The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response.

Organisms use several strategies to mitigate mitochondrial stress, including the activation of the mitochondrial unfolded protein response (UPRmt). The UPRmt in Caenorhabditis elegans, regulated by the transcription factor ATFS-1, expands on this recovery program by inducing an antimicrobial response against pathogens that target mitochondrial function. Here, we show that the mammalian ortholog of ATFS-1, ATF5, protects the host during infection with enteric pathogens but, unexpectedly, by maintaining the integrity of the intestinal barrier. Intriguingly, ATF5 supports intestinal barrier function by promoting a satiety response that prevents obesity and associated hyperglycemia. This consequently averts dysregulated glucose metabolism that is detrimental to barrier function. Mechanistically, we show that intestinal ATF5 stimulates the satiety response by transcriptionally regulating the gastrointestinal peptide hormone cholecystokinin, which promotes the secretion of the hormone leptin. We propose that ATF5 protects the host from enteric pathogens by promoting intestinal barrier function through a satiety-response-mediated metabolic control mechanism.

Snake venom gene expression is coordinated by novel regulatory architecture and the integration of multiple co-opted vertebrate pathways.

Understanding how regulatory mechanisms evolve is critical for understanding the processes that give rise to novel phenotypes. Snake venom systems represent a valuable and tractable model for testing hypotheses related to the evolution of novel regulatory networks, yet the regulatory mechanisms underlying venom production remain poorly understood. Here, we use functional genomics approaches to investigate venom regulatory architecture in the prairie rattlesnake and identify cis-regulatory sequences (enhancers and promoters), trans-regulatory transcription factors, and integrated signaling cascades involved in the regulation of snake venom genes. We find evidence that two conserved vertebrate pathways, the extracellular signal-regulated kinase and unfolded protein response pathways, were co-opted to regulate snake venom. In one large venom gene family (snake venom serine proteases), this co-option was likely facilitated by the activity of transposable elements. Patterns of snake venom gene enhancer conservation, in some cases spanning 50 million yr of lineage divergence, highlight early origins and subsequent lineage-specific adaptations that have accompanied the evolution of venom regulatory architecture. We also identify features of chromatin structure involved in venom regulation, including topologically associated domains and CTCF loops that underscore the potential importance of novel chromatin structure to coevolve when duplicated genes evolve new regulatory control. Our findings provide a model for understanding how novel regulatory systems may evolve through a combination of genomic processes, including tandem duplication of genes and regulatory sequences, cis-regulatory sequence seeding by transposable elements, and diverse transcriptional regulatory proteins controlled by a co-opted regulatory cascade.

Identification of an integrated stress and growth response signaling switch that directs vertebrate intestinal regeneration.

BACKGROUND: Snakes exhibit extreme intestinal regeneration following months-long fasts that involves unparalleled increases in metabolism, function, and tissue growth, but the specific molecular control of this process is unknown. Understanding the mechanisms that coordinate these regenerative phenotypes provides valuable opportunities to understand critical pathways that may control vertebrate regeneration and novel perspectives on vertebrate regenerative capacities. RESULTS: Here, we integrate a comprehensive set of phenotypic, transcriptomic, proteomic, and phosphoproteomic data from boa constrictors to identify the mechanisms that orchestrate shifts in metabolism, nutrient uptake, and cellular stress to direct phases of the regenerative response. We identify specific temporal patterns of metabolic, stress response, and growth pathway activation that direct regeneration and provide evidence for multiple key central regulatory molecules kinases that integrate these signals, including major conserved pathways like mTOR signaling and the unfolded protein response. CONCLUSION: Collectively, our results identify a novel switch-like role of stress responses in intestinal regeneration that forms a primary regulatory hub facilitating organ regeneration and could point to potential pathways to understand regenerative capacity in vertebrates.

On the offense and defense: mitochondrial recovery programs amidst targeted pathogenic assault.

