Biology subjects

Chien, P.

Publications and source records attributed to Chien, P..

At least 19 recordsLinked to original sources

bioRxiv2026

An NF2-wildtype malignant meningioma cell line for basic and translational science

BackgroundMeningiomas are the most common primary nervous system neoplasm in adults. There are few good cellular models, especially of high grade/malignant meningiomas, to use to identify new therapeutic agents and treatment regimens. The widely available, partially characterized, NF2-wildtype (NF2wt) Grade 3 malignant meningioma cell line IOMM-Lee can help meet this need. MethodsWe generated new data to better characterize IOMM-Lee genomic and mtDNA variants, proliferation rate and colony-forming efficiency and sensitivity to ionizing radiation as a function of ATM kinase activity. A screen of 349 anti-cancer drugs identified multiple, mechanistically distinct clinical use drugs with nanomolar IC50 values and high drug sensitivity prediction scores. ResultsExome sequencing confirmed that IOMM-Lee is NF2wt, and contains a pathogenic TERT-promoter (c.-124C>T) variant. Population doubling times (PDT) were short (19-21 hrs), and colony forming efficiency (CFE) high, of up to 87%. IOMM-Lee is comparatively radiosensitive with a D10 of [~]3.9 Gy, and could be radiosensitized by AZD-1390-mediated ATM kinase inhibition. Thirty-four anti-cancer compounds spanning several mechanistic classes were identified that potently suppressed cell proliferation at sub-micromolar IC50 values with high Breeze 2.0 Drug Sensitivity Scores. Importance of the StudyWe provide new data to better characterize IOMM-Lee, the most widely used cell line model of human Grade 3 malignant meningioma. These data identify and characterize IOMM-Lee genomic alterations and mtDNA variants; quantify growth kinetics and ionizing radiation sensitivity; and identify multiple mechanistically distinct, clinical use drugs with nanomolar IC50 values, high drug sensitivity prediction scores and potential as meningioma systemic therapies. Our data more clearly locate IOMM-Lee in the landscape of genomically-defined meningiomas, and will aid better use of this experimentally tractable cell line model to understand meningioma biology and identify more effective malignant meningioma therapies and treatment regimens. Key pointsO_LIIOMM-Lee lacks NF2 mutations, though is clearly related to but distinct from many other meningiomas and meningioma cell lines. C_LIO_LIIOMM-Lee grows rapidly, is comparatively radio-sensitive, and can be suppressed by several mechanistic classes of anti-cancer agents at clinically achievable, sub-micromolar IC50 values with high Drug Sensitivity Scores. C_LIO_LIThe experimental tractability, simplicity and versatility of IOMM-Lee can facilitate analyses of many aspects of meningioma biology and therapeutic development across a wide range of in vitro, high throughput and in vivo xenograft/organoid protocols. C_LI

cancer biology↗

bioRxiv2026

Collateral consequences of oxidative stress responses result in mitomycin C sensitivity

Bacterial survival depends on carefully balanced antioxidant defenses against reactive oxygen species. Caulobacter crescentus employs the transcription factor OxyR to activate hydrogen peroxide detoxification genes, a seemingly straightforward protective strategy. Here, we reveal an unexpected consequence of environment or genetic activation of peroxide resistance via OxyR resulting in vulnerability to reductively activated antibiotics. An activated OxyR allele protects against peroxide stress due to upregulation of the catalase-peroxidase KatG but simultaneous induction of the AhpCF reductase sensitizes cells to the reductively activated genotoxin mitomycin C. We show that this collateral vulnerability can be induced by brief exposure to oxidative stress and extends to bacteria beyond Caulobacter. Our findings illuminate a hidden cost of antioxidant signaling as robust defense against oxidative stress paradoxically creates exploitable vulnerabilities to chemotherapeutic prodrugs. SIGNIFICANCECellular stress responses are vital for survival, but our work reveals an underappreciated principle where activation creates trade-offs with far-reaching consequences. Using Caulobacter crescentus, we show that strengthened antioxidant defenses against peroxide simultaneously increase vulnerability to mitomycin C. Because these regulatory mechanisms are widespread across bacteria, our findings suggest a general model wherein stress-defense pathways unavoidably compromise resistance to alternative threats. This conceptual framework not only advances our understanding of stress-response regulation but also identifies new strategies for therapeutic exploitation of bacterial vulnerabilities.

