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A recent study in Scientific Reports identified glypican-3 (GPC3) as a novel extracellular interacting protein for FAT1 in hepato-cellular carcinoma (HCC) cells. FAT1 is a large transmembrane atypical cadherin with limited knowledge existing about its binding partners. While in Drosophila, dachsous (ds), another transmembrane member of the cadherin superfamily, is known to function as FAT1 ligand, no ligand is known in mammals so far. The revelation of GPC3 as a potential binding partner of FAT1 extracellular domain unfolds an opportunity to study potential triggers of FAT1 signaling in cancers. Available inhibitors of GPC3 in various phases of clinical trials also present an attractive option to curb GPC3-FAT1 signaling in tumors that overexpress these proteins., Graphical abstract Image, graphical abstract

Olson and Machesky discuss work from the Way laboratory that reveals how ARP3B isoform-specific ARP2/3 complexes lead to faster disassembly of branched actin filaments., The ARP2/3 complex promotes branched actin networks, but the importance of specific subunit isoforms is unclear. In this issue, Galloni, Carra, et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202102043) show that MICAL2 mediates methionine oxidation of ARP3B, thus destabilizing ARP2/3 complexes and leading to disassembly of branched actin filaments.

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant, cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increase the positive charge of the surface of this domain, ii) insertions into NTD of heterologous peptides, containing positively charged amino acids, and iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide and makes viruses less capable of syncytia formation. These viral adaptations result in higher affinity of viral particles to heparin sepharose, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers and two orders of magnitude lower GE:PFU ratios. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA+ viruses, evolution to HS binding may result in virus attenuation in vivo.IMPORTANCEThe spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to ACE2 receptor and later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the N-terminal domain (NTD) and in P1‘ position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells and decrease GE:PFU ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high affinity interaction of the spikes with the ACE2 receptor.

During growth, bacteria increase in size and divide. Division is initiated by the formation of the Z-ring, a ring-like cytoskeletal structure formed by treadmilling protofilaments of the tubulin homolog FtsZ. FtsZ localization is thought to be controlled by the Min and Noc systems, and here we explore why cell division fails at high temperature when the Min and Noc systems are simultaneously mutated. Microfluidic analysis of a

Lyme disease is a multistage inflammatory disease caused by the spirochete Borrelia burgdorferi transmitted through the bite of an infected Ixodes scapularis tick. We previously discovered a B. burgdorferi infectivity gene, bbk13, that facilitates mammalian infection by promoting spirochete population expansion in the skin inoculation site. Initial characterization of bbk13 was carried out using an intradermal needle inoculation model of mouse infection, which does not capture the complex interplay of the pathogen-vector-host triad of natural transmission. Here, we aimed to understand the role of bbk13 in the enzootic cycle of B. burgdorferi. B. burgdorferi spirochetes lacking bbk13 were unable to be acquired by naive larvae fed on needle-inoculated mice. Using a capsule feeding approach to restrict tick feeding activity to a defined skin site, we determined that delivery by tick bite alleviated the population expansion defect in the skin observed after needle inoculation of Δbbk13 B. burgdorferi. Despite overcoming the early barrier in the skin, Δbbk13 B. burgdorferi remained attenuated for distal tissue colonization after tick transmission. Disseminated infection by Δbbk13 B. burgdorferi was improved in needle-inoculated immunocompromised mice. Together, we established that bbk13 is crucial to the maintenance of B. burgdorferi in the enzootic cycle and that bbk13 is necessary beyond early infection in the skin, likely contributing to host immune evasion. Moreover, our data highlight the critical interplay between the pathogen, vector, and host as well as the distinct molecular genetic requirements for B. burgdorferi to survive at the pathogen-vector-host interface and achieve productive disseminated infection.