Bacterial pathogens employ a variety of tactics to persist in their host and promote infection. Pathogens often target host organelles in order to benefit their survival, either through manipulation or subversion of their function. Mitochondria are regularly targeted by bacterial pathogens owing to their diverse cellular roles, including energy production and regulation of programmed cell death. However, disruption of normal mitochondrial function during infection can be detrimental to cell viability because of their essential nature. In response, cells use multiple quality control programs to mitigate mitochondrial dysfunction and promote recovery. In this review, we will provide an overview of mitochondrial recovery programs including mitochondrial dynamics, the mitochondrial unfolded protein response (UPRmt ), and mitophagy. We will then discuss the various approaches used by bacterial pathogens to target mitochondria, which result in mitochondrial dysfunction. Lastly, we will discuss how cells leverage mitochondrial recovery programs beyond their role in organelle repair, to promote host defense against pathogen infection.

A nematode-derived, mitochondrial stress signaling-regulated peptide exhibits broad antibacterial activity.

A dramatic rise of infections with antibiotic-resistant bacterial pathogens continues to challenge the healthcare field due to the lack of effective treatment regimes. As such, there is an urgent need to develop new antimicrobial agents that can combat these multidrug-resistant superbugs. Mitochondria are central regulators of metabolism and other cellular functions, including the regulation of innate immunity pathways involved in the defense against infection. The mitochondrial unfolded protein response (UPRmt) is a stress-activated pathway that mitigates mitochondrial dysfunction through the regulation of genes that promote recovery of the organelle. In the model organism Caenorhabditis elegans, the UPRmt also mediates an antibacterial defense program that combats pathogen infection, which promotes host survival. We sought to identify and characterize antimicrobial effectors that are regulated during the UPRmt. From our search, we discovered that the antimicrobial peptide CNC-4 is upregulated during this stress response. CNC-4 belongs to the caenacin family of antimicrobial peptides, which are predominantly found in nematodes and are known to have anti-fungal properties. Here, we find that CNC-4 also possesses potent antimicrobial activity against a spectrum of bacterial species and report on its characterization.

A novel gene-diet interaction promotes organismal lifespan and host protection during infection via the mitochondrial UPR.

Cells use a variety of mechanisms to maintain optimal mitochondrial function including the mitochondrial unfolded protein response (UPRmt). The UPRmt mitigates mitochondrial dysfunction by differentially regulating mitoprotective gene expression through the transcription factor ATFS-1. Since UPRmt activation is commensurate with organismal benefits such as extended lifespan and host protection during infection, we sought to identify pathways that promote its stimulation. Using unbiased forward genetics screening, we isolated novel mutant alleles that could activate the UPRmt. Interestingly, we identified one reduction of function mutant allele (osa3) in the mitochondrial ribosomal gene mrpl-2 that activated the UPRmt in a diet-dependent manner. We find that mrpl-2(osa3) mutants lived longer and survived better during pathogen infection depending on the diet they were fed. A diet containing low levels of vitamin B12 could activate the UPRmt in mrpl-2(osa3) animals. Also, we find that the vitamin B12-dependent enzyme methionine synthase intersects with mrpl-2(osa3) to activate the UPRmt and confer animal lifespan extension at the level of ATFS-1. Thus, we present a novel gene-diet pairing that promotes animal longevity that is mediated by the UPRmt.

A pathogen branched-chain amino acid catabolic pathway subverts host survival by impairing energy metabolism and the mitochondrial UPR.

The mitochondrial unfolded protein response (UPRmt) is a stress-activated pathway promoting mitochondrial recovery and defense against infection. In C. elegans, the UPRmt is activated during infection with the pathogen Pseudomonas aeruginosa-but only transiently. As this may reflect a pathogenic strategy to target a pathway required for host survival, we conducted a P. aeruginosa genetic screen to uncover mechanisms associated with this temporary activation. Here, we find that loss of the P. aeruginosa acyl-CoA dehydrogenase FadE2 prolongs UPRmt activity and extends host survival. FadE2 shows substrate preferences for the coenzyme A intermediates produced during the breakdown of the branched-chain amino acids valine and leucine. Our data suggests that during infection, FadE2 restricts the supply of these catabolites to the host hindering host energy metabolism in addition to the UPRmt. Thus, a metabolic pathway in P. aeruginosa contributes to pathogenesis during infection through manipulation of host energy status and mitochondrial stress signaling potential.

