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Relatively little is known about the small subset of peroxisomal proteins with predicted protease activity. Here, we report that the peroxisomal LON2 (At5g47040) protease facilitates matrix protein import into Arabidopsis (Arabidopsis thaliana) peroxisomes. We identified T-DNA insertion alleles disrupted in five of the nine confirmed or predicted peroxisomal proteases and found only two—lon2 and deg15, a mutant defective in the previously described PTS2-processing protease (DEG15/At1g28320)—with phenotypes suggestive of peroxisome metabolism defects. Both lon2 and deg15 mutants were mildly resistant to the inhibitory effects of indole-3-butyric acid (IBA) on root elongation, but only lon2 mutants were resistant to the stimulatory effects of IBA on lateral root production or displayed Suc dependence during seedling growth. lon2 mutants displayed defects in removing the type 2 peroxisome targeting signal (PTS2) from peroxisomal malate dehydrogenase and reduced accumulation of 3-ketoacyl-CoA thiolase, another PTS2-containing protein; both defects were not apparent upon germination but appeared in 5- to 8-d-old seedlings. In lon2 cotyledon cells, matrix proteins were localized to peroxisomes in 4-d-old seedlings but mislocalized to the cytosol in 8-d-old seedlings. Moreover, a PTS2-GFP reporter sorted to peroxisomes in lon2 root tip cells but was largely cytosolic in more mature root cells. Our results indicate that LON2 is needed for sustained matrix protein import into peroxisomes. The delayed onset of matrix protein sorting defects may account for the relatively weak Suc dependence following germination, moderate IBA-resistant primary root elongation, and severe defects in IBA-induced lateral root formation observed in lon2 mutants.Peroxisomes are single-membrane-bound organelles found in most eukaryotes. Peroxin (PEX) proteins are necessary for various aspects of peroxisome biogenesis, including matrix protein import (for review, see Distel et al., 1996; Schrader and Fahimi, 2008). Most matrix proteins are imported into peroxisomes from the cytosol using one of two targeting signals, a C-terminal type 1 peroxisome-targeting signal (PTS1) or a cleavable N-terminal type 2 peroxisome-targeting signal (PTS2) (Reumann, 2004). PTS1- and PTS2-containing proteins are bound in the cytosol by soluble matrix protein receptors, escorted to the peroxisome membrane docking complex, and translocated into the peroxisome matrix (for review, see Platta and Erdmann, 2007). Once in the peroxisome, many matrix proteins participate in metabolic pathways, such as β-oxidation, hydrogen peroxide decomposition, and photorespiration (for review, see Gabaldon et al., 2006; Poirier et al., 2006).In addition to metabolic enzymes, several proteases are found in the peroxisome matrix. Only one protease, DEG15/Tysnd1, has a well-defined role in peroxisome biology. The rat Tysnd1 protease removes the targeting signal after PTS2-containing proteins enter the peroxisome and also processes certain PTS1-containing β-oxidation enzymes (Kurochkin et al., 2007). Similarly, the Arabidopsis (Arabidopsis thaliana) Tysnd1 homolog DEG15 (At1g28320) is a peroxisomal Ser protease that removes PTS2 targeting signals (Helm et al., 2007; Schuhmann et al., 2008).In contrast with DEG15, little is known about the other eight Arabidopsis proteins that are annotated as proteases in the AraPerox database of putative peroxisomal proteins (Reumann et al., 2004; Carter et al., 2004; Shimaoka et al., 2004), which, in combination with the minor PTS found in both of these predicted proteases (Reumann, 2004), suggests that these enzymes may not be peroxisomal. Along with DEG15, only two of the predicted peroxisomal proteases, an M16 metalloprotease (At2g41790), which we have named PXM16 for peroxisomal M16 protease, and a Lon-related protease (At5g47040/LON2; Ostersetzer et al., 2007), are found in the proteome of peroxisomes purified from Arabidopsis suspension cells (Eubel et al., 2008). DEG15 and LON2 also have been validated as peroxisomally targeted using GFP fusions (Ostersetzer et al., 2007; Schuhmann et al., 2008).