Antimicrobial peptides are evolving as novel therapeutic options against the increasing problem of multidrug-resistant microorganisms, and nisin is one such avenue. However, some bacteria possess a specific nisin resistance system (NSR), which cleaves the peptide reducing its bactericidal efficacy. NSR-based resistance was identified in strains of Streptococcus uberis, a ubiquitous pathogen that causes mastitis in dairy cattle. Previous studies have demonstrated that a nisin A derivative termed nisin PV, featuring S29P and I30V, exhibits enhanced resistance to proteolytic cleavage by NSR. Our objective was to investigate the ability of this nisin derivative to eradicate and inhibit biofilms of S. uberis DPC 5344 and S. uberis ATCC 700407 (nsr+) using crystal violet (biomass), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) (viability) assays, and confocal microscopy (viability and architecture). When preestablished biofilms were assessed, both peptides reduced biofilm biomass by over 60% compared to that of the untreated controls. However, a 42% higher reduction in viability was observed following treatment with nisin PV compared to that of nisin A. Accordingly, confocal microscopy analysis revealed significantly more dead cells on the biofilm upper surface and a reduced thickness following treatment with nisin PV. When biofilm inhibition was assessed, nisin PV inhibited biofilm formation and decreased viability up to 56% and 85% more than nisin A, respectively. Confocal microscopy analysis revealed a lack of biofilm for S. uberis ATCC 700407 and only dead cells for S. uberis DPC 5344. These results suggest that nisin PV is a promising alternative to effectively reduce the biofilm formation of S. uberis strains carrying NSR. IMPORTANCE One of the four most prevalent species of bovine mastitis-causing pathogens is S. uberis. Its ability to form biofilms confers on the bacteria greater resistance to antibiotics, requiring higher doses to be more effective. In a bid to limit antibiotic resistance development, the need for alternative antimicrobials is paramount. Bacteriocins such as nisin represent one such alternative that could alleviate the impact of mastitis caused by S. uberis. However, many strains of S. uberis have been shown to possess nisin resistance determinants, such as the nisin resistance protein (NSR). In this study, we demonstrate the ability of nisin and a nisin derivative termed PV that is insensitive to NSR to prevent and remove biofilms of NSR-producing S. uberis strains. These findings will add new information to the antimicrobial bacteriocins and control of S. uberis research fields specifically in relation to biofilms and nsr+ mastitis-associated strains.

Gas fermentation is a promising way to convert CO-rich gases to chemicals. We studied the use of synthetic cocultures composed of carboxydotrophic and propionigenic bacteria to convert CO to propionate. So far, isolated carboxydotrophs cannot directly ferment CO to propionate, and therefore, this cocultivation approach was investigated. Four distinct synthetic cocultures were constructed, consisting of Acetobacterium wieringae (DSM 1911T) and Pelobacter propionicus (DSM 2379T), Ac. wieringae (DSM 1911T) and Anaerotignum neopropionicum (DSM 3847T), Ac. wieringae strain JM and P. propionicus (DSM 2379T), and Ac. wieringae strain JM and An. neopropionicum (DSM 3847T). Propionate was produced by all the cocultures, with the highest titer (;24mM) being measured in the coculture composed of Ac. wieringae strain JM and An. neopropionicum, which also produced isovalerate (;4mM), butyrate (;1mM), and isobutyrate (0.3mM). This coculture was further studied using proteogenomics. As expected, enzymes involved in the Wood-Ljungdahl pathway in Ac. wieringae strain JM, which are responsible for the conversion of CO to ethanol and acetate, were detected; the proteome of An. neopropionicum confirmed the conversion of ethanol to propionate via the acrylate pathway. In addition, proteins related to amino acid metabolism and stress response were highly abundant during cocultivation, which raises the hypothesis that amino acids are exchanged by the two microorganisms, accompanied by isovalerate and isobutyrate production. This highlights the importance of explicitly looking at fortuitous microbial interactions during cocultivation to fully understand coculture behavior., This research was financially supported by Project NWO-GK-07 from the Netherlands Science Foundation (NWO), the Perspectief Programma P16-10 from NWO Applied and Engineering Sciences (AGS), by a Gravitation Grant (Project 024.002.002) of the Netherlands Ministry of Education, Culture and Science, and by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020— Programa Operacional Regional do Norte. The financial support from FCT and European Social Fund (POPH-QREN) through the grant PD/BD/128030/2016 (given to Ana Luísa Arantes) and PD/BD/150583/2020 (given to João P. C. Moreira) and through the project INNOVsyn—Innovative strategies for syngas fermentation (POCI-01-0145-FEDER-031377) are gratefully acknowledged., info:eu-repo/semantics/publishedVersion

