The ATP-driven extractor ATAD1/Msp1 proof-reads protein translocation into mitochondria.

The accumulation of translocation intermediates in the mitochondrial import machinery threatens cellular fitness and is associated with cancer and neurodegeneration. A recent study by Weidberg and colleagues identifies ATAD1 as an ATP-driven extraction machine on the mitochondrial surface that pulls precursors into the cytosol to prevent clogging of mitochondrial import pores.

Lessons Learned with Enhanced Recovery for Open Abdominal Aortic Aneurysm Surgery: A Long Term Regional Network Experience.

OBJECTIVE: This enhanced recovery programme (ERP) aimed to achieve early recovery for patients undergoing major surgery. Results of a standardised ERP protocol for open infrarenal abdominal aortic aneurysm (AAA) repair within a hub and spoke regional network are presented. METHODS: In this monocentric prospective study (January 2004 - December 2021), consecutive AAAs (≥ 55 mm) were included in the ERP (patient discharge on post-operative day [POD] 4). The four phases of the ERP were pre-admission, pre-operative, intra-operative, and post-operative. Exclusion criteria were BMI > 35 kg/m2, functional capacity < 4 MET, previous aortic or abdominal surgery, and life expectancy < 5 years. Transperitoneal surgery was undertaken with routine AAA resection, graft interposition, and closure. RESULTS: Consecutive patients (n = 778) were enrolled into the study (mean age 72.3 ± 3.2 years; n = 712 men); 160 (20.5%) were treated in spoke hospitals. Median follow up was 78 (IQR 28, 128) months; median length of stay, procedure time, and blood loss were 4 days (IQR 3, 5), 190 min (IQR 170, 225), and 564 mL (IQR 300, 600). Infrarenal clamping and tube graft configuration were used in 96.5% (n = 751) and 72.5% (n = 564) of patients; 30 day mortality and complication rates were 0.4% (n = 3) and 9.2% (n = 72). Discharge after POD 4 occurred in 15.0%, and most significant predictors for discharge after POD 4 were haemotransfusion, re-intervention, and ileus over 3 days. Overall survival was: 98.2% at 1 year, 85.0% at 5 years, and 59.9% at 10 years. Freedom from re-intervention was 97.9% at 1 year, 94.1% at 5 years, and 86.8% at 10 years. Short and long term outcomes were comparable between hub and spoke hospitals. CONCLUSION: The ERP protocol was associated with low short and long term mortality and complication rates. Future studies should apply the ERP protocol to other vascular centres.

Prohibitins, Phb1 and Phb2, function as Atg8 receptors to support yeast mitophagy and also play a negative regulatory role in Atg32 processing.

The prohibitins Phb1 and Phb2 assemble at the mitochondrial inner membrane to form a multi-dimeric complex. These scaffold proteins are highly conserved in eukaryotic cells, from yeast to mammals, and have been implicated in a variety of mitochondrial functions including aging, proliferation, and degenerative and metabolic diseases. In mammals, PHB2 regulates PINK1-PRKN mediated mitophagy by interacting with lipidated MAP1LC3B/LC3B. Despite their high conservation, prohibitins have not been linked to mitophagy in budding yeasts. In this study, we demonstrate that both Phb1 and Phb2 are required to sustain mitophagy in Saccharomyces cerevisiae. Prohibitin-dependent mitophagy requires formation of the Phb1-Phb2 complex and a conserved AIM/LIR-like motif identified in both yeast prohibitins. Furthermore, both Phb1 and Phb2 interact and exhibit mitochondrial colocalization with Atg8. Interestingly, we detected a basal C terminus processing of the mitophagy receptor Atg32 that depends on the presence of the i-AAA Yme1. In the absence of prohibitins this processing is highly enhanced but reverted by the inactivation of the rhomboid protease Pcp1. Together our results revealed a novel role of yeast prohibitins in mitophagy through its interaction with Atg8 and regulating an Atg32 proteolytic event. Abbreviation: AIM/LIR: Atg8-family interacting motif/LC3-interacting region; ANOVA: analysis of variance; ATG/Atg: autophagy related; C terminus/C-terminal: carboxyl terminus/carboxyl-terminal; GFP: green fluorescent protein; HA: human influenza hemagglutinin; Idh1: isocitrate dehydrogenase 1; MAP1C3B/LC3B: microtubule associated protein 1 light chain 3 beta; mCh: mCherry; MIM: mitochondrial inner membrane; MOM: mitochondrial outer membrane; N starvation: nitrogen starvation; N terminus: amino terminus; PARL: presenilin associated rhomboid like; Pcp1: processing of cytochrome c peroxidase 1; PCR: polymerase chain reaction; PGAM5: PGAM family member 5 mitochondrial serine/threonine protein phosphatase; PHBs/Phb: prohibitins; PINK1: PTEN induced kinase 1; PMSF: phenylmethylsulfonyl fluoride; PRKN: parkin RBR E3 ubiquitin protein ligase; SD: synthetic defined medium; SDS: sodium dodecyl sulfate; SMD-N: synthetic defined medium lacking nitrogen; WB: western blot; WT: wild type; Yme1: yeast mitochondrial escape 1; YPD: yeast extract-peptone-dextrose medium; YPLac: yeast extract-peptone-lactate medium.