microbiology↗

bioRxiv2026

Dynamic reorganization of Developmental to Adult Genome Topology Controls the Initiation and Stabilization of the Human Muscle Stem Cell State

Developmental gene expression is under tight temporal and spatial control. This regulation is imparted by tissue specific enhancers that integrate developmental signals into transcriptional responses to allow for developmental progression. Often species specific, the enhancers that regulate human muscle progenitor and stem cell gene expression are currently unknown. Here, we define the 3D chromatin organization of human muscle development and reveal key changes across the human genome that are associated with multiple layers of 3D genome reorganization during the transition from a more progenitor-like to muscle stem cell state, including a reduction of TAD numbers and an increase in CTCF binding at TAD boundaries and chromatin loops throughout developmental progression. Specifically, we found that increased CTCF occupancy at human enhancers of PAX7 in stem cells holds enhancer-promoter (e-p) loops for timely activation of PAX7 enhancers during early human development. These findings demonstrate that stem cell state acquisition is stabilized earlier than previously known and provide unprecedented insights into the initiation and control of the muscle stem cell state in humans.

developmental biology↗

bioRxiv2025

Stress testing reveals selective vulnerabilities in protein homeostasis

Protein quality control (PQC) systems are essential for cellular resilience to proteotoxic stress. Despite intensive study for decades, functional redundancies in the system obscure the contributions of the collectively important individual genes. Here, we leverage transposon sequencing across bacteria strains lacking key chaperones and proteases to reveal hidden determinants of stress response in protein homeostasis. By profiling fitness under multiple proteotoxic stresses, we uncover stress-specific vulnerabilities and reveal how major players of PQC mask correlations between transcriptomic responses and gene fitness. As an illustration of unexpected connections, we identify a heat-specific synthetic lethality between the disaggregase ClpB and DNA Polymerase I (PolA) mediated by persistent aggregation of the RecA recombinase and toxic persistence of the heat shock regulon. Our findings reveal that stress-induced aggregation is not broadly toxic. Rather, it becomes lethal in specific genetic or environmental contexts due to the depletion of components only needed in those specific circumstances. This work presents a framework to reveal normally hidden fragility in stress responses using gene fitness scores adaptable to a variety of systems.

molecular biology↗

bioRxiv2024

Allosteric modulation of the Lon protease by effector binding and local charges

The ATPase Associated with diverse cellular Activities (AAA+) family of proteases play crucial roles in cellular proteolysis and stress responses. Like other AAA+ proteases, the Lon protease is known to be allosterically regulated by nucleotide and substrate binding. Although it was originally classified as a DNA binding protein, the impact of DNA binding on Lon activity is unclear. In this study, we characterize the regulation of Lon by single-stranded DNA (ssDNA) binding and serendipitously identify general activation strategies for Lon. Upon binding to ssDNA, Lons ATP hydrolysis rate increases due to improved nucleotide binding, leading to enhanced degradation of protein substrates, including physiologically important targets. We demonstrate that mutations in basic residues that are crucial for Lons DNA binding not only reduces ssDNA binding but result in charge-specific consequences on Lon activity. Introducing negative charge at these sites induces activation akin to that induced by ssDNA binding, whereas neutralizing the charge reduces Lons activity. Based on single molecule measurements we find that this change in activity is correlated with changes in Lon oligomerization. Our study provides insights into the complex regulation of the Lon protease driven by electrostatic contributions from either DNA binding or mutations. HighlightsO_LIssDNA binding allosterically activates Lon ATP hydrolysis C_LIO_LINegative charge at DNA binding site is sufficient for Lon activation C_LIO_LINeutralization of charge at DNA binding site inhibits Lon ATP hydrolysis C_LIO_LILon activity is linked to formation of stable Lon hexamers C_LI SignificanceThe energy-dependent protease Lon is integral in both eukaryotic and prokaryotic physiology, contributing to protein quality control, stress management, developmental regulation, and pathogenicity. The ability to precisely regulate protein levels through targeted degradation underscores a need for tunability. We find that single-stranded DNA (ssDNA) acts as an allosteric regulator of Lon, leading to enhanced enzymatic activity. Mutations in basic residues crucial for DNA binding were found to affect Lon activity in a charge-specific manner highlighting the importance of electrostatic interactions regulating Lons function. Changes in Lon activity due to ssDNA binding or mutations were correlated with its oligomerization state. Our findings provide insights into the activation strategies of Lon, emphasizing the role of electrostatic contribution that modulate nucleotide affinity, oligomerization and proteolysis to advance our understanding of the complex regulatory mechanisms of the Lon protease.