Discovery and characterization of New Delhi metallo-ß-lactamase-1 inhibitor peptides that potentiate meropenem-dependent killing of carbapenemase-producing Enterobacteriaceae.

OBJECTIVES: Metallo-ß-lactamases (MBLs) are an emerging class of antimicrobial resistance enzymes that degrade ß-lactam antibiotics, including last-resort carbapenems. Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) are increasingly prevalent, but treatment options are limited. While several serine-dependent ß-lactamase inhibitors are formulated with commonly prescribed ß-lactams, no MBL inhibitors are currently approved for combinatorial therapies. New compounds that target MBLs to restore carbapenem activity against CPE are therefore urgently needed. Herein we identified and characterized novel synthetic peptide inhibitors that bound to and inhibited NDM-1, which is an emerging ß-lactam resistance mechanism in CPE. METHODS: We leveraged Surface Localized Antimicrobial displaY (SLAY) to identify and characterize peptides that inhibit NDM-1, which is a primary carbapenem resistance mechanism in CPE. Lead inhibitor sequences were chemically synthesized and MBCs and MICs were calculated in the presence/absence of carbapenems. Kinetic analysis with recombinant NDM-1 and select peptides tested direct binding and supported NDM-1 inhibitor mechanisms of action. Inhibitors were also tested for cytotoxicity. RESULTS: We identified approximately 1700 sequences that potentiated carbapenem-dependent killing against NDM-1 Escherichia coli. Several also enhanced meropenem-dependent killing of other CPE. Biochemical characterization of a subset indicated the peptides penetrated the bacterial periplasm and directly bound NDM-1 to inhibit enzymatic activity. Additionally, each demonstrated minimal haemolysis and cytotoxicity against mammalian cell lines. CONCLUSIONS: Our approach advances a molecular platform for antimicrobial discovery, which complements the growing need for alternative antimicrobials. We also discovered lead NDM-1 inhibitors, which serve as a starting point for further chemical optimization.

Multi-species comparisons of snakes identify coordinated signalling networks underlying post-feeding intestinal regeneration.

Several snake species that feed infrequently in nature have evolved the ability to massively upregulate intestinal form and function with each meal. While fasting, these snakes downregulate intestinal form and function, and upon feeding restore intestinal structure and function through major increases in cell growth and proliferation, metabolism and upregulation of digestive function. Previous studies have identified changes in gene expression that underlie this regenerative growth of the python intestine, but the unique features that differentiate this extreme regenerative growth from non-regenerative post-feeding responses exhibited by snakes that feed more frequently remain unclear. Here, we leveraged variation in regenerative capacity across three snake species-two distantly related lineages ( Crotalus and Python) that experience regenerative growth, and one ( Nerodia) that does not-to infer molecular mechanisms underlying intestinal regeneration using transcriptomic and proteomic approaches. Using a comparative approach, we identify a suite of growth, stress response and DNA damage response signalling pathways with inferred activity specifically in regenerating species, and propose a hypothesis model of interactivity between these pathways that may drive regenerative intestinal growth in snakes.

Uncovering a mitochondrial unfolded protein response in corals and its role in adapting to a changing world.