Polygenic risk score informed clinical model for improving abdominal aortic aneurysm screening.

INTRODUCTION: Abdominal aortic aneurysm (AAA) is a complex disease with environmental and genetic risk factors. Polygenic risk scores (PRS) based on disease-specific risk-associated single nucleotide variants (SNVs) have demonstrated effectiveness in stratifying individual-level disease risk for cardiovascular diseases. This prospective cohort study assessed associations of PRS of AAA (PRSAAA) with risk of incident AAA, analyzed the effectiveness of a combined clinical-genetic risk model, and explored the clinical utility of the model in identifying high-risk individuals for AAA screening. METHODS: PRSAAA was calculated using 911,440 SNVs and PRSCAD was calculated using 2,324,683 SNVs derived from mixed ancestry genome wide association studies. The UK Biobank was used as the study cohort. All individuals with complete genetic data available and no diagnosis of AAA at time of recruitment were included in the analysis and followed prospectively to assess for incident AAA. A PRS informed clinical model, Prob-AAA, was developed using clinically significant variables and PRSAAA. RESULTS: 481,105 individuals were included in the analysis with 2,668 incident AAA cases. Incident AAA increased from 0.30% to 0.93% between the lowest and highest decile of PRSAAA; similarly, severe AAA, requiring surgery and/or presenting with rupture, increased from 23% to 39% of incident AAA cases across deciles. PRSAAA was a predictor of incident AAA diagnosis (HR 2.06 [1.70 - 2.48]) independent of other clinical risk factors including male sex, older age, and smoking history. Prob-AAA was an independent predictor of incident AAA (HR 1.92 [1.69 - 2.20]), and identified 9.6% of cases of incident AAA compared to only 4.2% by PRSAAA. Current screening guidelines captured 5.7% of the overall cohort, with an incident AAA rate of approximately 3.2%. Amongst males not included by current guidelines, Prob-AAA identified an additional cohort, approximately 2% of the overall cohort, with a similar rate of incident AAA. CONCLUSIONS: Prob-AAA, a PRS informed clinical model for AAA, improved upon the predictive power of current, clinical risk factor informed, screening guidelines for AAA.

ATAD1 prevents clogging of TOM and damage caused by un-imported mitochondrial proteins.

Mitochondria require the constant import of nuclear-encoded proteins for proper functioning. Impaired protein import not only depletes mitochondria of essential factors but also leads to toxic accumulation of un-imported proteins outside the organelle. Here, we investigate the consequences of impaired mitochondrial protein import in human cells. We demonstrate that un-imported proteins can clog the mitochondrial translocase of the outer membrane (TOM). ATAD1, a mitochondrial ATPase, removes clogged proteins from TOM to clear the entry gate into the mitochondria. ATAD1 interacts with both TOM and stalled proteins, and its knockout results in extensive accumulation of mitochondrial precursors as well as decreased protein import. Increased ATAD1 expression contributes to improved fitness of cells with inefficient mitochondrial protein import. Overall, we demonstrate the importance of the ATAD1 quality control pathway in surveilling protein import and its contribution to cellular health.