RNA helicase A/DHX9 is required for diverse RNA-related essential cellular functions and antiviral responses and is hijacked by RNA viruses to support their replication. Here, we show that during the late replication stage in human cancer cells of myxoma virus (MYXV), a member of the double-stranded DNA (dsDNA) poxvirus family that is being developed as an oncolytic virus, DHX9, forms unique granular cytoplasmic structures, which we named “DHX9 antiviral granules.” These DHX9 antiviral granules are not formed if MYXV DNA replication and/or late protein synthesis is blocked. When formed, DHX9 antiviral granules significantly reduced nascent protein synthesis in the MYXV-infected cancer cells. MYXV late gene transcription and translation were also significantly compromised, particularly in nonpermissive or semipermissive human cancer cells where MYXV replication is partly or completely restricted. Directed knockdown of DHX9 significantly enhanced viral late protein synthesis and progeny virus formation in normally restrictive cancer cells. We further demonstrate that DHX9 is not a component of the canonical cellular stress granules. DHX9 antiviral granules are induced by MYXV, and other poxviruses, in human cells and are associated with other known cellular components of stress granules, dsRNA and virus encoded dsRNA-binding protein M029, a known interactor with DHX9. Thus, DHX9 antiviral granules function by hijacking poxviral elements needed for the cytoplasmic viral replication factories. These results demonstrate a novel antiviral function for DHX9 that is recruited from the nucleus into the cytoplasm, and this step can be exploited to enhance oncolytic virotherapy against the subset of human cancer cells that normally restrict MYXV. IMPORTANCE The cellular DHX9 has both proviral and antiviral roles against diverse RNA and DNA viruses. In this article, we demonstrate that DHX9 can form unique antiviral granules in the cytoplasm during myxoma virus (MYXV) replication in human cancer cells. These antiviral granules sequester viral proteins and reduce viral late protein synthesis and thus regulate MYXV, and other poxviruses, that replicate in the cytoplasm. In addition, we show that in the absence of DHX9, the formation of DHX9 antiviral granules can be inhibited, which significantly enhanced oncolytic MYXV replication in human cancer cell lines where the virus is normally restricted. Our results also show that DHX9 antiviral granules are formed after viral infection but not by common nonviral cellular stress inducers. Thus, our study suggests that DHX9 has antiviral activity in human cancer cells, and this pathway can be targeted for enhanced activity of oncolytic poxviruses against even restrictive cancer cells.

The transmission of SARS-CoV-2 is likely to occur through a number of routes, including contact with contaminated surfaces. Many studies have used reverse transcription-PCR (RT-PCR) analysis to detect SARS-CoV-2 RNA on surfaces, but seldom has viable virus been detected. This paper investigates the viability over time of SARS-CoV-2 dried onto a range of materials and compares viability of the virus to RNA copies recovered and whether virus viability is concentration dependent. Viable virus persisted for the longest time on surgical mask material and stainless steel, with a 99.9% reduction in viability by 122 and 114 h, respectively. Viability of SARS-CoV-2 reduced the fastest on a polyester shirt, with a 99.9% reduction within 2.5 h. Viability on the bank note was reduced second fastest, with 99.9% reduction in 75 h. RNA on all surfaces exhibited a 1-log reduction in genome copy number recovery over 21 days. The findings show that SARS-CoV-2 is most stable on nonporous hydrophobic surfaces. RNA is highly stable when dried on surfaces, with only 1-log reduction in recovery over 3 weeks. In comparison, SARS-CoV-2 viability reduced more rapidly, but this loss in viability was found to be independent of starting concentration. Expected levels of SARS-CoV-2 viable environmental surface contamination would lead to undetectable levels within 2 days. Therefore, when RNA is detected on surfaces, it does not directly indicate the presence of viable virus, even at low cycle threshold values. IMPORTANCE This study shows the impact of material type on the viability of SARS-CoV-2 on surfaces. It demonstrates that the decay rate of viable SARS-CoV-2 is independent of starting concentration. However, RNA shows high stability on surfaces over extended periods. This has implications for interpretation of surface sampling results using RT-PCR to determine the possibility of viable virus from a surface, where RT-PCR is not an appropriate technique to determine viable virus. Unless sampled immediately after contamination, it is difficult to align RNA copy numbers to quantity of viable virus on a surface.

