Lon Protease Research Articles

Mitochondrial protein homeostasis depends mainly on the efficient import and folding of nuclear-encoded proteins, and defects in this process can lead to proteotoxicity, which is harmful to the cell. Mitochondrial chaperones and proteases are essential defense mechanisms that ensure dysfunctional proteins' proper concentration, folding, and degradation. Lon protease 1 (Pim1 in yeast) is the mitochondrial matrix protease known to prevent protein aggregation by degrading unfolded proteins. Here, we show that two essential components of ATP-dependent presequence translocase and associated motor (PAM complex), Pam18 and Pam16, are specifically targeted for degradation by the proteolytically active Lon/Pim1, both in vitro and in vivo. Furthermore, overexpression of Pam18 and Pam16 exacerbates the growth defect of the delta pim1 strain. Hence, our study reveals, for the first time, that components involved in protein import are substrates of Pim1, which could have potential implications for regulating mitochondrial protein import and proteostasis.

Type III CRISPR systems typically generate cyclic oligoadenylate (cOA) second messengers such as cyclic tetra-adenylate (cA4) on detection of foreign RNA. These activate ancillary effector proteins which elicit a diverse range of immune responses. The CalpLTS system elicits a transcriptional response to infection when CalpL binds cA4 in its SAVED (SMODS associated and fused to various effectors domain) sensor domain, resulting in filament formation and activation of the Lon protease domain, which cleaves the anti-Sigma factor CalpT, releasing the CalpS Sigma factor for transcriptional remodelling. Here, we show that thermophilic viruses have appropriated the SAVED domain of CalpL as an anti-CRISPR, AcrIII-2, which they use to degrade cA4. AcrIII-2 dimers sandwich cA4, degrading it in a shared active site to short linear products, using a mechanism highly reminiscent of CalpL. This results in inhibition of a range of cA4 activated effectors in vitro. This is the first example of a virally encoded SAVED domain with ring nuclease activity, highlighting the complex interplay between viruses and cellular defences.

Large scale laboratory evolution uncovers clinically relevant collateral antibiotic sensitivity.

Mitochondrial and peroxisomal Lon proteases are functionally divergent in environmental adaptation and pathobiology of the entomopathogenic fungus Beauveria bassiana.

Lon protease 1 (LONP1), an adenosine triphosphate (ATP)-dependent protease encoded by nuclear DNA that is highly conserved, maintains the mitochondrial protein balance and regulates adaptive responses to cellular stress. LONP1 dysfunction ultimately results in various forms of cellular and tissue damage. The function of LONP1 in hepatocellular carcinoma (HCC) and how it affects HCC growth were investigated in this work. The RNA and protein expression levels of LONP1 were determined in paired HCC and adjacent tissue samples through real-time quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) staining. The correlation between LONP1 expression and clinical features was evaluated via statistical analysis. Overexpression (OE) and knockdown (KD) experiments, small RNA interference, Cell Counting Kit-8 (CCK8) and wound-healing assays, and animal experiments were employed to assess the potential mechanism by which LONP1 promotes the proliferation and migration of HCC cells both in vitro and in vivo. In HCC samples, LONP1 expression was higher than in the equivalent surrounding tissues. Compared to patients with low LONP1 expression, individuals with high LONP1 expression had shorter disease-free survival and overall survival periods. Functionally, LONP1 facilitated the proliferation and migration of HCC cells, whereas LONP1 knockdown mitigated the growth of HCC subcutaneous tumors. Mechanistically, LONP1 affects the processes of ferroptosis and cuproptosis processes by regulating the stability of aconitase 2 (ACO2). Histological analysis showed that the expression of LONP1 in liver cancer tissues was significantly upregulated, accompanied by a decrease in the level of ACO2 protein (Hematoxylin-Eosin (HE) staining and IHC verification). Mitochondrial function experiments indicated that overexpression of LONP1 led to a significant decrease in mitochondrial membrane potential suggesting mitochondrial dysfunction and reduced susceptibility to ferroptosis. Our results suggest that LONP1 promotes HCC proliferation and migration by inhibiting ferroptosis and cuproptosis through the degradation of ACO2. Therefore, targeting LONP1 might be an effective therapeutic strategy to inhibit HCC growth.