biochemistry↗

bioRxiv2024

Comparison of CcrM-dependent methylation in Caulobacter crescentus and Brucella abortus by nanopore sequencing

Bacteria rely on DNA methylation for restriction-modification systems and epigenetic control of gene expression. Here, we use direct detection of methylated bases by nanopore sequencing to monitor global DNA methylation in Alphaproteobacteria, where use of this technique has not yet been reported. One representative of this order, Caulobacter crescentus, relies on DNA methylation to control cell cycle progression, but it is unclear whether other members of this order, such as Brucella abortus, depend on the same systems. We addressed these questions by first measuring CcrM-dependent DNA methylation in Caulobacter and show excellent correlation between nanopore-based detection and previously published results. We then directly measure the impact of Lon-mediated CcrM degradation on the epigenome, verifying that loss of Lon results in pervasive methylation. We also show that the AlkB demethylase has no global impact on DNA methylation during normal growth. Next, we report on the global DNA methylation in Brucella abortus for the first time and find that CcrM-dependent methylation is reliant on Lon but impacts the two chromosomes differently. Finally, we explore the impact of the MucR transcription factor, known to compete with CcrM methylation, on the Brucella methylome and share the results with a publicly available visualization package. Our work demonstrates the utility of nanopore-based sequencing for epigenome measurements in Alphaproteobacteria and reveals new features of CcrM-dependent methylation in a zoonotic pathogen. ImportanceDNA methylation plays an important role in bacteria to maintain genome integrity and regulate of gene expression. We used nanopore sequencing to directly measure methylated bases in Caulobacter crescentus and Brucella abortus. In Caulobacter, we showed that stabilization of the CcrM methyltransferase upon loss of the Lon protease results in prolific methylation and discovered that the putative methylase AlkB is unlikely to have a global physiological effect. We measured genome-wide methylation in Brucella for the first time, revealing a similar role for CcrM in cell-cycle methylation but a more complex regulation by the Lon protease than in Caulobacter. Finally, we show how the virulence factor MucR impacts DNA methylation patterns in Brucella.

microbiology↗

bioRxiv2024

Discovering Genetic Modulators of the Protein Homeostasis System through Multilevel Analysis

Every protein progresses through a natural lifecycle from birth to maturation to death; this process is coordinated by the protein homeostasis system. Environmental or physiological conditions trigger pathways that maintain the homeostasis of the proteome. An open question is how these pathways are modulated to respond to the many stresses that an organism encounters during its lifetime. To address this question, we tested how the fitness landscape changes in response to environmental and genetic perturbations using directed and massively parallel transposon mutagenesis in Caulobacter crescentus. We developed a general computational pipeline for the analysis of gene-by-environment interactions in transposon mutagenesis experiments. This pipeline uses a combination of general linear models (GLMs), statistical knockoffs, and a nonparametric Bayesian statistical model to identify essential genetic network components that are shared across environmental perturbations. This analysis allows us to quantify the similarity of proteotoxic environmental perturbations from the perspective of the fitness landscape. We find that essential genes vary more by genetic background than by environmental conditions, with limited overlap among mutant strains targeting different facets of the protein homeostasis system. We also identified 146 unique fitness determinants across different strains, with 19 genes common to at least two strains, showing varying resilience to proteotoxic stresses. Experiments exposing cells to a combination of genetic perturbations and dual environmental stressors show that perturbations that are quantitatively dissimilar from the perspective of the fitness landscape are likely to have a synergistic effect on the growth defect. Significance StatementThis study provides critical insights into how cells adapt to environmental and genetic challenges affecting protein homeostasis. Using multilevel statistical analysis and transposon mutagenesis, we find that a model organism, Caulobacter crescentus, lacks a universal redundancy mechanism for coping with stress, as evidenced by the limited overlap in essential genes across different environmental and genetic perturbations. Our methods also pinpoint key fitness determinants and enable the prediction of perturbation combinations that synergistically affect cell growth.

