism and its potential application in the food industry for cheese production.

To Explore Potential Natural Inhibitors of Cysteine Proteases to Combat Human African Trypanosomiasis and Chagas Disease.

BACKGROUND: Human African Trypanosomiasis (HAT), also known as sleeping sickness, and Chagas disease are neglected tropical diseases caused by Trypanosoma brucei and Trypanosoma cruzi, respectively. These diseases present significant challenges in treatment due to the toxicity, low efficacy, and drug-resistant strains associated with current therapies. INTRODUCTION: Cysteine proteases play vital roles in the life cycles of these parasites, making them potential targets for therapeutic intervention. Natural inhibitors sourced from plants, marine organisms, and microorganisms show promise for developing novel therapies. METHODS: This review surveys the potential of natural inhibitors as therapeutic agents against HAT and Chagas disease. It compiles PubMed and PubChem information from various studies to provide an overview of their activities and characteristics, including their ability to inhibit cysteine proteases, modulate the host immune response, and interfere with other parasite proteins. RESULTS: Several natural inhibitors, such as berberine, curcumin, and tannins, have been identified and characterized. These inhibitors have demonstrated encouraging outcomes in both in vitro and in vivo experiments, indicating their potential as therapeutic agents for HAT and Chagas disease. CONCLUSION: Natural inhibitors of cysteine proteases offer a promising avenue for developing novel therapies against HAT and Chagas disease. Further research is needed to identify additional natural inhibitors and optimize their efficacy and safety for human use. The significance of this study lies in its potential to contribute to the discovery of effective, safe, and affordable treatments for these neglected tropical diseases.

Characterization of the extracellular proteases from Bacillus inaquosorum strain E1-8 and its application in the preparation of hydrolysates from plant and animal proteins with antioxidant, antifreeze and anti-browning properties.

BACKGROUND: Bacillus inaquosorum strains is widely recognized for their plant-growth-promoting and biocontrol capabilities, yet their roles in protease production remain unclear. The present study aimed to comprehensively assess the protease-producing performance of B. inaquosorum strain E1-8, at the same time as exploring the novel application of agricultural Bacillus proteases in the preparation of protein hydrolysates for fresh-cut fruits preservation. RESULTS: First, genomic sequencing revealed the diversity of E1-8 proteases, indicating 15 putative extracellular proteases. Subsequently, the fermentation conditions for E1-8 protease production were optimized, with sweet potato powder and soybean meal identified as the most suitable carbon and nitrogen sources, respectively, resulting in a maximum protease activity of 321.48 U mL-1. Upon culturing the strain under these optimized conditions, only an S8 family serine protease and an M48 family metalloprotease were revealed by secretomic analysis and protease inhibitor assays. Additionally, the optimal protease conditions for generating protein hydrolysates from soy, pea, fish and porcine proteins were determined. The molecular weight of the hydrolysates primarily ranged from 2000 to 180 Da, with a total of 17 amino acids identified. The application of these hydrolysates demonstrated a 2,2-diphenyl-1-picrylhydrazyl (i.e. DPPH) scavenging activity ranging from 58.64% to 84.12%, significantly reducing of the melting peaks and the freezing points. Furthermore, the browning index of apple slices stored at 4 °C decreased by 14.81% to 22.15% on the second day, and similar effects were observed in fresh-cut banana stored at 4 °C for 7 days. CONCLUSION: The protein hydrolysates obtained exhibit remarkable antioxidant, antifreeze and anti-browning properties for fresh-cut fruits. © 2024 Society of Chemical Industry.

No signs of mast cell involvement in long-COVID: A case-control study.

Long-COVID caused by SARS-CoV-2 infection has significant and increasing effects on human health worldwide. Although a unifying molecular or biological explanation is lacking, several pathophysiological mechanisms have been proposed. Involvement of mast cells-evolutionary old "multipurpose" innate immune cells-was reported recently in studies of acute infection and post-acute-COVID-19 syndrome. Mast cell activity has been suggested in long-COVID. In this case-control study, we compared data from 24 individuals with long-COVID (according to the NICE criteria) and 24 age- and sex-matched healthy individuals with a history of SARS-CoV-2 infection without developing sequelae. Serum levels of the proteases beta-tryptase (TPSB2) and carboxypeptidase (CPA3), which are mast cell specific, were measured using immunoassays. The values were compared between the two groups and correlated to measures of physical exertional intolerance. TPSB2 and CPA3 levels were median (range) 26.9 (2.0-1000) and 5.8 (1.5-14.0) ng/mL, respectively, in the long-COVID group. The corresponding values in the control group were 10.9 (2.0-1000) (p = 0.93) and 5.3 (3.5-12.9) ng/mL (p = 0.82). No significant correlations between TPSB2 or CPA3 levels and scores on the ten physical subscales of SF-36, 3.1-3.10 were revealed. We found no significant differences in the levels of mast cell activation markers TPSB2 and CPA3 between the long-COVID and control groups and no correlations with proxy markers of exercise intolerance. Mast cell activation does not appear to be part of long-term pathogenesis of long-COVID, at least in the majority of patients.

Comparative bioinformatic analysis and biochemical characterization of digestive proteinases from Pacific whiting (Merluccius productus).