Although accumulating research has indicated the link between mitochondrial function and osteomyelitis, the nature of this relationship has not yet been fully clarified, therefore, this present 2-sample Mendelian randomization (MR) study was designed to identify the causal link between mitochondrial function and osteomyelitis. In this study, inverse variance weighting (IVW), MR-Egger, weighted median, simple mode, and weighted mode analyses were utilized to assess this causal relationship and possible targets for osteomyelitis treatment. On the basis of the IVW results, Lon protease homolog increased the risk of osteomyelitis by 14.08% (OR=1.1408, P =0.0061). Although ribosomal protein L34, hydroxymethylglutaryl-CoA synthase, and pyruvate carboxylase can reduce the incidence of osteomyelitis by 14.78% (OR=0.8522, P =0.0236), 13.01% (OR=0.8699, P =0.0170), and 11.38% (OR=0.8862, P =0.0478), respectively. This study indicates a causal association between mitochondrial function and osteomyelitis, and such insights may offer novel insights into exploring strategies for prevention or curing of osteomyelitis.

The ATP-dependent cytoplasmic protease Lon has critical functions in protein quality control and cellular regulation in organisms across the three domains of life. In the opportunistic pathogen Pseudomonas aeruginosa, lon loss-of-function mutants exhibit multiple phenotypic defects in motility, virulence, antibiotic tolerance and biofilm formation. However, only a couple of native substrate proteins of Lon are described in P. aeruginosa until now and most of the phenotypes associated with Lon remain unexplained. Here, we searched for novel Lon substrates in P. aeruginosa by analyzing proteome-wide changes in protein levels and stabilities following lon overexpression. Our search yielded a large number of putative Lon substrates with diverse cellular functions, including metabolic enzymes, stress proteins and a significant fraction of motility-related proteins. In vitro degradation assays confirmed the metabolic protein SpeH, the heat shock protein IbpA as well as seven proteins involved in flagella- and type IV pilus-mediated motility as novel substrates of Lon. The new motility-associated substrates include both key regulators of motility (FliA, RpoN, AmrZ) as well as structural flagellar components (FliG, FliS and FlgE). Further, by isolating suppressor mutations bypassing the motility defect of lon- cells, we reveal that Lon-dependent degradation of the specific substrate SulA, a cell division inhibitor, is crucial for ensuring proper cell division and motility under optimal conditions. In sum, our work highlights Lon's regulatory role in degrading functional proteins involved in critical cellular processes and contributes to a better molecular understanding of the pathways underlying Pseudomonas pathogenicity.

The ATP-dependent cytoplasmic protease Lon has critical functions in protein quality control and cellular regulation in organisms across the three domains of life. In the opportunistic pathogen Pseudomonas aeruginosa, lon loss-of-function mutants exhibit multiple phenotypic defects in motility, virulence, antibiotic tolerance and biofilm formation. However, only a couple of native substrate proteins of Lon are described in P. aeruginosa until now and most of the phenotypes associated with Lon remain unexplained. Here, we searched for novel Lon substrates in P. aeruginosa by analyzing proteome-wide changes in protein levels and stabilities following lon overexpression. Our search yielded a large number of putative Lon substrates with diverse cellular functions, including metabolic enzymes, stress proteins and a significant fraction of motility-related proteins. In vitro degradation assays confirmed the metabolic protein SpeH, the heat shock protein IbpA as well as seven proteins involved in flagella- and type IV pilus-mediated motility as novel substrates of Lon. The new motility-associated substrates include both key regulators of motility (FliA, RpoN, AmrZ) as well as structural flagellar components (FliG, FliS and FlgE). Further, by isolating suppressor mutations bypassing the motility defect of lon- cells, we reveal that Lon-dependent degradation of the specific substrate SulA, a cell division inhibitor, is crucial for ensuring proper cell division and motility under optimal conditions. In sum, our work highlights Lon’s regulatory role in degrading functional proteins involved in critical cellular processes and contributes to a better molecular understanding of the pathways underlying Pseudomonas pathogenicity.