systems biology↗

bioRxiv2024

Proteolytic control of FixT by the Lon protease impacts FixLJ signaling in Caulobacter crescentus

Responding to changes in oxygen levels is critical for aerobic microbes. In Caulobacter crescentus, low oxygen is sensed by the FixL-FixJ two-component system which induces multiple genes, including heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address if the degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that {Delta}lon strains are deficient in FixLJ-dependent heme biosynthesis, consistent with elevated FixT levels as deletion of fixT suppresses this defect. Transcriptomics validate this result as there is diminished expression of many FixLJ-activated genes in {Delta}lon. However, no physiological changes in response to microaerobic conditions occurred upon loss of Lon, suggesting that FixT dynamics are not a major contributor to fitness in oxygen limiting conditions. Similarly, stabilization of FixT in {Delta}lon strains does not contribute to any known Lon-related fitness defect, such as cell morphology defects or stress sensitivity. In fact, cells lacking both FixT and Lon are compromised in viability during adaptation to long term aerobic growth. Our work highlights the complexity of protease-dependent regulation of transcription factors and explains the molecular basis of defective heme accumulation in Lon-deficient Caulobacter. ImportanceThe Lon protease shapes protein quality control, signaling pathways, and stress responses in many bacteria species. Loss of Lon often results in multiple phenotypic consequences. In this work, we found a connection between the Lon protease and deficiencies in heme accumulation that then led to our finding of a global change in gene expression due to degradation of a regulator of the hypoxic response. However, loss of degradation of this regulator did not explain other phenotypes associated with Lon deficiencies demonstrating the complex and multiple pathways that this highly conserved protease can impact.

microbiology↗

bioRxiv2023

Regulation of the transcription factor CdnL promotes adaptation to nutrient stress in Caulobacter

In response to nutrient deprivation, bacteria activate a conserved stress response pathway called the stringent response (SR). During SR activation in Caulobacter crescentus, SpoT synthesizes the secondary messengers (p)ppGpp, which affect transcription by binding RNA polymerase to downregulate anabolic genes. (p)ppGpp also impacts expression of anabolic genes by controlling the levels and activities of their transcriptional regulators. In Caulobacter, a major regulator of anabolic genes is the transcription factor CdnL. If and how CdnL is controlled during the SR and why that might be functionally important is unclear. Here, we show that CdnL is downregulated post-translationally during starvation in a manner dependent on SpoT and the ClpXP protease. Inappropriate stabilization of CdnL during starvation causes misregulation of ribosomal and metabolic genes. Functionally, we demonstrate that the combined action of SR transcriptional regulators and CdnL clearance allows for rapid adaptation to nutrient repletion. Moreover, cells that are unable to clear CdnL during starvation are outcompeted by wild-type cells when subjected to nutrient fluctuations. We hypothesize that clearance of CdnL during the SR, in conjunction with direct binding of (p)ppGpp and DksA to RNAP, is critical for altering the transcriptome in order to permit cell survival during nutrient stress. SIGNIFICANCEThe stringent response (SR) is a ubiquitous bacterial stress response that promotes adaptation to nutrient deprivation. While it is known that SR activation affects RNA polymerase activity to reprogram the transcriptome, the impact of the SR on other transcriptional regulators is not well understood. Here, we show that a conserved transcription factor, CdnL, is cleared upon activation of the SR, and that its clearance is important for cells to efficiently adapt to nutrient fluctuations. Our results suggest that CdnL regulation enables adaptation by transcriptionally downregulating ribosome biosynthesis and flux through metabolic pathways, thereby promoting survival during nutrient stress. As CdnL homologs are broadly found, we hypothesize that CdnL regulation is a conserved mechanism of bacterial adaptation to stress.

microbiology↗

bioRxiv2023

Proteomic survey of the DNA damage response in Caulobacter crescentus reveals evidence of post-transcriptional control