Fungal dissemination into the bloodstream is a critical step leading to invasive fungal infections. Here, using intravital imaging, we show that Kupffer cells (KCs) in the liver have a prominent function in the capture of circulating Cryptococcus neoformans and Candida albicans, thereby reducing fungal dissemination to target organs. Complement C3 but not C5, and complement receptor CRIg but not CR3, are involved in capture of C. neoformans. Internalization of C. neoformans by KCs is subsequently mediated by multiple receptors, including CR3, CRIg, and scavenger receptors, which work synergistically along with C5aR signaling. Following phagocytosis, the growth of C. neoformans is inhibited by KCs in an IFN-γ independent manner. Thus, the liver filters disseminating fungi from circulation via KCs, providing a mechanistic explanation for the enhanced risk of cryptococcosis among individuals with liver diseases, and suggesting a therapeutic strategy to prevent fungal dissemination through enhancing KC functions., Patients with liver diseases are at increased risk of fungal infections. Here the authors show that Kupffer cells are critical for the filtration of fungi out of the blood and thereby for liver-mediated protection against disseminating fungal infection.

Linda Wordeman discusses work from Strothman et al. that sheds new light on the differences between plus- and minus-end microtubule dynamics., Microtubule minus ends are inherently more stable than plus ends despite the fact that free tubulin associates more avidly to the plus end. In this issue, Strothman et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201905019) measure, for the first time, the off-rate for GTP-tubulin and find that it is different for the two ends, suggesting that this parameter may control the transition to disassembly at microtubule ends.

Chen et al. show that XLF functions to limit fork reversal during DNA replication. H2AX prevents MRE11-dependent replication stress in XLF-deficient cells, suggesting that H2AX prevents the resection of regressed arms at reversed forks., XRCC4-like factor (XLF) is a non-homologous end joining (NHEJ) DNA double strand break repair protein. However, XLF deficiency leads to phenotypes in mice and humans that are not necessarily consistent with an isolated defect in NHEJ. Here we show that XLF functions during DNA replication. XLF undergoes cell division cycle 7–dependent phosphorylation; associates with the replication factor C complex, a critical component of the replisome; and is found at replication forks. XLF deficiency leads to defects in replication fork progression and an increase in fork reversal. The additional loss of H2AX, which protects DNA ends from resection, leads to a requirement for ATR to prevent an MRE11-dependent loss of newly synthesized DNA and activation of DNA damage response. Moreover, H2ax−/−:Xlf−/− cells exhibit a marked dependence on the ATR kinase for survival. We propose that XLF and H2AX function in series to prevent replication stress induced by the MRE11-dependent resection of regressed arms at reversed replication forks.

The RIPK3-MLKL pathway protects epithelial cells from Listeria monocytogenes invasion. Sai et al. find that Listeria infection activates MLKL but does not induce its oligomerization or necroptotic cell death. Instead, Listeria-activated MLKL directly targets intracellular bacteria and suppresses their replication., RIPK3, a key mediator of necroptosis, has been implicated in the host defense against viral infection primary in immune cells. However, gene expression analysis revealed that RIPK3 is abundantly expressed not only in immune organs but also in the gastrointestinal tract, particularly in the small intestine. We found that orally inoculated Listeria monocytogenes, a bacterial foodborne pathogen, efficiently spread and caused systemic infection in Ripk3-deficient mice while almost no dissemination was observed in wild-type mice. Listeria infection activated the RIPK3-MLKL pathway in cultured cells, which resulted in suppression of intracellular replication of Listeria. Surprisingly, Listeria infection–induced phosphorylation of MLKL did not result in host cell killing. We found that MLKL directly binds to Listeria and inhibits their replication in the cytosol. Our findings have revealed a novel functional role of the RIPK3-MLKL pathway in nonimmune cell-derived host defense against Listeria invasion, which is mediated through cell death–independent mechanisms.

Transport of intracellular cargo generally requires coiled-coil adaptor proteins that connect cargo-bound receptors, usually GTPases, to dynein motor complexes. Wang et al. report that two Rab GTPases, CRACR2a and Rab45, contain coiled-coil domains and can directly act as dynein adaptors with CRACR2a–dynein participating in calcium-regulated endocytic trafficking., Cytoplasmic dynein is a minus end–directed microtubule motor that transports intracellular cargoes. Transport is initiated by coiled-coil adaptors that (a) join dynein and its cofactor dynactin into a motile complex and (b) interact with a cargo-bound receptor, which is frequently a Rab GTPase on an organelle. Here, we report two novel dynein adaptors, CRACR2a and Rab45, that have a coiled-coil adaptor domain, a pair of EF-hands, and a Rab GTPase fused into a single polypeptide. CRACR2a-mediated, but not Rab45-mediated, dynein motility is activated by calcium in vitro. In Jurkat T cells, elevation of intracellular calcium activates CRACR2a-mediated dynein transport. We further found that T cell receptor activation induces the formation of CRACR2a puncta at the plasma membrane, which initially associate with the actin cortex and subsequently detach and travel along microtubules, suggestive of an endocytic process. These results provide the first examples of Rab GTPases that directly act as dynein adaptors and implicate CRACR2a–dynein in calcium-regulated endocytic trafficking.