[This retracts the article DOI: 10.3389/fcimb.2016.00011.].

Accumulation of reactive oxygen species (ROS) induces oxidative stress, leading to substantial damage to cellular macromolecules, necessitating efficient protein quality control mechanisms. The Lon protease, a highly conserved ATP-dependent protease, is thought to play a central role in mitigating oxidative stress by targeting damaged and misfolded proteins for degradation. This review examines the role of Lon in oxidative stress responses, including its role in degrading oxidized proteins, regulating antioxidant pathways, and modulating heme and Fe-S cluster homeostasis. We highlight cases of substrate recognition through structural changes and describe situations where Lon activity is further regulated by redox conditions. By synthesizing studies across a range of organisms, we find that despite the clear importance of Lon for oxidative stress tolerance, universal rules for Lon degradation of damaged proteins during this response remain unclear.

Nanopollutants (nZnO) amplify hypoxia-induced cellular stress in a keystone marine bivalve, Mytilus edulis.

Pulmonary hypertension (PH) is a chronic and life-threatening disease characterized by pulmonary vascular remodeling (PVR), which involves the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). These cells exhibit metabolic characteristics akin to cancer cells, particularly in their shift toward glycolysis. The Lon protease 1 (LONP1) has been shown to promote glycolytic reprogramming of tumor cells, conferring a malignant proliferative phenotype. However, the precise role of LONP1 in PH remains unclear. In the present study, Su5416/hypoxia-induced and monocrotaline (MCT)-induced PH rodent models and platelet-derived growth factor BB (PDGF-BB)-induced PASMCs were used to investigate the role and mechanism of LONP1 in PH. The results revealed an up-regulation of LONP1 expression in lung tissues from two PH rodent models, as well as in PDGF-BB-induced PASMCs. In vivo knockdown of LONP1 significantly alleviated PASMC mitochondrial dysfunction, reduced glycolytic enzyme expression, and decreased lactate accumulation, thereby mitigating PVR. Additionally, in vitro experiments demonstrated that knockdown or inhibition of LONP1 attenuated glycolytic reprogramming, proliferation, and migration of PASMCs, whereas overexpression of LONP1 had converse effects. Mechanistic studies confirmed that mitochondrial pyruvate carrier 1 (MPC1) was a direct substrate for LONP1-mediated degradation. Functional experiments with MPC1 knockdown and overexpression further elucidated its role in the proliferation and migration of PASMCs. Rescue experiments indicated that MPC1 knockdown abrogated the suppressive effects of LONP1 knockdown on glycolytic reprogramming, proliferation, and migration in PASMCs. Therapeutically, knockdown or pharmacological inhibition of LONP1 significantly reversed MCT-induced PH in rats. Thus, targeting LONP1 may represent a promising therapeutic strategy for PH.