The bacterial DNA damage response is a critical, coordinated response to endogenous and exogenous sources of DNA damage. Response dynamics are dependent on coordinated synthesis and loss of relevant proteins. While much is known about its global transcriptional control, changes in protein abundance that occur upon DNA damage are less well characterized at the system level. Here, we perform a proteome-wide survey of the DNA damage response in Caulobacter crescentus. We find that while most protein abundance changes upon DNA damage are readily explained by changes in transcription, there are exceptions. The survey also allowed us to identify the novel DNA damage response factor, YaaA, which has been overlooked by previously published, transcription- focused studies. A similar survey in a {Delta}lon strain was performed to explore lons role in DNA damage survival. The {Delta}lon strain had a smaller dynamic range of protein abundance changes in general upon DNA damage compared to the wild type strain. This system-wide change to the dynamics of the response may explain this strains sensitivity to DNA damage. Our proteome survey of the DNA damage response provides additional insight into the complex regulation of stress response and nominates a novel response factor that was overlooked in prior studies. IMPORTANCEThe DNA damage response helps bacteria to react to and potentially survive DNA damage. The mutagenesis induced during this stress response contributes to the development of antibiotic resistance. Understanding how bacteria coordinate their response to DNA damage could help us to combat this growing threat to human health. While the transcriptional regulation of the bacterial DNA damage response has been characterized, this study is the first to our knowledge to assess the proteomic response to DNA damage in Caulobacter.

microbiology↗

bioRxiv2022

Clamp loader processing is important during DNA replication stress

The DNA clamp loader is critical to the processivity of the DNA polymerase and coordinating synthesis on the leading and lagging strands. In bacteria the major subunit of the clamp loader, DnaX, has two forms: the essential full-length {tau} and shorter {gamma}. These are conserved across bacterial species and three distinct mechanisms have been found to create them: ribosomal frameshift, transcriptional slippage, and, in Caulobacter crescentus, proteolysis. This conservation suggests that DnaX processing is evolutionarily important, but its role remains unknown. Here we find a bias against switching from expression of a wild type dnaX to a nonprocessable{tau} -only allele in Caulobacter. Despite this bias, cells are able to adapt to the{tau} -only allele with little effect on growth or morphology and only minor defects during DNA damage. Motivated by transposon sequencing, we find that loss of the gene sidA in the{tau} -only strain slows growth and increases filamentation. Even in the absence of exogenous DNA damage treatment, the{Delta} sidA {tau}-only double mutant shows induction of and dependance on recA, likely due to a defect in resolution of DNA replication fork stalling. We find that some of the phenotypes of the{Delta} sidA {tau}-only can be complemented by expression of {gamma} but that an overabundance of {tau}-only dnaX is also detrimental. The data presented here suggest that DnaX processing is important during resolution of replication fork stalling events during DNA replication stress. IMPORTANCEThough the presence of DnaX {tau} and {gamma} forms is conserved across bacteria, different species have developed different mechanisms to make these forms. This conservation and independent evolution of mechanisms suggest that having two forms of DnaX is important. Despite having been discovered more than 30 years ago, the purpose of expressing both {tau} and {gamma} is still unclear. Here, we present evidence that expressing two forms of DnaX and controlling the abundance and/or ratio of the forms is important during the resolution of replication fork stalling.

microbiology↗

bioRxiv2022

EstG is a novel esterase required for cell envelope integrity

Proper regulation of the bacterial cell envelope is critical for cell survival. Identification and characterization of enzymes that maintain cell envelope homeostasis is crucial, as they can be targets for effective antibiotics. In this study, we have identified a novel enzyme, called EstG, whose activity protects cells from a variety of lethal assaults in the -proteobacterium Caulobacter crescentus. Despite homology to transpeptidase family cell wall enzymes and an ability to protect against cell wall-targeting antibiotics, EstG does not demonstrate biochemical activity towards cell wall substrates. Instead, EstG is genetically connected to the periplasmic enzymes OpgH and BglX, responsible for synthesis and hydrolysis of osmoregulated periplasmic glucans (OPGs), respectively. The crystal structure of EstG revealed similarities to esterases and transesterases, and we demonstrated esterase activity of EstG in vitro. Using biochemical fractionation, we identified a cyclic hexamer of glucose as a likely substrate of EstG. This molecule is the first OPG described in Caulobacter and establishes a novel class of OPGs, the regulation and modification of which is important for stress survival and adaptation to fluctuating environments. Our data indicate that EstG, BglX, and OpgH comprise a previously unknown OPG pathway in Caulobacter. Ultimately, we propose that EstG is a novel enzyme that, instead of acting on the cell wall, acts on cyclic OPGs to provide resistance to a variety of cellular stresses.