Although centromeres usually associate with heterochromatic repetitive sequences, such repetitive sequences are not detected around neocentromeres. Nishimura et al. performed systematic 4C analysis of cells containing differently positioned neocentromeres and demonstrate that these neocentromeres commonly associate with distant heterochromatin-rich regions at the 3D level., The centromere is an important genomic locus for chromosomal segregation. Although the centromere is specified by sequence-independent epigenetic mechanisms in most organisms, it is usually composed of highly repetitive sequences, which associate with heterochromatin. We have previously generated various chicken DT40 cell lines containing differently positioned neocentromeres, which do not contain repetitive sequences and do not associate with heterochromatin. In this study, we performed systematic 4C analysis using three cell lines containing differently positioned neocentromeres to identify neocentromere-associated regions at the 3D level. This analysis reveals that these neocentromeres commonly associate with specific heterochromatin-rich regions, which were distantly located from neocentromeres. In addition, we demonstrate that centromeric chromatin adopts a compact structure, and centromere clustering also occurs in vertebrate interphase nuclei. Interestingly, the occurrence of centromere–heterochromatin associations depend on CENP-H, but not CENP-C. Our analyses provide an insight into understanding the 3D architecture of the genome, including the centromeres., Graphical Abstract

Toyama et al. monitor the replacement of long-lived components of nuclear pore complexes (NPCs) and nucleosomes in postmitotic cells. They describe age mosaicism at the level of chromatin organization and find that NPCs are maintained by piecemeal replacement in postmitotic nondividing cells but by entire complex replacement in an ESCRT-dependent manner in nondividing, starved quiescent cells., Many adult tissues contain postmitotic cells as old as the host organism. The only organelle that does not turn over in these cells is the nucleus, and its maintenance represents a formidable challenge, as it harbors regulatory proteins that persist throughout adulthood. Here we developed strategies to visualize two classes of such long-lived proteins, histones and nucleoporins, to understand the function of protein longevity in nuclear maintenance. Genome-wide mapping of histones revealed specific enrichment of long-lived variants at silent gene loci. Interestingly, nuclear pores are maintained by piecemeal replacement of subunits, resulting in mosaic complexes composed of polypeptides with vastly different ages. In contrast, nondividing quiescent cells remove old nuclear pores in an ESCRT-dependent manner. Our findings reveal distinct molecular strategies of nuclear maintenance, linking lifelong protein persistence to gene regulation and nuclear integrity.

Florence Niedergang introduces work by Turan et al. demonstrating that herpes virus egress from dendritic cells is differentially regulated by kinesin-dependent positioning of lysosomes and autophagic degradation of nuclear lamins., Herpes simplex viruses bud into the nuclear membrane of infected cells. Turan et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201801151) demonstrate that mature dendritic cells control the peripheral location of lysosomes, reducing autophagic degradation of lamins and inhibiting viral release.

Defective lysine catabolism leads to two types of hyperlysinemia, but the mechanisms are unclear. Zhou et al. reveal that accumulation of saccharopine, an intermediate of lysine catabolism, leads to defective development of Caenorhbditis elegans and mice and that this correlates with disrupted mitochondrial dynamics, damage, and functional loss., Amino acid catabolism is frequently executed in mitochondria; however, it is largely unknown how aberrant amino acid metabolism affects mitochondria. Here we report the requirement for mitochondrial saccharopine degradation in mitochondrial homeostasis and animal development. In Caenorhbditis elegans, mutations in the saccharopine dehydrogenase (SDH) domain of the bi-functional enzyme α-aminoadipic semialdehyde synthase AASS-1 greatly elevate the lysine catabolic intermediate saccharopine, which causes mitochondrial damage by disrupting mitochondrial dynamics, leading to reduced adult animal growth. In mice, failure of mitochondrial saccharopine oxidation causes lethal mitochondrial damage in the liver, leading to postnatal developmental retardation and death. Importantly, genetic inactivation of genes that raise the mitochondrial saccharopine precursors lysine and α-ketoglutarate strongly suppresses SDH mutation-induced saccharopine accumulation and mitochondrial abnormalities in C. elegans. Thus, adequate saccharopine catabolism is essential for mitochondrial homeostasis. Our study provides mechanistic and therapeutic insights for understanding and treating hyperlysinemia II (saccharopinuria), an aminoacidopathy with severe developmental defects.