The Anthropocene will be characterized by increased environmental disturbances, leading to the survival of stress-tolerant organisms, particularly in the oceans, where novel marine diseases and elevated temperatures are re-shaping ecosystems. These environmental changes underscore the importance of identifying mechanisms which promote stress tolerance in ecologically important non-model species such as reef-building corals. Mitochondria are central regulators of cellular stress and have dedicated recovery pathways including the mitochondrial unfolded protein response, which increases the transcription of protective genes promoting protein homeostasis, free radical detoxification and innate immunity. In this investigation, we identify a mitochondrial unfolded protein response in the endangered Caribbean coral Orbicella faveolata, by performing in vivo functional replacement using a transcription factor (Of-ATF5) originating from a coral in the model organism Caenorhabditis elegans. In addition, we use RNA-seq network analysis and transcription factor-binding predictions to identify a transcriptional network of genes likely to be regulated by Of-ATF5 which is induced during the immune challenge and temperature stress. Overall, our findings uncover a conserved cellular pathway which may promote the ability of reef-building corals to survive increasing levels of environmental stress.

The mitochondrial unfolded protein response: Signaling from the powerhouse.

Mitochondria are multifaceted and indispensable organelles required for cell performance. Accordingly, dysfunction to mitochondria can result in cellular decline and possibly the onset of disease. Cells use a variety of means to recover mitochondria and restore homeostasis, including the activation of retrograde pathways such as the mitochondrial unfolded protein response (UPRmt). In this Minireview, we will discuss how cells adapt to mitochondrial stress through UPRmt regulation. Furthermore, we will explore the current repertoire of biological functions that are associated with this essential stress-response pathway.

The Transcription Factor ATF5 Mediates a Mammalian Mitochondrial UPR.

Mitochondrial dysfunction is pervasive in human pathologies such as neurodegeneration, diabetes, cancer, and pathogen infections as well as during normal aging. Cells sense and respond to mitochondrial dysfunction by activating a protective transcriptional program known as the mitochondrial unfolded protein response (UPR(mt)), which includes genes that promote mitochondrial protein homeostasis and the recovery of defective organelles [1, 2]. Work in Caenorhabditis elegans has shown that the UPR(mt) is regulated by the transcription factor ATFS-1, which is regulated by organelle partitioning. Normally, ATFS-1 accumulates within mitochondria, but during respiratory chain dysfunction, high levels of reactive oxygen species (ROS), or mitochondrial protein folding stress, a percentage of ATFS-1 accumulates in the cytosol and traffics to the nucleus where it activates the UPR(mt) [2]. While similar transcriptional responses have been described in mammals [3, 4], how the UPR(mt) is regulated remains unclear. Here, we describe a mammalian transcription factor, ATF5, which is regulated similarly to ATFS-1 and induces a similar transcriptional response. ATF5 expression can rescue UPR(mt) signaling in atfs-1-deficient worms requiring the same UPR(mt) promoter element identified in C. elegans. Furthermore, mammalian cells require ATF5 to maintain mitochondrial activity during mitochondrial stress and promote organelle recovery. Combined, these data suggest that regulation of the UPR(mt) is conserved from worms to mammals.

Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response.

Mitochondrial genomes (mitochondrial DNA, mtDNA) encode essential oxidative phosphorylation (OXPHOS) components. Because hundreds of mtDNAs exist per cell, a deletion in a single mtDNA has little impact. However, if the deletion genome is enriched, OXPHOS declines, resulting in cellular dysfunction. For example, Kearns-Sayre syndrome is caused by a single heteroplasmic mtDNA deletion. More broadly, mtDNA deletion accumulation has been observed in individual muscle cells and dopaminergic neurons during ageing. It is unclear how mtDNA deletions are tolerated or how they are propagated in somatic cells. One mechanism by which cells respond to OXPHOS dysfunction is by activating the mitochondrial unfolded protein response (UPR(mt)), a transcriptional response mediated by the transcription factor ATFS-1 that promotes the recovery and regeneration of defective mitochondria. Here we investigate the role of ATFS-1 in the maintenance and propagation of a deleterious mtDNA in a heteroplasmic Caenorhabditis elegans strain that stably expresses wild-type mtDNA and mtDNA with a 3.1-kilobase deletion (∆mtDNA) lacking four essential genes. The heteroplasmic strain, which has 60% ∆mtDNA, displays modest mitochondrial dysfunction and constitutive UPR(mt) activation. ATFS-1 impairment reduced the ∆mtDNA nearly tenfold, decreasing the total percentage to 7%. We propose that in the context of mtDNA heteroplasmy, UPR(mt) activation caused by OXPHOS defects propagates or maintains the deleterious mtDNA in an attempt to recover OXPHOS activity by promoting mitochondrial biogenesis and dynamics.