TRIP13 - a potential drug target in cancer pharmacotherapy.

ATPases Associated with Diverse Cellular Activity (AAA+ATPases) are important enzymatic functional proteins in human cells. Thyroid Hormone Receptor Interacting Protein-13 (TRIP13) is a member of this protein superfamily, that partly regulates DNA repair pathways and spindle assembly checkpoints during mitosis. TRIP13 is reported as an oncogene involving multiple pathways in many human malignancies, including multiple myeloma, brain tumors, etc. The structure of TRIP13 reveals the mechanisms for ATP binding and how TRIP13 recognizes the Mitotic Arrest Deficiency-2 (MAD2) protein, with p31comet acting as an adapter protein. DCZ0415, TI17, DCZ5417, and DCZ5418 are the reported small-molecule inhibitors of TRIP13, which have been demonstrated to inhibit TRIP13's biological functions significantly and effective in suppressing various types of malignant cells, indicating that TRIP13 is a significant anticancer drug target. Currently, no systematic reviews are cutting across the functions, structure, and novel inhibitors of TRIP13. This review provides a comprehensive overview of TRIP13's biological functions, its roles in eighteen different cancers, four small molecule inhibitors, different underlying molecular mechanisms, and its functionality as a potential anticancer drug target.

Effect of modulation factor and low dose threshold level on gamma pass rates of single isocenter multi-target SRT treatment plans.

PURPOSE: SRS MapCHECK (SMC) is a commercially available patient-specific quality assurance (PSQA) tool for stereotactic radiosurgery (SRS) applications. This study investigates the effects of degree of modulation, location off-axis, and low dose threshold (LDT) selection on gamma pass rates (GPRs) between SMC and treatment planning system, Analytical Anisotropic Algorithm (AAA), or Vancouver Island Monte Carlo (VMC++ algorithm) system calculated dose distributions. METHODS: Volumetric-modulated arc therapy (VMAT) plans with modulation factors (MFs) ranging from 2.7 to 10.2 MU/cGy were delivered to SMC at isocenter and 6 cm off-axis. SMC measured dose distributions were compared against AAA and VMC++ via gamma analysis (3%/1 mm) with LDT of 10% to 80% using SNC Patient software. RESULTS: Comparing on-axis SMC dose against AAA and VMC++ with LDT of 10%, all AAA-calculated plans met the acceptance criteria of GPR ≥ 90%, and only one VMC++ calculated plan was marginally outside the acceptance criteria with pass rate of 89.1%. Using LDT of 80% revealed decreasing GPR with increasing MF. For AAA, GPRs reduced from 100% at MF of 2.7 MU/cGy to 57% at MF of 10.2 MU/cGy, and for VMC++ calculated plans, the GPRs reduced from 89% to 60% in the same MF range. Comparison of SMC dose off-axis against AAA and VMC++ showed more pronounced reduction of GPR with increasing MF. For LDT of 10%, AAA GPRs reduced from 100% to 83% in the MF range of 2.7 to 9.8 MU/cGy, and VMC++ GPR reduced from 100% to 91% in the same range. With 80% LDT, GPRs dropped from 100% to 42% for both algorithms. CONCLUSIONS: MF, dose calculation algorithm, and LDT selections are vital in VMAT-based SRT PSQA. LDT of 80% enhances sensitivity of gamma analysis for detecting dose differences compared to 10% LDT. To achieve better agreement between calculated and SMC dose, it is recommended to limit the MF to 4.6 MU/cGy on-axis and 3.6 MU/cGy off-axis.

A pro-metastatic tRNA fragment drives aldolase A oligomerization to enhance aerobic glycolysis in lung adenocarcinoma.