Recombinant protein production is a known source of oxidative stress. However, knowledge of which reactive oxygen species are involved or the specific growth phase in which stress occurs remains lacking. Using modern, hypersensitive genetic H2O2-specific probes, microcultivation, and continuous measurements in batch culture, we observed H2O2 accumulation during and following the diauxic shift in engineered Saccharomyces cerevisiae, correlating with peak α-amylase production. In agreement with previous studies supporting a role of the translation initiation factor kinase Gcn2 in the response to H2O2, we find that Gcn2-dependent phosphorylation of eIF2α increases alongside translational attenuation in strains engineered to produce large amounts of α-amylase. Gcn2 removal significantly improved α-amylase production in two previously optimized high-producing strains but not in the wild type. Gcn2 deficiency furthermore reduced intracellular H2O2 levels and the Hac1 splicing ratio, while expression of antioxidants and the endoplasmic reticulum (ER) disulfide isomerase PDI1 increased. These results suggest protein synthesis and ER oxidative folding are coupled and subject to feedback inhibition by H2O2. IMPORTANCE Recombinant protein production is a multibillion dollar industry. Optimizing the productivity of host cells is, therefore, of great interest. In several hosts, oxidants are produced as an unwanted side product of recombinant protein production. The buildup of oxidants can result in intracellular stress responses that could compromise the productivity of the host cell. Here, we document a novel protein synthesis inhibitory mechanism that is activated by the buildup of a specific oxidant (H2O2) in the cytosol of yeast cells upon the production of recombinant proteins. At the center of this inhibitory mechanism lies the protein kinase Gcn2. By removing Gcn2, we observed a doubling of recombinant protein productivity in addition to reduced H2O2 levels in the cytosol. In this study, we want to raise awareness of this inhibitory mechanism in eukaryotic cells to further improve protein production and contribute to the development of novel protein-based therapeutic strategies.

In this JCB issue, work from Yang et al. highlights how the ESCRT pathway localizes to the vacuole surface to execute protein sorting of its resident proteins., Lysosomes (vacuoles in yeast) are master regulators of metabolism and protein turnover, but how they degrade their own resident proteins is unclear. Recently, multiple models have been proposed explaining yeast vacuole protein sorting, but the role of the ESCRT pathway was unclear. In this JCB issue, work from Yang et al. (https://doi.org/10.1083/jcb.202012104) highlights how the ESCRT pathway localizes to the vacuole surface to execute protein sorting of its resident proteins.

Homocitrate synthase (HCS) catalyzes the aldol condensation of 2-oxoglutarate (2-OG) and acetyl coenzyme A (AcCoA) to form homocitrate, which is the first enzyme of the lysine biosynthetic pathway in the yeast Saccharomyces cerevisiae. The HCS activity is tightly regulated via feedback inhibition by the end product lysine. Here, we designed a feedback inhibition-insensitive HCS of S. cerevisiae (ScLys20) for high-level production of lysine in yeast cells. In silico docking of the substrate 2-OG and the inhibitor lysine to ScLys20 predicted that the substitution of serine with glutamate at position 385 would be more suitable for desensitization of the lysine feedback inhibition than the substitution from serine to phenylalanine in the already known Ser385Phe variant. Enzymatic analysis revealed that the Ser385Glu variant is far more insensitive to feedback inhibition than the Ser385Phe variant. We also found that the lysine contents in yeast cells expressing the Ser385Glu variant were 4.62- and 1.47-fold higher than those of cells expressing the wild-type HCS and Ser385Phe variant, respectively, due to the extreme desensitization to feedback inhibition. In this study, we obtained highly feedback inhibition-insensitive HCS using in silico docking and enzymatic analysis. Our results indicate that the rational engineering of HCS for feedback inhibition desensitization by lysine could be useful for constructing new yeast strains with higher lysine productivity. IMPORTANCE A traditional method for screening toxic analogue-resistant mutants has been established for the breeding of microbes that produce high levels of amino acids, including lysine. However, another efficient strategy is required to further improve their productivity. Homocitrate synthase (HCS) catalyzes the first step of lysine biosynthesis in the yeast Saccharomyces cerevisiae, and its activity is subject to feedback inhibition by lysine. Here, in silico design of a key enzyme that regulates the biosynthesis of lysine was utilized to increase the productivity of lysine. We designed HCS for the high-level production of lysine in yeast cells by in silico docking simulation. The engineered HCS exhibited much less sensitivity to lysine and conferred higher production of lysine than the already known variant obtained by traditional breeding. The combination of in silico design and experimental analysis of a key enzyme will contribute to advances in metabolic engineering for the construction of industrial microorganisms.

