Mammalian ATG8 proteins maintain autophagosomal membrane integrity through ESCRTs.

The canonical autophagy pathway in mammalian cells sequesters diverse cytoplasmic cargo within the double membrane autophagosomes that eventually convert into degradative compartments via fusion with endolysosomal intermediates. Here, we report that autophagosomal membranes show permeability in cells lacking principal ATG8 proteins (mATG8s) and are unable to mature into autolysosomes. Using a combination of methods including a novel in vitro assay to measure membrane sealing, we uncovered a previously unappreciated function of mATG8s to maintain autophagosomal membranes in a sealed state. The mATG8 proteins GABARAP and LC3A bind to key ESCRT-I components contributing, along with other ESCRTs, to the integrity and imperviousness of autophagic membranes. Autophagic organelles in cells lacking mATG8s are permeant, are arrested as amphisomes, and do not progress to functional autolysosomes. Thus, autophagosomal organelles need to be maintained in a sealed state in order to become lytic autolysosomes.

Plague vaccines: new developments in an ongoing search.

As the reality of pandemic threats challenges humanity, exemplified during the ongoing SARS-CoV-2 infections, the development of vaccines targeting these etiological agents of disease has become increasingly critical. Of paramount concern are novel and reemerging pathogens that could trigger such events, including the plague bacterium Yersinia pestis. Y. pestis is responsible for more human deaths than any other known pathogen and exists globally in endemic regions of the world, including the four corners region and Northern California in the USA. Recent cases have been scattered throughout the world, including China and the USA, with serious outbreaks in Madagascar during 2008, 2013-2014, and, most recently, 2017-2018. This review will focus on recent advances in plague vaccine development, a seemingly necessary endeavor, as there is no Food and Drug Administration-licensed vaccine available for human distribution in western nations, and that antibiotic-resistant strains are recovered clinically or intentionally developed. Progress and recent development involving subunit, live-attenuated, and nucleic acid-based plague vaccine candidates will be discussed in this review. KEY POINTS: • Plague vaccine development remains elusive yet critical. • DNA, animal, and live-attenuated vaccine candidates gain traction.

Utilizing AfDesign for Developing a Small Molecule Inhibitor of PICK 1-PDZ.

INTRODUCTION: The PICK1 PDZ domain has been identified as a potential drug target for neurological disorders. After many years of effort, a few inhibitors, such as TAT-C5 and mPD5, have been discovered experimentally to bind to the PDZ domain with a relatively high binding affinity. With the rapid growth of computational research, there is an urgent need for more efficient computational methods to design viable ligands that target proteins. METHOD: Recently, a newly developed program called AfDesign (part of ColabDesign) at https:// github.com/sokrypton/ColabDesign), an open-source software built on AlphaFold, has been suggested to be capable of generating ligands that bind to targeted proteins, thus potentially facilitating the ligand development process. To evaluate the performance of this program, we explored its ability to target the PICK1 PDZ domain, given our current understanding of it. We found that the designated length of the ligand and the number of recycles play vital roles in generating ligands with optimal properties. RESULTS: Utilizing AfDesign with a sequence length of 5 for the ligand produced the highest comparable ligands to that of prior identified ligands. Moreover, these designed ligands displayed significantly lower binding energy compared to manually created sequences. CONCLUSION: This work demonstrated that AfDesign can potentially be a powerful tool to facilitate the exploration of the ligand space for the purpose of targeting PDZ domains.

Progress on the research and development of plague vaccines with a call to action.

There is a compelling demand for approved plague vaccines due to the endemicity of Yersinia pestis and its potential for pandemic spread. Whilst substantial progress has been made, we recommend that the global funding and health security systems should work urgently to translate some of the efficacious vaccines reviewed herein to expedite clinical development and to prevent future disastrous plague outbreaks, particularly caused by antimicrobial resistant Y. pestis strains.Content includes material subject to Crown Copyright © 2024.This is an open access article under the Open Government License ( http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/ ).

A bacteriophage cocktail targeting Yersinia pestis provides strong post-exposure protection in a rat pneumonic plague model.