Open in a separate windowaMajor PTS1 (Reumann, 2004).bMinor PTS1 (Reumann, 2004).cValidated PTS1 (Reumann et al., 2007).dMinor PTS2 (Reumann, 2004).PXM16 is the only one of the nine Arabidopsis M16 (pitrilysin family) metalloproteases (García-Lorenzo et al., 2006; Rawlings et al., 2008) containing a predicted PTS. M16 subfamilies B and C contain the plastid and mitochondrial processing peptidases (for review, see Schaller, 2004), whereas PXM16 belongs to M16 subfamily A, which includes insulin-degrading peptidases (Schaller, 2004). A tomato (Solanum lycopersicum) M16 subfamily A protease similar to insulin-degrading enzymes with a putative PTS1 was identified in a screen for proteases that cleave the wound response peptide hormone systemin (Strassner et al., 2002), but the role of Arabidopsis PXM16 is unknown.Arabidopsis LON2 is a typical Lon protease with three conserved domains: an N-terminal domain, a central ATPase domain in the AAA family, and a C-terminal protease domain with a Ser-Lys catalytic dyad (Fig. 1A; Lee and Suzuki, 2008). Lon proteases are found in prokaryotes and in some eukaryotic organelles (Fig. 1C) and participate in protein quality control by cleaving unfolded proteins and can regulate metabolism by controlling levels of enzymes from many pathways, including cell cycle, metabolism, and stress responses (for review, see Tsilibaris et al., 2006). Four Lon homologs are encoded in the Arabidopsis genome; isoforms have been identified in mitochondria, plastids, and peroxisomes (Ostersetzer et al., 2007; Eubel et al., 2008; Rawlings et al., 2008). Mitochondrial Lon protesases are found in a variety of eukaryotes (Fig. 1A) and function both as ATP-dependent proteases and as chaperones promoting protein complex assemblies (Lee and Suzuki, 2008). LON2 is the only Arabidopsis Lon isoform with a canonical C-terminal PTS1 (SKL-COOH; Ostersetzer et al., 2007) or found in the peroxisome proteome (Eubel et al., 2008; Reumann et al., 2009). Functional studies have been conducted with peroxisomal Lon isoforms found in the proteome of peroxisomes purified from rat hepatic cells (pLon; Kikuchi et al., 2004) and the methylotrophic yeast Hansenula polymorpha (Pln; Aksam et al., 2007). Rat pLon interacts with β-oxidation enzymes, and a cell line expressing a dominant negative pLon variant has decreased β-oxidation activity, displays defects in the activation processing of PTS1-containing acyl-CoA oxidase, and missorts catalase to the cytosol (Omi et al., 2008). H. polymorpha Pln is necessary for degradation of a misfolded, peroxisome-targeted version of dihydrofolate reductase and for degradation of in vitro-synthesized alcohol oxidase in peroxisomal matrix extracts, but does not contribute to degradation of peroxisomally targeted GFP (Aksam et al., 2007).Open in a separate windowFigure 1.Diagram of LON2 protein domains, gene models for LON2, PXM16, DEG15, PED1, PEX5, and PEX6, and phylogenetic relationships of LON family members. A, Organization of the 888-amino acid LON2 protein. Locations of the N-terminal domain conserved among Lon proteins, predicted ATP-binding Walker A and B domains (black circles), active site Ser (S) and Lys (K) residues (asterisks), and the C-terminal Ser-Lys-Leu (SKL) peroxisomal targeting signal (PTS1) are shown (Lee and Suzuki, 2008). B, Gene models for LON2, PXM16, DEG15, PED1, PEX5, and PEX6 and locations of T-DNA insertions (triangles) or missense alleles (arrows) used in this study. Exons are depicted by black boxes, introns by black lines, and untranslated regions by gray lines. C, Phylogenetic relationships among LON homologs. Sequences were aligned using MegAlign (DNAStar) and the ClustalW method. The PAUP 4.0b10 program (Swofford, 2001) was used to generate an unrooted phylogram from a trimmed alignment corresponding to Arabidopsis LON2 residues 400 to 888 (from the beginning