Despite being the leading cause of lung cancer-related deaths, the underlying molecular mechanisms driving metastasis progression are still not fully understood. Transfer RNA-derived fragments (tRFs) have been implicated in various biological processes in cancer. However, the role of tRFs in lung adenocarcinoma (LUAD) remains unclear. Our study identified a tRF, tRF-Val-CAC-024, associated with the high-risk component of LUAD, through validation using 3 cohorts. Our findings demonstrated that tRF-Val-CAC-024 acts as an oncogene in LUAD. Mechanistically, tRF-Val-CAC-024 was revealed to bind to aldolase A (ALDOA) dependent on Q125/E224 and promote the oligomerization of ALDOA, resulting in increased enzyme activity and enhanced aerobic glycolysis in LUAD cells. Additionally, we provide preliminary evidence of its potential clinical value by investigating the therapeutic effects of tRF-Val-CAC-024 antagomir-loaded lipid nanoparticles (LNPs) in cell-line-derived xenograft models. These results could enhance our understanding of the regulatory mechanisms of tRFs in LUAD and provide a potential therapeutic target.

Diet and Microbiome-Directed Therapy 2.0 for IBD.

Inflammatory bowel disease (IBD) comprises chronic and relapsing disorders of the gastrointestinal tract, characterized by dysregulated immune responses to the gut microbiome. The gut microbiome and diet are key environmental factors that influence the onset and progression of IBD and can be leveraged for treatment. In this review, we summarize the current evidence on the role of the gut microbiome and diet in IBD pathogenesis, and the potential of microbiome-directed therapies and dietary interventions to improve IBD outcomes. We discuss available data and the advantages and drawbacks of the different approaches to manipulate the gut microbiome, such as fecal microbiota transplantation, next-generation and conventional probiotics, and postbiotics. We also review the use of diet as a therapeutic tool in IBD, including the effects in induction and maintenance, special diets, and exclusive enteral nutrition. Finally, we highlight the challenges and opportunities for the translation of diet and microbiome interventions into clinical practice, such as the need for personalization, manufacturing and regulatory hurdles, and the specificity to take into account for clinical trial design.

Assembly of the Tn7 targeting complex by a regulated stepwise process.

The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements.

Mother-infant bonding: The role of postpartum depression, violence, and bonding established with one's own mother during childhood.

Mother-infant bonding is influenced by several risk and protective factors, and the literature has investigated the relationships between these factors independently. This study aimed to verify the interrelationships of some of these factors and how they influence mother-infant bonding in Brazil. In this study, 361 mothers participated, and the outcome variable of mother-infant bonding was assessed using the Postpartum Bonding Questionnaire (PBQ). Multivariate regression analysis was performed using a hierarchical model with three blocks structured according to the influence exerted on mother-infant bonding. The PBQ's factor scores were estimated and used in the subsequent analyses to decrease measurement error. The variable "violence experienced by mothers" was statistically significant for explaining the second block model but not significant for the third block. Network analysis was performed after multiple regression, showing that the violence experienced by mothers does not directly influence mother-infant bonding but rather is mediated by postpartum depression. This explains why violence is not significant in the hierarchical multiple regression when maternal depression is added to the model. This study's strengths lie in its utilization of PBQ factor scores and network analysis, enabling the estimation of conditional relationships among variables. This approach provides deeper insights into factors affecting mother-infant bonding.

Varios factores de riesgo y de protección ejercen influencia sobre la unión afectiva madre­infante; la literatura disponible ha investigado las relaciones entre estos factores de una manera independiente. Este estudio se propuso verificar las interrelaciones de algunos de estos factores y cómo ellos influyen en la unión afectiva madre­infante. Se consultó un total de 361 madres y el variable resultado de afectividad madre­infante se evaluó por medio del Cuestionario de Afectividad de Postparto (PBQ). Se llevaron a cabo análisis de regresión multivariados usando un modelo jerárquico con tres estructuras de bloques de acuerdo con la influencia ejercida sobre la unión afectiva madre­infante. Se estimaron y usaron los puntajes de factores del PBQ en los análisis subsecuentes para disminuir el error en la medida. La variable "violencia experimentada por las madres" fue estadísticamente significativa para explicar el segundo modelo de bloque, pero no significativa para el tercer bloque. Se llevó a cabo un análisis de interrelaciones después de la regresión múltiple, demostrando que la violencia experimentada por las madres no influye directamente la afectividad madre­infante, sino que la misma es mediada por la depresión posterior al parto. Esto explica por qué la violencia no es significativa en la jerárquica regresión múltiple cuando la depresión materna se le agrega al modelo. Entre los puntos fuertes de este estudio se incluye el uso de los puntajes de factores del PBQ y el análisis de interrelaciones, lo cual permitió que se estimaran las relaciones condicionales existente dentro del grupo de variables, aportando una mayor comprensión de algunos factores que interfieren en la unión afectiva madre­infante.