Haemophilus ducreyi causes cutaneous ulcers in children who live in yaws-endemic countries and the genital ulcer disease chancroid. In the human host, H. ducreyi resides in an abscess and may need to resist both heat and oxidative stress, which result in aggregation and misfolding of bacterial proteins. In Escherichia coli, the hslUV (clpYQ) operon encodes a proteasome-like complex that degrades misfolded proteins and is upregulated during heat shock. In previous studies, we showed that hslUV transcripts are upregulated in experimental lesions caused by H. ducreyi in human volunteers, suggesting that HslUV may help H. ducreyi adapt to the abscess environment. Here, we constructed an unmarked hslUV operon deletion mutant, 35000HPΔhslUV, in H. ducreyi. Whole-genome sequencing showed that compared to its parent (35000HP), the mutant contained only the deletion of interest. Six volunteers were inoculated at three sites on skin overlying the deltoid on opposite arms with 35000HP and 35000HPΔhslUV. Within 24 h, papules formed at 88.9% (95% CI [69%, 100%]) at both parent and mutant-inoculated sites (P = 1.0). Pustules formed at 44.4% (95% CI [25.6%, 64.3%]) at parent-inoculated sites and 33.3% (95% CI [2.5%, 64.1%]) at mutant-inoculated sites (P = 0.17). Thus, the proteosome-like complex encoded by hslUV was dispensable for H. ducreyi virulence in humans. In the absence of hslUV, H. ducreyi likely utilizes other systems such as the Lon protease, ClpXP, and ClpB/DnaK to combat protein aggregation and misfolding, underscoring the importance of the functional redundancy of such systems in gram-negative pathogens.

The ability of bacteria to commit to surface colonization and biofilm formation is a highly regulated process. In this study, we characterized the activity and structure of SadB, initially identified as a key regulator in the transition from reversible to irreversible surface attachment. Our results show that SadB acts as an adaptor protein that tightly regulates the master regulator AmrZ at the post-translational level. SadB directly binds to the C-terminal domain of AmrZ, leading to its rapid degradation, primarily by the Lon protease. Structural analysis suggests that SadB does not directly interact with small molecules upon signal transduction, differing from previous findings in Pseudomonas fluorescens. Instead, the SadB structure supports its role in mediating protein-protein interactions, establishing it as a major checkpoint for biofilm commitment.

Abstract 2018 Lon Protease: The Jaws of Life

BackgroundMitochondria play a multifaceted role in tumorigenesis, influencing energy metabolism, redox balance, and apoptosis. However, whether mitochondrial traits causally affect cancer risk remains unclear. This study aimed to evaluate the potential causal effects of 82 mitochondrial-related exposures on six major cancers—hepatic, colorectal, lung, esophageal, thyroid, and breast—using Mendelian randomization (MR).MethodsTwo-sample MR analysis was performed using the inverse variance weighted (IVW) method, with MR-Egger regression and weighted median as complementary approaches. Sensitivity analyses (Cochran’s Q test, MR-Egger intercept, leave-one-out) and the Steiger test were applied to assess heterogeneity, pleiotropy, and causal directionality.ResultsWe observed a negative correlation between “39S ribosomal protein L34, mitochondrial”, and others, with hepatic cancer, while “[Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 2, mitochondrial”, and others exhibited a positive correlation with hepatic cancer. “Phenylalanine-tRNA ligase, mitochondrial”, and others demonstrated a negative association with colorectal cancer, whereas “Methylmalonyl-CoA epimerase, mitochondrial”, and others exhibited a positive correlation with colorectal cancer. “Succinate dehydrogenase assembly factor 2, mitochondrial” exhibited a negative correlation with lung cancer, while “Superoxide dismutase [Mn], mitochondrial levels” showed a positive correlation with lung cancer. “Lon protease homolog, mitochondrial” demonstrated a positive correlation with esophageal cancer. “Iron-sulfur cluster assembly enzyme ISCU, mitochondrial”, and others exhibited a negative correlation with thyroid cancer, while “Diablo homolog, mitochondrial”, and others showed a positive correlation with thyroid cancer. “ADP-ribose pyrophosphatase, mitochondrial”, and others exhibited a negative correlation with breast cancer, while “39S ribosomal protein L34, mitochondrial”, and others showed a positive correlation with breast cancer.ConclusionsThis study provides MR-based evidence that specific mitochondrial-related traits have causal effects on the risk of several common cancers. Notably, certain single-nucleotide polymorphisms (SNPs) acted as instrumental variables across multiple cancer types through shared mitochondrial mechanisms, such as oxidative stress regulation and metabolic reprogramming. These findings highlight mitochondria as cross-cutting contributors to cancer susceptibility and suggest potential avenues for mitochondrial-targeted prevention and therapy. The identification of pleiotropic genetic variants also offers insights for developing shared biomarkers and therapeutic targets across malignancies.