of the ATPase domain to the end of the protein). The bootstrap method was performed for 500 replicates with distance as the optimality criterion. Bootstrap values are indicated at the nodes. Predicted peroxisomal proteins have C-terminal PTS1 signals in parentheses and are in light-gray ovals. Proteins in the darker gray oval have N-terminal extensions and include mitochondrial and chloroplastic proteins. Sequence identifiers are listed in Supplemental Table S2.In this work, we examined the roles of several putative peroxisomal proteases in Arabidopsis. We found that lon2 mutants displayed peroxisome-deficient phenotypes, including resistance to the protoauxin indole-3-butyric acid (IBA) and age-dependent defects in peroxisomal import of PTS1- and PTS2-targeted matrix proteins. Our results indicate that LON2 contributes to matrix protein import into Arabidopsis peroxisomes. 相似文献
Table I.
Putative Arabidopsis proteases predicted or demonstrated to be peroxisomalAGI Identifier | Alias | Protein Class | T-DNA Insertion Alleles | PTS | Localization Evidence | Localization References |
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At1g28320 | DEG15 | PTS2-processing protease | SALK_007184 (deg15-1) | SKL>a | GFP | Reumann et al., 2004; Helm et al., 2007; Eubel et al., 2008; Schuhmann et al., 2008) |
Proteomics | ||||||
Bioinformatics | ||||||
At2g41790 | PXM16 | Peptidase M16 family protein | SALK_019128 (pxm16-1) | PKL>b | Proteomics | Reumann et al., 2004, 2009; Eubel et al., 2008) |
SALK_023917 (pxm16-2) | Bioinformatics | |||||
At5g47040 | LON2 | Lon protease homolog | SALK_128438 (lon2-1) | SKL>a | GFP | Reumann et al., 2004, 2009; Ostersetzer et al., 2007; Eubel et al., 2008) |
SALK_043857 (lon2-2) | Proteomics | |||||
Bioinformatics | ||||||
At2g18080 | Ser-type peptidase | SALK_020628 | SSI>c | Bioinformatics | (Reumann et al., 2004) | |
SALK_102239 | ||||||
At2g35615 | Aspartyl protease | SALK_090795 | ANL>b | Bioinformatics | (Reumann et al., 2004) | |
SALK_036333 | ||||||
At3g57810 | Ovarian tumor-like Cys protease | SKL>a | Bioinformatics | (Reumann et al., 2004) | ||
At4g14570 | Acylaminoacyl-peptidase protein | CKL>b | Bioinformatics (peroxisome) | (Reumann et al., 2004; Shimaoka et al., 2004) | ||
Proteomics (vacuole) | ||||||
At4g20310 | Peptidase M50 family protein | RMx5HLd | Bioinformatics | (Reumann et al., 2004) | ||
At4g36195 | Ser carboxypeptidase S28 family | SSM>b | Bioinformatics (peroxisome) | (Carter et al., 2004; Reumann et al., 2004) | ||
Proteomics (vacuole) |
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Sabine Drevet Bertrand Favier Emmanuel Brun Gaëtan Gavazzi Bernard Lardy 《Comparative medicine》2022,72(1):3
Osteoarthritis (OA) is a multidimensional health problem and a common chronic disease. It has a substantial impact on patient quality of life and is a common cause of pain and mobility issues in older adults. The functional limitations, lack of curative treatments, and cost to society all demonstrate the need for translational and clinical research. The use of OA models in mice is important for achieving a better understanding of the disease. Models with clinical relevance are needed to achieve 2 main goals: to assess the impact of the OA disease (pain and function) and to study the efficacy of potential treatments. However, few OA models include practical strategies for functional assessment of the mice. OA signs in mice incorporate complex interrelations between pain and dysfunction. The current review provides a comprehensive compilation of mouse models of OA and animal evaluations that include static and dynamic clinical assessment of the mice, merging evaluation of pain and function by using automatic and noninvasive techniques. These new techniques allow simultaneous recording of spontaneous activity from thousands of home cages and also monitor environment