Mitophagy and the mitochondrial unfolded protein response in neurodegeneration and bacterial infection.

Mitochondria are highly dynamic and structurally complex organelles that provide multiple essential metabolic functions. Mitochondrial dysfunction is associated with neurodegenerative conditions such as Parkinson's disease, as well as bacterial infection. Here, we explore the roles of mitochondrial autophagy (mitophagy) and the mitochondrial unfolded protein response (UPR(mt)) in the response to mitochondrial dysfunction, focusing in particular on recent evidence on the role of mitochondrial import efficiency in the regulation of these stress pathways and how they may interact to protect the mitochondrial pool while initiating an innate immune response to protect against bacterial pathogens.

Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 promotes OXPHOS recovery during the UPR(mt).

Mitochondrial diseases and aging are associated with defects in the oxidative phosphorylation machinery (OXPHOS), which are the only complexes composed of proteins encoded by separate genomes. To better understand genome coordination and OXPHOS recovery during mitochondrial dysfunction, we examined ATFS-1, a transcription factor that regulates mitochondria-to-nuclear communication during the mitochondrial UPR, via ChIP-sequencing. Surprisingly, in addition to regulating mitochondrial chaperone, OXPHOS complex assembly factor, and glycolysis genes, ATFS-1 bound directly to OXPHOS gene promoters in both the nuclear and mitochondrial genomes. Interestingly, atfs-1 was required to limit the accumulation of OXPHOS transcripts during mitochondrial stress, which required accumulation of ATFS-1 in the nucleus and mitochondria. Because balanced ATFS-1 accumulation promoted OXPHOS complex assembly and function, our data suggest that ATFS-1 stimulates respiratory recovery by fine-tuning OXPHOS expression to match the capacity of the suboptimal protein-folding environment in stressed mitochondria, while simultaneously increasing proteostasis capacity.

Mitochondrial UPR-regulated innate immunity provides resistance to pathogen infection.

Metazoans identify and eliminate bacterial pathogens in microbe-rich environments such as the intestinal lumen; however, the mechanisms are unclear. Host cells could potentially use intracellular surveillance or stress response programs to detect pathogens that target monitored cellular activities and then initiate innate immune responses. Mitochondrial function is evaluated by monitoring mitochondrial protein import efficiency of the transcription factor ATFS-1, which mediates the mitochondrial unfolded protein response (UPR(mt)). During mitochondrial stress, mitochondrial import is impaired, allowing ATFS-1 to traffic to the nucleus where it mediates a transcriptional response to re-establish mitochondrial homeostasis. Here we examined the role of ATFS-1 in Caenorhabditis elegans during pathogen exposure, because during mitochondrial stress ATFS-1 induced not only mitochondrial protective genes but also innate immune genes that included a secreted lysozyme and anti-microbial peptides. Exposure to the pathogen Pseudomonas aeruginosa caused mitochondrial dysfunction and activation of the UPR(mt). C. elegans lacking atfs-1 were susceptible to P. aeruginosa, whereas hyper-activation of ATFS-1 and the UPR(mt) improved clearance of P. aeruginosa from the intestine and prolonged C. elegans survival in a manner mainly independent of known innate immune pathways. We propose that ATFS-1 import efficiency and the UPR(mt) is a means to detect pathogens that target mitochondria and initiate a protective innate immune response.

Signaling the mitochondrial unfolded protein response.