Treponema pallidum, an obligate human pathogen, has an outer membrane (OM) whose physical properties, ultrastructure, and composition differ markedly from those of phylogenetically distant Gram-negative bacteria. We developed structural models for the outer membrane protein (OMP) repertoire (OMPeome) of T. pallidum Nichols using solved Gram-negative structures, computational tools, and small-angle X-ray scattering (SAXS) of selected recombinant periplasmic domains. The T. pallidum "OMPeome" harbors two "stand-alone" proteins (BamA and LptD) involved in OM biogenesis and four paralogous families involved in the influx/efflux of small molecules: 8-stranded β-barrels, long-chain-fatty-acid transporters (FadLs), OM factors (OMFs) for efflux pumps, and T. pallidum repeat proteins (Tprs). BamA (TP0326), the central component of a β-barrel assembly machine (BAM)/translocation and assembly module (TAM) hybrid, possesses a highly flexible polypeptide-transport-associated (POTRA) 1-5 arm predicted to interact with TamB (TP0325). TP0515, an LptD ortholog, contains a novel, unstructured C-terminal domain that models inside the β-barrel. T. pallidum has four 8-stranded β-barrels, each containing positively charged extracellular loops that could contribute to pathogenesis. Three of five FadL-like orthologs have a novel α-helical, presumptively periplasmic C-terminal extension. SAXS and structural modeling further supported the bipartite membrane topology and tridomain architecture of full-length members of the Tpr family. T. pallidum's two efflux pumps presumably extrude noxious small molecules via four coexpressed OMFs with variably charged tunnels. For BamA, LptD, and OMFs, we modeled the molecular machines that deliver their substrates into the OM or external milieu. The spirochete's extended families of OM transporters collectively confer a broad capacity for nutrient uptake. The models also furnish a structural road map for vaccine development. IMPORTANCE The unusual outer membrane (OM) of T. pallidum, the syphilis spirochete, is the ultrastructural basis for its well-recognized capacity for invasiveness, immune evasion, and persistence. In recent years, we have made considerable progress in identifying T. pallidum's repertoire of OMPs. Here, we developed three-dimensional (3D) models for the T. pallidum Nichols OMPeome using structural modeling, bioinformatics, and solution scattering. The OM contains three families of OMP transporters, an OMP family involved in the extrusion of noxious molecules, and two "stand-alone" proteins involved in OM biogenesis. This work represents a major advance toward elucidating host-pathogen interactions during syphilis; understanding how T. pallidum, an extreme auxotroph, obtains a wide array of biomolecules from its obligate human host; and developing a vaccine with global efficacy.