conditions. Technologies such as videography and computational approaches can also be used to improve pain assessment in rodents but these new tools must first be validated experimentally. An example of a new tool is the digital ventilated cage, which is an automated home-cage monitor that records spontaneous activity in the cages.Osteoarthritis (OA) is a multidimensional health problem and a common chronic disease.36 Functional limitations, the absence of curative treatments, and the considerable cost to society result in a substantial impact on quality of life.76 Historically, OA has been described as whole joint and whole peri-articular diseases and as a systemic comorbidity.9,111 OA consists of a disruption of articular joint cartilage homeostasis leading to a catabolic pathway characterized by chondrocyte degeneration and destruction of the extracellular matrix (ECM). Low-grade chronic systemic inflammation is also actively involved in the process.42,92 In clinical practice, mechanical pain, often accompanied by a functional decline, is the main reason for consultations. Recommendations to patients provide guidance for OA management.22, 33,49,86 Evidence-based consensus has led to a variety of pharmacologic and nonpharmacologic modalities that are intended to guide health care providers in managing symptomatic patients. Animal-based research is of tremendous importance for the study of early diagnosis and treatment, which are crucial to prevent the disease progression and provide better care to patients.The purpose of animal-based OA research is 2-fold: to assess the impact of the OA disease (pain and function) and to study the efficacy of a potential treatment.18,67 OA model species include large animals such as the horse, goat, sheep, and dog, whose size and anatomy are expected to better reflect human joint conditions. However, small animals such as guinea pig, rabbit, mouse, and rat represent 77% of the species used.1,87 In recent years, mice have become the most commonly used model for studying OA. Mice have several advantageous characteristics: a short development and life span, easy and low-cost breeding and maintenance, easy handling, small joints that allow histologic analysis of the whole joint,32 and the availability of genetically modified lines.108 Standardized housing, genetically defined strains and SPF animals reduce the genetic and interindividual acquired variability. Mice are considered the best vertebrate model in terms of monitoring and controlling environmental conditions.7,14,15,87 Mouse skeletal maturation is reached at 10 wk, which theoretically constitutes the minimal age at which mice should be entered into an OA study.64,87,102 However, many studies violate this limit by testing mice at 8 wk of age.Available models for OA include the following (32,111 physical activity and exercise induced OA; noninvasive mechanical loading (repetitive mild loading and single-impact injury); and surgically induced (meniscectomy models or anterior cruciate ligament transection). The specific model used would be based on the goal of the study.7 For example, OA pathophysiology, OA progression, and OA therapies studies could use spontaneous, genetic, surgical, or noninvasive models. In addition, pain studies could use chemical models. Lastly, post-traumatic studies would use surgical or noninvasive models; the most frequently used method is currently destabilization of the medial meniscus,32 which involves transection of the medial meniscotibial ligament, thereby destabilizing the joint and causing instability-driven OA. An important caveat for mouse models is that the mouse and human knee differ in terms of joint size, joint biomechanics, and histologic characteristics (layers, cellularity),32,64 and joint differences could confound clinical translation.10 Table 1. Mouse models of osteoarthritis.