Diabetes and Abdominal Aortic Aneurysm: Is the Protective Effect on AAA Due to Antidiabetic Medications Alone, Due to the Disease Alone, or Both?

Diabetes is a metabolic disease that may result in multiple microvascular and macrovascular diseases. Interestingly, many studies have demonstrated the inverse relationship between diabetes and the development and expansion of abdominal aortic aneurysm (AAA). One hypothesis is that the aortic wall stiffness resulting from hyperglycemia and advanced glycation end products could delay the development and growth of AAA. Other studies have proposed that the concurrent use of antidiabetic medications which promote anti-inflammatory cytokines while hindering pro-inflammatory cytokines may potentially be the reason for this protective effect of diabetes on AAA. Contrastingly, the presence of diabetes has been found to have a negative effect on the outcome of AAA following its repair which may be due to elevated blood glucose negatively affecting the healing process. The current literature has also demonstrated the negative impact of the use of fluoroquinolones on AAA. This comprehensive review critically reviewed and summarized the role of diabetes, anti-diabetes medications and fluoroquinolones on AAA, and on the effect of diabetes and certain anti-diabetes medications on outcomes following its repair.

Role of Yme1 in mitochondrial protein homeostasis: from regulation of protein import, OXPHOS function to lipid synthesis and mitochondrial dynamics.

Mitochondria are essential organelles of eukaryotic cells and thus mitochondrial proteome is under constant quality control and remodelling. Yme1 is a multi-functional protein and subunit of the homo-hexametric complex i-AAA proteinase. Yme1 plays vital roles in the regulation of mitochondrial protein homeostasis and mitochondrial plasticity, ranging from substrate degradation to the regulation of protein functions involved in mitochondrial protein biosynthesis, energy production, mitochondrial dynamics, and lipid biosynthesis and signalling. In this mini review, we focus on discussing the current understanding of the roles of Yme1 in mitochondrial protein import via TIM22 and TIM23 pathways, oxidative phosphorylation complex function, as well as mitochondrial lipid biosynthesis and signalling, as well as a brief discussion of the role of Yme1 in modulating mitochondrial dynamics.

Allosteric activation of VCP, an AAA unfoldase, by small molecule mimicry.

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme function, activator binding must be permissive to different conformational states needed for mechanochemistry. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, an AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP ATPase Activator 1 (VAA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~threefold. Our cryo-EM studies resulted in structures (ranging from ~2.9 to 3.7 Å-resolution) of VCP in apo and ADP-bound states and revealed that VAA1 binds an allosteric pocket near the C-terminus in both states. Engineered mutations in the VAA1-binding site confer resistance to VAA1, and furthermore, modulate VCP activity. Mutation of a phenylalanine residue in the VCP C-terminal tail that can occupy the VAA1 binding site also stimulates ATPase activity, suggesting that VAA1 acts by mimicking this interaction. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry.

Endovascular Treatment of Abdominal Aortic Aneurysm: Impact of Diabetes on Endoleaks and Reintervention.