The hake fishery plays a crucial role due to its significant economic impact. The genus Merluccius includes 12 extant species found along the coasts of the Americas, Europe, and Africa. However, research on their digestive physiology and the enzymes involved in digestion, including proteases, remains limited. Proteases play a key role in protein digestion, a vital process for all living organisms. This study focused on screening the genomes of eight Merluccius spp. for eight specific proteases previously identified in Merluccius polli. Additionally, the study conducted biochemical analyses of proteases found in the stomach and intestine of Pacific whiting fish (Merluccius productus), comparing the results with the genomic findings. The analysis revealed that proteases across Merluccius spp. are conserved, although with slight variations, particularly in chymotrypsin and aspartic proteases. Biochemical characterization of M. productus identified at least three main proteases in the stomach, active at acidic pH, and at least seven proteases in the intestine, active at alkaline pH, as determined by electrophoresis. Further investigation, including specific inhibition studies, determination of molecular mass, and assessment of pH and temperature preferences for catalysis, revealed that one of the stomach proteases functioning at acidic pH likely belongs to the acid peptidase class, likely pepsin. Similarly, analysis of proteases active at alkaline pH indicated the presence of a chymotrypsin and a trypsin, consistent with genomic findings in M. productus. These results are important as they provide insights into the digestive physiology of Merluccius spp., contributing to a better understanding of their nutritional needs.

Transcriptional Profiling of Staphylococcus aureus during the Transition from Asymptomatic Nasal Colonization to Skin Colonization/Infection in Patients with Atopic Dermatitis.

Staphylococcus aureus acts both as a colonizing commensal bacterium and invasive pathogen. Nasal colonization is associated with an increased risk of infection caused by the identical strain. In patients with atopic dermatitis (AD), the degree of S. aureus colonization is associated with the severity of the disease. Here, we comparatively analyzed the in vivo transcriptional profile of S. aureus colonizing the nose and non-diseased skin (non-lesional skin) as opposed to the diseased skin (lesional skin-defined here as infection) of 12 patients with AD. The transcriptional profile during the asymptomatic colonization of the nose closely resembled that of the lesional skin samples for many of the genes studied, with an elevated expression of the genes encoding adhesion-related proteins and proteases. In addition, the genes that modify and remodel the cell wall and encode proteins that facilitate immune evasion showed increased transcriptional activity. Notably, in a subgroup of patients, the global virulence regulator Agr (accessory gene regulator) and downstream target genes were inactive during nasal colonization but upregulated in the lesional and non-lesional skin samples. Taken together, our results demonstrate a colonization-like transcriptional profile on diseased skin and suggest a role for the peptide quorum sensing system Agr during the transition from asymptomatic nasal colonization to skin colonization/infection.

Verticillium dahliae Elicitor VdSP8 Enhances Disease Resistance Through Increasing Lignin Biosynthesis in Cotton.

Verticillium wilt caused by the soil-borne fungus Verticillium dahliae Kleb., is a destructive plant disease that instigates severe losses in many crops. Improving plant resistance to Verticillium wilt has been a challenge in most crops. In this study, a V. dahliae secreted protein VdSP8 was identified and shown to activate hyper-sensitive response (HR) and systemic acquired resistance (SAR) to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) and Botrytis cinerea in tobacco plants. We identified a ß-glucosidase named GhBGLU46 as a cotton plant target of VdSP8. VdSP8 interacts with GhBGLU46 both in vivo and in vitro and promotes the ß-glucosidase activity of GhBGLU46. Silencing of GhBGLU46 reduced the expression of genes involved in lignin biosynthesis, such as GhCCR4, GhCCoAOMT2, GhCAD3 and GhCAD6, thus decreasing lignin deposition and increasing Verticillium wilt susceptibility. We have shown that GhBGLU46 is indispensable for the function of VdSP8 in plant resistance. These results suggest that plants have also evolved a strategy to exploit the invading effector protein VdSP8 to enhance plant resistance.

Rhodesain inhibitors on the edge of reversibility-irreversibility.

A comparative study of Michael acceptor and keto-Michael acceptor inhibitors of the cysteine protease rhodesain has been performed. Five new inhibitors have been prepared bearing the peptide structure of the known cysteine protease inhibitor K11777 and differing on the warhead. For the preparation of the Michael acceptor warhead, a Horner-Wadsworth-Emmons reaction was used. In the synthetic routes of the keto-Michael acceptor warheads, keto-enoate and keto-vinyl sulfone, a metathesis reaction and a radical sulfonylation were the key steps, respectively. Interestingly, keto-Michael acceptors inhibited rhodesain through a dual mode of action, showing reversibility at low inhibitor concentrations and irreversibility at high inhibitor concentrations.

Assessing AF2's ability to predict structural ensembles of proteins.

Recent breakthroughs in protein structure prediction have enhanced the precision and speed at which protein configurations can be determined. Additionally, molecular dynamics (MD) simulations serve as a crucial tool for capturing the conformational space of proteins, providing valuable insights into their structural fluctuations. However, the scope of MD simulations is often limited by the accessible timescales and the computational resources available, posing challenges to comprehensively exploring protein behaviors. Recently emerging approaches have focused on expanding the capability of AlphaFold2 (AF2) to predict conformational substates of protein. Here, we benchmark the performance of various workflows that have adapted AF2 for ensemble prediction and compare the obtained structures with ensembles obtained from MD simulations and NMR. We provide an overview of the levels of performance and accessible timescales that can currently be achieved with machine learning (ML) based ensemble generation. Significant minima of the free energy surfaces remain undetected.