Open in a separate windowSince all animal models have strengths and weaknesses, it is often best to plan using a number of models and techniques together to combine the results.In humans, the lack of correlation between OA imaging assessment and clinical signs highlights the need to consider the functional data and the quality of life to personalize OA management. Clinical outcomes are needed to achieve 2 main goals: to assess the impact of the OA in terms of pain and function and to study the efficacy of treatments.65 Recent reviews offer few practical approaches to mouse functional assessment and novel approaches to OA models in mice.7,32,67,75,79,83,87, 100,120 This review will focus on static and dynamic clinical assessment of OA using automatic and noninvasive emerging techniques (Test name Techniques Kind of assessment Output Specific equipment required Static measurement Von Frey filament testing Calibrated nylon filaments of various thickness (and applied force) are pressed against the skin of the plantar surface of the paw in ascending order of force Stimulus- evoked pain-like behavior
Mechanical stimuli - Tactile allodynia
The most commonly used test Latency to paw withdrawal
and
Force exerted are recorded Yes Knee extension test Apply a knee extension on both the intact and affected knee
or
Passive extension range of the operated knee joint under anesthesia Stimulus-evoked pain-like behavior Number of vocalizations evoked in 5 extensions None Hotplate Mouse placed on hotplate. A cutoff latency has been determined to avoid lesions Stimulus-evoked pain-like behavior
Heat stimuli- thermal sensitivity Latency of paw withdrawal Yes Righting ability Mouse placed on its back Neuromuscular screening Latency to regain its footing None Cotton swab test Bringing a cotton swab into contact with eyelashes, pinna, and whiskers Stimulus-evoked pain-like behavior
Neuromuscular screening Withdrawal or twitching response None Spontaneous activity Spontaneous cage activity One by one the cages must be laid out in a specific platform Spontaneous pain behavior
Nonstimulus evoked pain
Activity Vibrations evoked by animal movements Yes Open field analysis Experiment is performed in a clear chamber and mice can freely explore Spontaneous pain behavior
Nonstimulus evoked pain
Locomotor analysis Paw print assessment
Distance traveled, average walking speed, rest time, rearing Yes Gait analysis Mouse is placed in a specific cage equipped with a fluorescent tube and a glass plate allowing an automated quantitative gait analysis Nonstimulus evoked pain
Gait analysis
Indirect nociception Intensity of the paw contact area, velocity, stride frequency, length, symmetry, step width Yes Dynamic weight bearing system Mouse placed is a specific cage. This method is a computerized capacitance meter (similar to gait analysis) Nonstimulus evoked pain
Weight-bearing deficits
Indirect nociception Body weight redistribution to a portion of the paw surface Yes Voluntary wheel running Mouse placed is a specific cage with free access to stainless steel activity wheels. The wheel is connected to a computer that automatically record data Nonstimulus evoked pain
Activity Distance traveled in the wheel Yes Burrowing analysis Mouse placed is a specific cage equipped with steel tubes (32 cm in length and 10 cm in diameter) and quartz sand in Plexiglas cages (600 · 340x200 mm) Nonstimulus evoked pain
Activity Amount of sand burrowed Yes Digital video recordings Mouse placed is a specific cage according to the tool Nonstimulus evoked pain
Or
Evoked pain Scale of pain or specific outcome Yes Digital ventilated cage system Nondisrupting capacitive-based technique: records spontaneous activity 24/7, during both light and dark phases directly from the home cage rack Spontaneous pain behavior
Nonstimulus evoked pain
Activity-behavior Distance walked, average speed, occupation front, occupation rear, activation density.