Highlights • Human lung cancer cells injected through mouse-tail vein colonize mouse lungs within 24 h and form lung tumors in about 30 days. • Pretreatment of mice with weekly or daily dosing of erlotinib reduces but does not abolish tail vein injected lung cancer cells to form lung tumors. • Pretreatment of mice with weekly or daily dosing of osimertinib abolish lung cancer cells injected through a tail vein to form lung tumors. • The low-dose once-a-week adjuvant osimertinib could have lower toxicity, higher affordability, and potentially affect acquired resistance., The recently conducted ADAURA trial concludes daily dosing of adjuvant osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), improves disease-free survival with stage IB/II/IIIA EGFR -mutated non-small cell lung cancer patients in comparison to placebo. We have developed a preclinical orthotopic mouse model, using luciferase tagged lung adenocarcinoma cells harboring EGFR TKI sensitive exon 19 deletion to model and extend trial implications comparing a weekly vs daily dosing outcome of osimertinib to a first-generation TKI- erlotinib. We find that 100% of mice in both the groups receiving osimertinib daily or weekly before injection of cells show a complete absence of homing of cells in mice's lungs from day three until day 18 post-injection of cells. On the other hand, 25% and 75% of mice receiving erlotinib daily and weekly before injecting cells show homing of cells to the lungs. The tumors observed in the lungs, when dissected at day 30, confirmed the colonization of the injected cells homing to the organ. Thus, our study establishes the efficacy of pretreatment with osimertinib in reducing tumor cells' homing to mouse lungs in an in vivo mouse model., Graphical abstract Image, graphical abstract

Yamamoto highlights work from Nthiga et al. that shows that Calcoco1 binds Golgi-resident ZDHHC17 to facilitate autophagic degradation of the Golgi., Degradation by macroautophagy can be highly selective, but given the promiscuity of cargo receptors, questions remain surrounding how this selectivity is achieved. In this issue, Nthiga et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202006128) show how the adaptor Calcoco1 distinguishes cargo by how it binds.

Trichinellosis is one of most neglected foodborne zoonoses worldwide. During Trichinella spiralis infection, the intestinal immune response is the first line of defense and plays a vital role in the host’s resistance., Trichinellosis is one of most neglected foodborne zoonoses worldwide. During Trichinella spiralis infection, the intestinal immune response is the first line of defense and plays a vital role in the host’s resistance. Previous studies indicate that purinergic P2X7 receptor (P2X7R) and pyrin domain-containing protein 3 (NLRP3) inflammasome are involved in the intestinal immune response in T. spiralis infection. However, the precise role of P2X7R and its effect on NLRP3 remains largely underdetermined. In this study, we aimed to investigate the role of P2X7R in the activation of NLRP3 in macrophages during the intestinal immune response against T. spiralis. We found that T. spiralis infection upregulated expression of P2X7R and activation of NLRP3 in macrophages in mice. In vivo, P2X7R deficiency resulted in increased intestinal adult and muscle larval burdens, along with decreased expression of NLRP3/interleukin-1β (IL-1β) in macrophages from the infected mice with T. spiralis. In In vitro experiments, P2X7R blockade inhibited activation of NLRP3/IL-1β via NF-κB and thus reduced the capacity of macrophages to kill newborn larvae of T. spiralis. These results indicate that P2X7R mediates the elimination of T. spiralis by activating the NF-κB/NLRP3/IL-1β pathway in macrophages. Our findings contribute to the understanding of the intestinal immune mechanism of T. spiralis infection.

Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield., Pseudomonas putida S12 is inherently solvent tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of P. putida S12 carries several gene clusters involved in solvent tolerance, and the removal of this megaplasmid caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in plasmid-cured P. putida S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent tolerance of this strain. Whole-genome sequencing identified several single nucleotide polymorphisms (SNPs) and a mobile element insertion enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% [vol/vol]). We identified mutations in an RND efflux pump regulator, arpR, that resulted in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit β′, a global two-component regulatory system (GacA/GacS), and a putative AraC family transcriptional regulator, Afr. Transcriptomic analysis further revealed a constitutive downregulation of energy-consuming activities in ALE-derived strains, such as flagellar assembly, FoF1 ATP synthase, and membrane transport proteins. In summary, constitutive expression of a solvent extrusion pump in combination with high metabolic flexibility enabled the restoration of the solvent tolerance trait in P. putida S12 lacking its megaplasmid. IMPORTANCE Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield. Having a solvent tolerance trait is highly advantageous for microbial hosts used in the biobased production of aromatics. The presence of a megaplasmid has been linked to the solvent tolerance trait of Pseudomonas putida; however, the extent of innate, intrinsic solvent tolerance in this bacterium remained unclear. Using adaptive laboratory evolution, we successfully adapted the plasmid-cured P. putida S12 strain to regain its solvent tolerance. Through these adapted strains, we began to clarify the causes, origins, limitations, and trade-offs of the intrinsic solvent tolerance in P. putida. This work sheds light on the possible genetic engineering targets to enhance solvent tolerance in Pseudomonas putida as well as other bacteria.