microbiology↗

bioRxiv2021

Plasticity in AAA+ proteases reveals ATP-dependent substrate specificity principles

In bacteria, AAA+ proteases such as Lon and ClpXP degrade substrates with exquisite specificity. These machines capture the energy of ATP hydrolysis to power unfolding and degradation of target substrates. Here, we show that a mutation in the ATP binding site of ClpX shifts protease specificity to promote degradation of normally Lon-restricted substrates. However, this ClpX mutant is worse at degrading ClpXP targets, suggesting an optimal balance in substrate preference for a given protease that is surprisingly easy to alter. In vitro, wildtype ClpXP also degrades Lon-restricted substrates more readily when ATP levels are reduced, similar to the shifted specificity of mutant ClpXP, which has altered ATP hydrolysis kinetics. Based on these results, we suggest that rates of ATP hydrolysis not only power substrate unfolding and degradation, but also tune protease specificity. We consider various models for this effect based on emerging structures of AAA+ machines showing conformationally distinct states. O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/456811v2_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1b72081org.highwire.dtl.DTLVardef@1b713c7org.highwire.dtl.DTLVardef@73a92aorg.highwire.dtl.DTLVardef@167422d_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG eTOCAAA+ proteases, such as Lon and ClpXP, select distinct targets for degradation to maintain proteostasis. Mahmoud et al. show that ATP hydrolysis can tune substrate specificity of ClpX, allowing ClpX to degrade Lon-restricted substrates under limiting ATP conditions or in the presence of a ClpX mutant. HighlightsO_LIA Walker B mutation of the AAA+ protease ClpX alters substrate specificity C_LIO_LIClpX mutant degrades new substrates but degrades canonical substrates less well C_LIO_LIDecreasing ATP levels enhances ClpXP mediated degradation of some classes of substrates C_LIO_LIATP-induced changes in conformational states accompany alterations in ClpX specificity C_LI

molecular biology↗

bioRxiv2021

Model-based identification of conditionally-essential genes from transposon-insertion sequencing data

The understanding of bacterial gene function has been greatly enhanced by recent advancements in the deep sequencing of microbial genomes. Transposon insertion sequencing methods combines next-generation sequencing techniques with transposon mutagenesis for the exploration of the essentiality of genes under different environmental conditions. We propose a model-based method that uses regularized negative binomial regression to estimate the change in transposon insertions attributable to gene-environment changes without transformations or uniform normalization. An empirical Bayes model for estimating the local false discovery rate combines unique and total count information to test for genes that show a statistically significant change in transposon counts. When applied to RB-TnSeq (randomized barcode transposon sequencing) and Tn-seq (transposon sequencing) libraries made in strains of Caulobacter crescentus using both total and unique count data the model was able to identify a set of conditionally essential genes for each target condition that shed light on their functions and roles during various stress conditions. Author summaryTransposon insertion sequencing allows the study of bacterial gene function by combining next-generation sequencing techniques with transposon mutagenesis under different genetic and environmental perturbations. Our proposed regularized negative binomial regression method improves the quality of analysis of this data.

bioinformatics↗

bioRxiv2021

Live-Cell Imaging of (p)ppGpp with RNA-based Fluorescent Sensors

Guanosine tetra- and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to directly image and measure cellular (p)ppGpp. Here, we report a type of RNA-based fluorescent sensors for live-cell imaging of (p)ppGpp. Our sensor is engineered by conjugating a recently identified (p)ppGpp-specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell-to-cell variation and cellular dynamics of (p)ppGpp levels can now be obtained under different nutritional conditions. We predict that these RNA-based sensors will be broadly adapted to study bacterial stringent response.

cell biology↗

bioRxiv2020

Degradation of ATFS-1 by LONP-1 promotes deleterious mitochondrial genome heteroplasmy