Animal locomotion index, animal tracking distance, animal tracking speed, animal running wheel distance and speed or rotation Yes Challenged activity Rotarod test Gradual and continued acceleration of a rotating rod onto which mice are placed Motor coordination
Indirect nociception Rotarod latency: riding time and speed with a maximum cut off. Yes Hind limb and fore grip strength Mouse placed over a base plate in front of a connected grasping tool Muscle strength of limbs Peak force, time resistance Yes Wire hang analysis Suspension of the mouse on the wire and start the time Muscle strength of limbs: muscle function and coordination Latency to fall gripping None
(self -constructed)
Models | Pros | Cons | |
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Spontaneous | Wild type mice7,9,59,67,68,70,72,74,80,85,87,115,118,119,120 | - Model of aging phenotype - The less invasive model - Physiological relevance: mimics human pathogenesis - No need for technical expertise - No need for specific equipment | - Variability in incidence - Large number of animals at baseline - Long-term study: Time consuming (time of onset: 4 -15 mo) - Expensive (husbandry) |
Genetically modified mice2,7,25,40,50,52,67,72,79,80, 89,120 | - High incidence - Earlier time of onset: 18 wk - No need for specific equipment - Combination with other models | - Time consuming for the strain development - Expensive | |
Chemical- induced | Mono-iodoacetate injection7,11,46,47,60,66,90,91,101,128 | - Model of pain-like phenotype - To study mechanism of pain and antalgic drugs - Short-term study: Rapid progression (2-7 wk) - Reproducible - Low cost | - Need for technical expertise - Need for specific equipment - Systemic injection is lethal - Destructive effect: does not allow to study the early phase of pathogenesis |
Papain injection66,67,120 | - Short-term study: rapid progression - Low cost | - Need for technical expertise - Need for specific equipment - Does not mimic natural pathogenesis | |
Collagenase injection7,65,67,98 | - Short-term study: rapid progression (3 wk) - Low cost | - Need for technical expertise - Need for specific equipment - Does not mimic natural pathogenesis | |
Non-invasive | High-fat diet (Alimentary induced obesity model)5,8,43,45,57,96,124 | Model of metabolic phenotype No need for technical expertise No need for specific equipment Reproducible | Long-term study: Time consuming (8 wk–9 mo delay) Expensive |
Physical activity and exercise model45,73 | Model of post traumatic phenotype No need for technical expertise | Long-term study: time consuming (18 mo delay) Expensive Disparity of results | |
Mechanical loading models Repetitive mild loading models Single-impact injury model7,16,23,24, 32,35,104,105,106 | Model of post traumatic phenotype Allow to study OA development Time of onset: 8-10 wk post injury Noninvasive | Need for technical expertise Need for specific equipment Heterogeneity in protocol practices Repetitive anesthesia required or ethical issues | |
Surgical | Ovariectomy114 | Contested. | |
Meniscectomy model7,32,63,67,87 | Model of post traumatic phenotype High incidence Short-term study: early time of onset (4 wk from surgery) To study therapies | Need for technical expertise Need for specific equipment Surgical risks Rapid progression compared to human | |
Anterior cruciate ligament transection (ACLT)7,39,40,61,48,67,70,87,126 | Model of posttraumatic phenotype High incidence Short-term study: early time of onset (3-10 wk from surgery) Reproducible To study therapies | Need for technical expertise Need for specific equipment Surgical risks Rapid progression compared to human | |
Destabilization of medial meniscus (DMM)7,32,39,40 | Model of post traumatic phenotype High incidence Short-term study: early time of onset (4 wk from surgery) To study therapies The most frequently used method | Need for technical expertise Need for specific equipment Surgical risks Rapid progression compared to human |
Mechanical stimuli - Tactile allodynia
The most commonly used test
and
Force exerted are recorded
or
Passive extension range of the operated knee joint under anesthesia
Heat stimuli- thermal sensitivity
Neuromuscular screening
Nonstimulus evoked pain
Activity
Nonstimulus evoked pain
Locomotor analysis
Distance traveled, average walking speed, rest time, rearing
Gait analysis
Indirect nociception
Weight-bearing deficits
Indirect nociception
Activity
Activity
Or
Evoked pain
Nonstimulus evoked pain
Activity-behavior
Animal locomotion index, animal tracking distance, animal tracking speed, animal running wheel distance and speed or rotation
Indirect nociception
(self -constructed)