Yersinia pestis, one of the deadliest bacterial pathogens ever known, is responsible for three plague pandemics and several epidemics, with over 200 million deaths during recorded history. Due to high genomic plasticity, Y. pestis is amenable to genetic mutations as well as genetic engineering that can lead to the emergence or intentional development of pan-drug-resistant strains. Indeed, antibiotic-resistant strains (e.g., strains carrying multidrug-resistant or MDR plasmids) have been isolated in various countries and endemic areas. Thus, there is an urgent need to develop novel, safe, and effective treatment approaches for managing Y. pestis infections. This includes infections by antigenically distinct strains for which vaccines (none FDA approved yet) may not be effective and those that cannot be managed by currently available antibiotics. Lytic bacteriophages provide one such alternative approach. In this study, we examined post-exposure efficacy of a bacteriophage cocktail, YPP-401, to combat pneumonic plague caused by Y. pestis CO92. YPP-401 is a four-phage preparation effective against a panel of at least 68 genetically diverse Y. pestis strains. Using a pneumonic plague aerosol challenge model in gender-balanced Brown Norway rats, YPP-401 demonstrated ~88% protection when delivered 18 h post-exposure for each of two administration routes (i.e., intraperitoneal and intranasal) in a dose-dependent manner. Our studies provide proof-of-concept that YPP-401 could be an innovative, safe, and effective approach for managing Y. pestis infections, including those caused by naturally occurring or intentionally developed multidrug-resistant strains.IMPORTANCECurrently, there are no FDA-approved plague vaccines. Since antibiotic-resistant strains of Y. pestis have emerged or are being intentionally developed to be used as a biothreat agent, new treatment modalities are direly needed. Phage therapy provides a viable option against potentially antibiotic-resistant strains. Additionally, phages are nontoxic and have been approved by the FDA for use in the food industry. Our study provides the first evidence of the protective effect of a cocktail of four phages against pneumonic plague, the most severe form of disease. When treatment was initiated 18 h post infection by either the intranasal or intraperitoneal route in Brown Norway rats, up to 87.5% protection was observed. The phage cocktail had a minimal impact on a representative human microbiome panel, unlike antibiotics. This study provides strong proof-of-concept data for the further development of phage-based therapy to treat plague.

Noncanonical roles of ATG5 and membrane atg8ylation in retromer assembly and function.

ATG5 is one of the core autophagy proteins with additional functions such as noncanonical membrane atg8ylation, which among a growing number of biological outputs includes control of tuberculosis in animal models. Here we show that ATG5 associates with retromer's core components VPS26, VPS29 and VPS35 and modulates retromer function. Knockout of ATG5 blocked trafficking of a key glucose transporter sorted by the retromer, GLUT1, to the plasma membrane. Knockouts of other genes essential for membrane atg8ylation, of which ATG5 is a component, affected GLUT1 sorting, indicating that membrane atg8ylation as a process affects retromer function and endosomal sorting. The contribution of membrane atg8ylation to retromer function in GLUT1 sorting was independent of canonical autophagy. These findings expand the scope of membrane atg8ylation to specific sorting processes in the cell dependent on the retromer and its known interactors.

A Bacteriophage Cocktail Targeting Yersinia pestis Provides Strong Post-Exposure Protection in a Rat Pneumonic Plague Model.

Yersinia pestis , one of the deadliest bacterial pathogens ever known, is responsible for three plague pandemics and several epidemics, with over 200 million deaths during recorded history. Due to high genomic plasticity, Y. pestis is amenable to genetic mutations as well as genetic engineering that can lead to the emergence or intentional development of pan-drug resistant strains. The dissemination of such Y. pestis strains could be catastrophic, with public health consequences far more daunting than those caused by the recent COVID-19 pandemic. Thus, there is an urgent need to develop novel, safe, and effective treatment approaches for managing Y. pestis infections. This includes infections by antigenically distinct strains for which vaccines, none FDA approved yet, may not be effective, and those that cannot be controlled by approved antibiotics. Lytic bacteriophages provide one such alternative approach. In this study, we examined post-exposure efficacy of a bacteriophage cocktail, YPP-401, to combat pneumonic plague caused by Y. pestis CO92. YPP-401 is a four-phage preparation with a 100% lytic activity against a panel of 68 genetically diverse Y. pestis strains. Using a pneumonic plague aerosol challenge model in gender-balanced Brown Norway rats, YPP-401 demonstrated ∼88% protection when delivered 18 hours post-exposure for each of two administration routes (i.e., intraperitoneal and intranasal) in a dose-dependent manner. Our studies suggest that YPP-401 could provide an innovative, safe, and effective approach for managing Y. pestis infections, including those caused by naturally occurring or intentionally developed strains that cannot be managed by vaccines in development and antibiotics.