Mitochondria are compartmentalized organelles essential for numerous cellular functions including ATP generation, iron-sulfur cluster biogenesis, nucleotide and amino acid metabolism as well as apoptosis. To promote biogenesis and proper function, mitochondria have a dedicated repertoire of molecular chaperones to facilitate protein folding and quality control proteases to degrade those proteins that fail to fold correctly. Mitochondrial protein folding is challenged by the complex organelle architecture, the deleterious effects of electron transport chain-generated reactive oxygen species and the mitochondrial genome's susceptibility to acquiring mutations. In response to the accumulation of unfolded or misfolded proteins beyond the organelle's chaperone capacity, cells mount a mitochondrial unfolded protein response (UPR(mt)). The UPR(mt) is a mitochondria-to-nuclear signal transduction pathway resulting in the induction of mitochondrial protective genes including mitochondrial molecular chaperones and proteases to re-establish protein homeostasis within the mitochondrial protein-folding environment. Here, we review the current understanding of UPR(mt) signal transduction and the impact of the UPR(mt) on diseased cells. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

Coordinated lumen contraction and expansion during vulval tube morphogenesis in Caenorhabditis elegans.

Morphogenesis is a developmental phase during which cell fates are executed. Mechanical forces shaping individual cells play a key role during tissue morphogenesis. By investigating morphogenesis of the Caenorhabditis elegans hermaphrodite vulva, we show that the force-generating actomyosin network is differentially regulated by NOTCH and EGFR/RAS/MAPK signaling to shape the vulval tube. NOTCH signaling activates expression of the RHO kinase LET-502 in the secondary cell lineage through the ETS-family transcription factor LIN-1. LET-502 induces actomyosin-mediated contraction of the apical lumen in the secondary toroids, thereby generating a dorsal pushing force. In contrast, MAPK signaling in the primary lineage downregulates LET-502 RHO kinase expression to prevent toroid contraction and allow the gonadal anchor cell to expand the dorsal lumen of the primary toroids. The antagonistic action of the MAPK and NOTCH pathways thus controls vulval tube morphogenesis linking cell fate specification to morphogenesis.

Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation.

To better understand the response to mitochondrial dysfunction, we examined the mechanism by which ATFS-1 (activating transcription factor associated with stress-1) senses mitochondrial stress and communicates with the nucleus during the mitochondrial unfolded protein response (UPR(mt)) in Caenorhabditis elegans. We found that the key point of regulation is the mitochondrial import efficiency of ATFS-1. In addition to a nuclear localization sequence, ATFS-1 has an N-terminal mitochondrial targeting sequence that is essential for UPR(mt) repression. Normally, ATFS-1 is imported into mitochondria and degraded. However, during mitochondrial stress, we found that import efficiency was reduced, allowing a percentage of ATFS-1 to accumulate in the cytosol and traffic to the nucleus. Our results show that cells monitor mitochondrial import efficiency via ATFS-1 to coordinate the level of mitochondrial dysfunction with the protective transcriptional response.

The transcription factor VAB-23 links vulval cell fate specification and morphogenesis.

During organogenesis, individual cells must commit to and execute specific cell fates. However, the molecular mechanisms linking cell fate specification to fate execution and morphogenesis remain a largely unexplored area in developmental biology. The Caenorhabditis elegans vulva is an excellent model to dissect the molecular pathways linking cell fate specification and execution during organogenesis. We have recently identified a conserved nuclear zinc finger transcription factor called VAB-23 that plays essential roles during vulval torid formation in the larva and ventral epidermal closure in the embryo. VAB-23 regulates the transcription of specific target genes including smp-1 Semaphorin. EGFR/RAS/MAPK signaling upregulates via the HOX protein LIN-39 the expression of VAB-23 in the 1° vulval cell lineage, indicating that cell fate specification and execution are temporally overlapping and tightly linked processes. Here, we discuss the roles of VAB-23 in morphogenesis and the implications of its regulation on the spatio-temporal control of organogenesis.