The accumulation of deleterious mitochondrial genomes ({Delta}mtDNAs) underlies inherited mitochondrial diseases and contributes to the aging-associated decline in mitochondrial function. In heteroplasmic cells, oxidative phosphorylation (OXPHOS) function declines as the population of {Delta}mtDNAs increase relative to wildtype mtDNAs. In response to mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcription program to promote the recovery of mitochondrial function. Paradoxically, ATFS-1 is also required to maintain {Delta}mtDNAs in heteroplasmic worms. However, the mechanism(s) by which ATFS-1 promotes {Delta}mtDNA accumulation relative to wildtype mtDNAs is unclear. Here, we show that mitochondrial-localized ATFS-1 binds almost exclusively to {Delta}mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the preferential binding of the mtDNA replicative polymerase (POLG) to {Delta}mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wildtype mtDNAs. Furthermore, LONP-1 inhibition in C. elegans and human cybrid cells improved the heteroplasmy ratio and restored OXPHOS function. Our findings suggest that ATFS-1 promotes mtDNA replication by recruiting POLG to mtDNA in a manner that is antagonized by LONP-1. We speculate that this mechanism promotes the repair and expansion of the mitochondrial network by synchronizing mtDNA replication with UPRmt activation driven by nuclear ATFS-1 activity. However, this repair mechanism cannot resolve OXPHOS defects in mitochondria harboring {Delta}mtDNAs, resulting in an accumulation of ATFS-1 in dysfunctional mitochondria and constitutive replication of {Delta}mtDNAs.

cell biology↗

bioRxiv2020

Cargo competition for a dimerization interface stabilizes a protease adaptor in Caulobacter crescentus

Bacterial protein degradation is a regulated process aided by protease adaptors that alter specificity of energy dependent proteases. In Caulobacter crescentus, cell-cycle dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric RcdA adaptor which binds multiple cargo and delivers substrates to the ClpXP protease. RcdA itself is degraded in the absence of cargo and how RcdA recognizes its targets is unknown. Here we show that RcdA dimerization and cargo binding compete for a common interface. Cargo binding separates RcdA dimers and a monomeric variant of RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. Based on HDX-MS studies showing that different cargo rely on different regions of the dimerization interface, we generate RcdA variants that are selective for specific cargo and show cellular defects consistent with changes in selectivity. Using the same interface for dimerization and cargo binding offers an ability to limit excess protease adaptors by self-degradation, while providing capacity for binding a range of substrates. Significance StatementEnergy-dependent proteases broadly regulate bacterial physiology and development. Adaptor proteins tune the substrate specificity of proteases to only degrade selective substrates during the bacterial life cycle and during times of cellular stress. In the process of delivering cargo to their respective proteases, adaptor proteins are inherently protected from degradation until the delivery is complete. How protease adaptors can recognize a wide range of cargo while maintaining stringent specificity and how this process results in stabilization of adaptors remains unclear. Here, we show that direct competition for distinct regions of the dimer interface of the RcdA adaptor by its cargo protects RcdA from degradation by the ClpXP protease, and that this interface can be selectively perturbed in a rational manner with biochemical and physiological consequences. HighlightsO_LICargo binding of RcdA cargo competes with dimerization C_LIO_LIDimerization of RcdA is necessary for self-degradation by ClpXP C_LIO_LIRcdA can deliver either cargo or other RcdA subunits to ClpXP C_LIO_LIDifferent regions of the dimerization interface are needed for different cargo C_LI

biochemistry↗

bioRxiv2020

Degradation of Lon in Caulobacter crescentus

Protein degradation is an essential process in all organisms. This process is irreversible and energetically costly; therefore, protein destruction must be tightly controlled. While environmental stresses often lead to upregulation of proteases at the transcriptional level, little is known about post-translational control of these critical machines. In this study we show that in Caulobacter crescentus levels of the Lon protease are controlled through proteolysis. Lon turnover requires active Lon and ClpAP proteases. We show that specific determinants dictate Lon stability with a key carboxy-terminal histidine residue driving recognition. Expression of stabilized Lon variants results in toxic levels of protease that deplete normal Lon substrates such as the replication initiator DnaA to lethally low levels. Taken together, this work demonstrates a feedback mechanism in which ClpAP and Lon collaborate to tune Lon proteolytic capacity for the cell. ImportanceProteases are essential, but unrestrained activity can also kill cells by degrading essential proteins. The quality control protease Lon must degrade many misfolded and native substrates. We show that Lon is itself controlled through proteolysis and that bypassing this control results in toxic consequences for the cell.

microbiology↗