Simple and Effective Conformational Sampling Strategy for Intrinsically Disordered Proteins Using the UNRES Web Server.

Intrinsically disordered proteins (IDPs) contain more charged amino acids than folded proteins, resulting in a lack of hydrophobic core(s) and a tendency to adopt rapidly interconverting structures rather than well-defined structures. The structural heterogeneity of IDPs, encoded by the amino acid sequence, is closely related to their unique roles in biological pathways, which require them to interact with different binding partners. Recently, Robustelli and co-workers have demonstrated that a balanced all-atom force field can be used to sample heterogeneous structures of disordered proteins ( Proc. Natl. Acad. Sci. U.S.A. 2018, 115, E4758-E4766). However, such a solution requires extensive computational resources, such as Anton supercomputers. Here, we propose a simple and effective solution to sample the conformational space of IDPs using a publicly available web server, namely, the UNited-RESidue (UNRES) web server. Our proposed solution requires no investment in computational resources and no prior knowledge of UNRES. UNRES Replica Exchange Molecular Dynamics (REMD) simulations were carried out on a set of eight disordered proteins at temperatures spanning from 270 to 430 K. Utilizing the latest UNRES force field designed for structured proteins, with proper selections of temperatures, we were able to produce comparable results to all-atom force fields as reported in work done by Robustelli and co-workers. In addition, NMR observables and the radius of gyration calculated from UNRES ensembles were directly compared with the experimental data to further evaluate the accuracy of the UNRES model at all temperatures. Our results suggest that carrying out the UNRES simulations at optimal temperatures using the UNRES web server can be a good alternative to sample heterogeneous structures of IDPs.

AlphaFold Accurately Predicts the Structure of Ribosomally Synthesized and Post-Translationally Modified Peptide Biosynthetic Enzymes.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products biosynthesized from a genetically encoded precursor peptide. The enzymes that install the post-translational modifications on these peptides have the potential to be useful catalysts in the production of natural-product-like compounds and can install non-proteogenic amino acids in peptides and proteins. However, engineering these enzymes has been somewhat limited, due in part to limited structural information on enzymes in the same families that nonetheless exhibit different substrate selectivities. Despite AlphaFold2's superior performance in single-chain protein structure prediction, its multimer version lacks accuracy and requires high-end GPUs, which are not typically available to most research groups. Additionally, the default parameters of AlphaFold2 may not be optimal for predicting complex structures like RiPP biosynthetic enzymes, due to their dynamic binding and substrate-modifying mechanisms. This study assessed the efficacy of the structure prediction program ColabFold (a variant of AlphaFold2) in modeling RiPP biosynthetic enzymes in both monomeric and dimeric forms. After extensive benchmarking, it was found that there were no statistically significant differences in the accuracy of the predicted structures, regardless of the various possible prediction parameters that were examined, and that with the default parameters, ColabFold was able to produce accurate models. We then generated additional structural predictions for select RiPP biosynthetic enzymes from multiple protein families and biosynthetic pathways. Our findings can serve as a reference for future enzyme engineering complemented by AlphaFold-related tools.

Insight into the Initial Stages of the Folding Process in Onconase Revealed by UNRES.

The unfolded state of proteins presents many challenges to elucidate the structural basis for biological function. This state is characterized by a large degree of structural heterogeneity which makes it difficult to generate structural models. However, recent experiments into the initial folding events of the 104-residue ribonuclease homologue onconase (ONC) were able to identify the regions in the protein that participate in the initial folding of this protein. Therefore, to gain additional structural insight into the unfolded state of proteins, this study utilized molecular dynamics simulations using the UNited-RESidue (UNRES) force field to evaluate whether there is a good agreement between the experimentally determined initial structures and the structures identified by computer simulations along a folding pathway. Indeed, these UNRES simulations accurately identified the two regions experimentally observed to form the initial native structure along the folding pathway of ONC. In addition, these regions are determined to be chain folding initiation sites (CFIS) according to methods developed previously. Subsequent self-organization maps (SOM) analysis has revealed key structural states involved in these early folding events.