Background: Diabetes has a protective effect on abdominal aortic aneurysms (AAAs); however, there are contrasting reports on the impact of diabetes on endovascular aortic repair (EVAR) outcomes, endoleaks (ELs) being the major negative outcome. The present study characterizes ELs and their outcomes in AAA patients, diabetic or not. Methods: This single-center, retrospective, comparative study was carried out on 324 AAA patients who underwent elective EVARs between 2007 and 2016 at the University Hospital of Liège (Belgium). The primary endpoint was the incidence and effect of ELs on the evolution of the aneurysmal sac; the secondary endpoints were surgical reintervention and mortality rate. Diabetic and non-diabetic patients were compared with respect to various risk factors by logistic regression, while a Cox regression was used to analyze survival. Results: In AAA patients meeting the inclusion criteria (n = 248), 23% were diabetic. EL incidence was comparable (p = 0.74) in diabetic (38.7%) vs. non-diabetic (43.9%) patients. EL risk factors were age (HR = 1.04, p = 0.014) and fibrate intake (HR = 3.12, p = 0.043). A significant association was observed between ELs and aneurysm sac enlargement (p < 0.001), regardless of group (p = 0.46). Aneurysm sac regression per month for non-diabetic patients was -0.24 ± 0.013, while for diabetics it was -0.18 ± 0.027 (p = 0.059). Dyslipidemia (HR = 3.01, p = 0.0060) and sulfonylureas (HR = 8.43, p = 0.043) were associated with shorter EL duration, while diabetes (HR = 0.080, p = 0.038) and beta blockers (HR = 0.46, p = 0.036) were associated with longer EL duration. The likelihood of reoperation decreased with more recent surgery (OR = 0.90, p = 0.040), regardless of diabetic status. All-cause mortality was higher for the non-diabetic group (45.5% vs. 26.3%, p = 0.0096). Conclusions: Endoleak occurrence is a known risk factor for sac expansion. In diabetic patients, endoleaks lasted longer, and regression of the aneurysm sac tended to be slower. The number and type of reintervention was not related to the diabetic status of AAA patients, but overall survival was higher in patients with diabetes.

Advancements in unravelling the fundamental function of the ATAD3 protein in multicellular organisms.

ATPase family AAA domain containing protein 3, commonly known as ATAD3 is a versatile mitochondrial protein that is involved in a large number of pathways. ATAD3 is a transmembrane protein that spans both the inner mitochondrial membrane and outer mitochondrial membrane. It, therefore, functions as a connecting link between the mitochondrial lumen and endoplasmic reticulum facilitating their cross-talk. ATAD3 contains an N-terminal domain which is amphipathic in nature and is inserted into the membranous space of the mitochondria, while the C-terminal domain is present towards the lumen of the mitochondria and contains the ATPase domain. ATAD3 is known to be involved in mitochondrial biogenesis, cholesterol transport, hormone synthesis, apoptosis and several other pathways. It has also been implicated to be involved in cancer and many neurological disorders making it an interesting target for extensive studies. This review aims to provide an updated comprehensive account of the role of ATAD3 in the mitochondria especially in lipid transport, mitochondrial-endoplasmic reticulum interactions, cancer and inhibition of mitophagy.

ACR Appropriateness Criteria® Screening for Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a significant vascular disease found in 4% to 8% of the screening population. If ruptured, its mortality rate is between 75% and 90%, and it accounts for up to 5% of sudden deaths in the United States. Therefore, screening of AAA while asymptomatic has been a crucial portion of preventive health care worldwide. Ultrasound of the abdominal aorta is the primary imaging modality for screening of AAA recommended for asymptomatic adults regardless of their family history or smoking history. Alternatively, duplex ultrasound and CT abdomen and pelvis without contrast may be appropriate for screening. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision process support the systematic analysis of the medical literature from peer reviewed journals. Established methodology principles such as Grading of Recommendations Assessment, Development, and Evaluation or GRADE are adapted to evaluate the evidence. The RAND/UCLA Appropriateness Method User Manual provides the methodology to determine the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where peer reviewed literature is lacking or equivocal, experts may be the primary evidentiary source available to formulate a recommendation.

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 those involved in heme biosynthesis, to accommodate microaerobic conditions. The FixLJ inhibitor FixT is also induced under low oxygen conditions and is degraded by the Lon protease when the oxygen levels are sufficient, which together provides negative feedback proposed to adjust FixLJ signaling thresholds during changing conditions. Here, we address whether degradation of FixT by the Lon protease contributes to phenotypic defects associated with loss of Lon. We find that ∆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, along with heme biosynthesis, there is diminished expression of many FixL-activated genes in ∆lon. However, stabilization of FixT in ∆lon strains does not contribute to restoring 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 growth in standard aerobic conditions. 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. IMPORTANCE: The 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 in part 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.