The Lon protease homolog 1 (LONP1) is an ATP-dependent mitochondrial protease essential for maintaining proteostasis, bioenergetics, and cellular homeostasis. LONP1 plays a pivotal role in protein quality control, mitochondrial DNA maintenance, and oxidative phosphorylation system (OXPHOS) regulation, particularly under stress conditions. Dysregulation of LONP1 has been implicated in various pathologies, including cancer, metabolic disorders, and reproductive diseases, positioning it as a promising pharmacological target. This review examines compounds that modulate LONP1 activity, categorizing them into inhibitors and activators. Inhibitors such as CDDO and its derivatives selectively target LONP1, impairing mitochondrial proteolysis, inducing protein aggregation, and promoting apoptosis, particularly in cancer cells. Compounds like Obtusilactone A and proteasome inhibitors (e.g., MG262) demonstrate potent cytotoxicity, further expanding the therapeutic landscape. Conversely, LONP1 activators, including Artemisinin derivatives and 84-B10, restore mitochondrial function and protect against conditions such as polycystic ovary syndrome (PCOS) and acute kidney injury (AKI). Future research should focus on improving the specificity, bioavailability, and pharmacokinetics of these modulators. Advances in structural biology and drug discovery will enable the development of novel LONP1-targeted therapies, addressing diseases driven by mitochondrial dysfunction and proteostasis imbalance.

Substrate recognition and cleavage-site preferences of Lon protease.

BackgroundCerebral ischemia-reperfusion injury (CIRI) leads to cognitive dysfunction, neuronal death, and inflammation. Understanding the molecular mechanisms underlying CIRI is crucial for developing effective therapeutic strategies.ObjectiveThis study aims to investigate the...

Lon is an AAA+ serine protease that plays a crucial role in protein quality control in both eukaryotes and prokaryotes. Protein degradation is tightly regulated and clearing the right protein at a specific cell cycle stage is crucial. Dysregulation of the amount of Lon in the cellular milieu leads to mitochondrial encephalomyopathy, cancer, lactic acidosis, Friedrich ataxia, and CODAS syndrome. Despite the ubiquity of Lon protease in the cell, the degradation of proteins is effectively controlled, and the regulation of Lon protease activity remains a long-standing question. Lon is observed to be functionally active as a hexamer; however, previous studies have suggested the existence of a dodecameric state that acts on peptide substrates rather than proteins. Thus, we hypothesize that dodecamer formation might be a regulatory state of the protein and our study aims to elucidate its structural basis. Here we report the formation of higher-order oligomers in solution and further identify different states of the E. coli Lon (EcLon) oligomer. Utilizing Mass Photometry, we demonstrate the formation of various higher-order oligomeric states, including the dodecamer (12mer), undecamer (11mer), and decamer (10mer). Furthermore, to stabilize these states for structural elucidation, we have developed seven nanobodies that bind to EcLon. Using CryoEM we determined high-resolution structures of the 12mer, 11mer, and 10mer oligomeric states formed by 6+6mer, 6+5mer, and 5+5mer, respectively. The interface between these oligomeric arrangements is formed by the intricate interaction of the N-terminal domain (NTD) of Lon. Interestingly, the arrangement of the NTD interface differs distinctly among the three configurations. While the 12mer and 11mer states were identified in the apo form, the NTD-binding nanobody enriched the 10mer state in our CryoEM studies. The 12mer forms a symmetrical arrangement of the two hexamers with a contiguous channel connecting them, whereas the 11mer and 10mer states have the two oligomers arranged at an angle, with the NTD blocking the formation of a contiguous channel between them. Based on our CryoEM structures we hypothesize that the different states achieved by Lon may regulate its activity and the number of active proteins in the system. Furthermore, we anticipate trapping different states of EcLon with other nanobodies and conducting experiments to assign functional relevance to these states.