Correction: Gajurel et al. Production and Secretion of Isowighteone in Hairy Root Cultures of Pigeon Pea (Cajanus cajan) Co-Treated with Multiple Elicitors. Plants 2022, 11, 834.

In the original publication [...].

Production and Secretion of Isowighteone in Hairy Root Cultures of Pigeon Pea (Cajanus cajan) Co-Treated with Multiple Elicitors.

Isowighteone (3'-isoprenyl genistein) is a prenylated stilbenoid derivative that exhibits neuroprotective, antibacterial, and anti-inflammatory properties. To establish a bioproduction system for this bioactive compound, hairy root cultures of pigeon pea (Cajanus cajan (L.) Millsp.) were developed via Agrobacterium rhizogenes-mediated transformation. The cultures were co-treated with methyl jasmonate, cyclodextrin, hydrogen peroxide, and magnesium chloride to enhance the production of isowighteone. The amount of isowighteone that accumulated in the culture medium upon elicitation varied with the period of elicitation. Isowighteone was purified from extracts of the culture medium by semi-preparative HPLC, and its identity was confirmed by tandem mass spectrometry. After 144 h of elicitation in 12-day-old hairy root culture, the total yield of isowighteone was 8058.618 ± 445.78 µg/g DW, of which approximately 96% was found in the culture medium. The yield of isowighteone in the elicited hairy root culture was approximately 277-fold higher than in the non-elicited root culture. The difference between the phenotypes of the elicited and non-elicited pigeon pea hairy roots was studied using scanning electron microscopy. The non-elicited hairy roots had uniform surfaces whereas the elicited roots had non-uniform shapes. Pigeon pea hairy roots provide a sustainable platform for producing and studying the biosynthesis of isowighteone.

Recognizing the Binding Pattern and Dissociation Pathways of the p300 Taz2-p53 TAD2 Complex.

The dynamic association and dissociation between proteins are the basis of cellular signal transduction. This process becomes much more complicated if one or both interaction partners are intrinsically disordered because intrinsically disordered proteins can undergo disorder-to-order transitions upon binding to their partners. p53, a transcription factor with disordered regions, plays significant roles in many cellular signaling pathways. It is critical to understand the binding/unbinding mechanism involving these disordered regions of p53 at the residue level to reveal how p53 performs its biological functions. Here, we studied the dissociation process of the intrinsically disordered N-terminal transactivation domain 2 (TAD2) of p53 and the transcriptional adaptor zinc-binding 2 (Taz2) domain of transcriptional coactivator p300 using a combination of classical molecular dynamics, steered molecular dynamics, self-organizing maps, and time-resolved force distribution analysis (TRFDA). We observed two different dissociation pathways with different probabilities. One dissociation pathway starts from the TAD2 N-terminus and propagates to the α-helix and finally the C-terminus. The other dissociation pathway is in the opposite order. Subsequent TRFDA results reveal that key residues in TAD2 play critical roles. Besides the residues in agreement with previous experimental results, we also highlighted some other residues that play important roles in the disassociation process. In the dissociation process, non-native interactions were formed to partially compensate for the energy loss due to the breaking of surrounding native interactions. Moreover, our statistical analysis results of other experimentally determined complex structures involving either Taz2 or TAD2 suggest that the binding of the Taz2-TAD2 complex is mainly governed by the binding site of Taz2, which includes three main binding regions. Therefore, the complexes involving Taz2 may follow similar binding/unbinding behaviors, which could be studied together to generate common principles.

A Bacteriophage-Based, Highly Efficacious, Needle- and Adjuvant-Free, Mucosal COVID-19 Vaccine.

The U.S. Food and Drug Administration-authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected in two doses and effective in preventing COVID-19, but they do not induce efficient mucosal immunity or prevent viral transmission. Here, we report the first noninfectious, bacteriophage T4-based, multicomponent, needle- and adjuvant-free, mucosal vaccine harboring engineered Spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of two doses of this T4 SARS-CoV-2 vaccine 21 days apart induced robust mucosal immunity, in addition to strong systemic humoral and cellular immune responses. The intranasal vaccine induced broad virus neutralization antibody titers against multiple variants, Th1-biased cytokine responses, strong CD4+ and CD8+ T cell immunity, and high secretory IgA titers in sera and bronchoalveolar lavage specimens from vaccinated mice. All of these responses were much stronger in intranasally vaccinated mice than those induced by the injected vaccine. Furthermore, the nasal vaccine provided complete protection and sterilizing immunity against the mouse-adapted SARS-CoV-2 MA10 strain, the ancestral WA-1/2020 strain, and the most lethal Delta variant in both BALB/c and human angiotensin converting enzyme (hACE2) knock-in transgenic mouse models. In addition, the vaccine elicited virus-neutralizing antibodies against SARS-CoV-2 variants in bronchoalveolar lavage specimens, did not affect the gut microbiota, exhibited minimal lung lesions in vaccinated and challenged mice, and is completely stable at ambient temperature. This modular, needle-free, phage T4 mucosal vaccine delivery platform is therefore an excellent candidate for designing efficacious mucosal vaccines against other respiratory infections and for emergency preparedness against emerging epidemic and pandemic pathogens. IMPORTANCE According to the World Health Organization, COVID-19 may have caused ~15-million deaths across the globe and is still ravaging the world. Another wave of ~100 million infections is predicted in the United States due to the emergence of highly transmissible immune-escaped Omicron variants. The authorized vaccines would not prevent these transmissions since they do not trigger mucosal immunity. We circumvented this limitation by developing a needle-free, bacteriophage T4-based, mucosal vaccine. This intranasally administered vaccine generates superior mucosal immunity in mice, in addition to inducing robust humoral and cell-mediated immune responses, and provides complete protection and sterilizing immunity against SARS-CoV-2 variants. The vaccine is stable, adjuvant-free, and cost-effectively manufactured and distributed, making it a strategically important next-generation COVID vaccine for ending this pandemic.

Combinatorial Viral Vector-Based and Live Attenuated Vaccines without an Adjuvant to Generate Broader Immune Responses to Effectively Combat Pneumonic Plague.

Mice immunized with a combination of an adenovirus vector (Ad5-YFV) and live-attenuated (LMA)-based vaccines were evaluated for protective efficacy against pneumonic plague. While the Ad5-YFV vaccine harbors a fusion cassette of three genes encoding YscF, F1, and LcrV, LMA represents a mutant of parental Yersinia pestis CO92 deleted for genes encoding Lpp, MsbB, and Ail. Ad5-YFV and LMA were either administered simultaneously (1-dose regimen) or 21 days apart in various orders and route of administration combinations (2-dose regimen). The 2-dose regimen induced robust immune responses to provide full protection to animals against parental CO92 and its isogenic F1 deletion mutant (CAF-) challenges during both short- and long-term studies. Mice intranasally (i.n.) immunized with Ad5-YFV first followed by LMA (i.n. or intramuscularly [i.m.]) had higher T- and B-cell proliferative responses and LcrV antibody titers than those in mice vaccinated with LMA (i.n. or i.m.) first ahead of Ad5-YFV (i.n.) during the long-term study. Specifically, the needle- and adjuvant-free vaccine combination (i.n.) is ideal for use in plague regions of endemicity. Conversely, with a 1-dose regimen, mice vaccinated with Ad5-YFV i.n. and LMA by the i.m. route provided complete protection to animals against CO92 and its CAF- mutant challenges and elicited Th1/Th2, as well as Th17 responses, making it suitable for emergency vaccination during a plague outbreak or bioterrorist attack. This is a first study in which a viral vector-based and live-attenuated vaccines were effectively used in combination, representing adjuvant- and/or needle-free immunization, with each vaccine triggering a distinct cellular immune response. IMPORTANCE Yersinia pestis, the causative agent of plague, is a Tier-1 select agent and a reemerging human pathogen. A 2017 outbreak in Madagascar with >75% of cases being pneumonic and 8.6% causalities emphasized the importance of the disease. The World Health Organization has indicated an urgent need to develop new-generation subunit and live-attenuated plague vaccines. We have developed a subunit vaccine, including three components (YscF, F1, and LcrV) using an adenovirus platform (Ad5-YFV). In addition, we have deleted virulence genes of Y. pestis (e.g., lpp, msbB, and ail) to develop a live-attenuated vaccine (LMA). Both of these vaccines generated robust humoral and cellular immunity and were highly efficacious in several animal models. We hypothesized the use of a heterologous prime-boost strategy or administrating both vaccines simultaneously could provide an adjuvant- and/or a needle-free vaccine(s) that has attributes of both vaccines for use in regions of endemicity and during an emergency situation.