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Rhizomelic chondrodysplasia punctata type 1 (RCDP1) is a peroxisomal disorder characterized by skeletal shortening, intellectual disability, seizures, cataracts, and reduced lifespans. RCDP1 is caused by biallelic loss-of-function variants in PEX7, which encodes a protein required for importing select enzymes into the peroxisome matrix, including those essential for ether lipid synthesis (e.g., plasmalogens) and the branched-chain fatty acid catabolism. Plasmalogen deficiency is a hallmark of RCDP1 and other peroxisomal disorders, including RCDP types 2-5 (RCDP2-5) and Zellweger spectrum disorders (ZSD). Here, we performed comprehensive metabolomic profiling of clinical samples from RCDP patients and Pex7-deficient mouse models. We identified profound neurometabolic disturbances in the cerebral cortex and cerebellum of Pex7-deficient mice involving multiple lipid classes, including phosphatidylethanolamines (PEs), phosphatidylcholines (PCs), acylcarnitines, and sphingomyelins. Notably, many of these neurometabolic alterations were absent in patient and Pex7-deficient mouse plasma, indicating that plasma-based profiling can underrepresent the extent of CNS lipid remodeling. Overall, these findings reveal novel insights into neurometabolic adaptations to plasmalogen deficiency and suggest the potential involvement of additional pathways that may contribute to neurological dysfunction in RCDP.

Peroxisomes are organelles that carry out β-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction 1 . Among disease-causing variant genes are those required for protein transport into peroxisomes. The peroxisomal protein import machinery, which also shares similarities with chloroplasts 2 , is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes 3 . Current models postulate a large pore formed by transmembrane proteins 4 ; however, so far, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae , the minimum transport machinery includes the membrane proteins Pex13 and Pex14 and the cargo-protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid–liquid phase separation (LLPS) with Pex5–cargo. Intrinsically disordered regions in Pex13 and Pex5 resemble those found in nuclear pore complex proteins. Peroxisomal protein import depends on both the number and pattern of aromatic residues in these intrinsically disordered regions, consistent with their roles as ‘stickers’ in associative polymer models of LLPS 5 , 6 . Finally, imaging fluorescence cross-correlation spectroscopy shows that cargo import correlates with transient focusing of GFP–Pex13 and GFP–Pex14 on the peroxisome membrane. Pex13 and Pex14 form foci in distinct time frames, suggesting that they may form channels at different saturating concentrations of Pex5–cargo. Our findings lead us to suggest a model in which LLPS of Pex5–cargo with Pex13 and Pex14 results in transient protein transport channels 7 . A study presents evidence to support a model in which liquid–liquid phase separation of components of the transport machinery mediates formation of transient protein transport channels on peroxisomes.

Lipid droplets (LDs) are key subcellular organelles for regulating lipid metabolism. Although several subcellular organelles participate in lipid metabolism, it remains elusive whether physical contacts between subcellular organelles and LDs might be involved in lipolysis upon nutritional deprivation. Here, we demonstrate that peroxisomes and peroxisomal protein PEX5 mediate fasting-induced lipolysis by stimulating adipose triglyceride lipase (ATGL) translocation onto LDs. During fasting, physical contacts between peroxisomes and LDs are increased by KIFC3-dependent movement of peroxisomes toward LDs, which facilitates spatial translocations of ATGL onto LDs. In addition, PEX5 could escort ATGL to contact points between peroxisomes and LDs in the presence of fasting cues. Moreover, in adipocyte-specific PEX5-knockout mice, the recruitment of ATGL onto LDs was defective and fasting-induced lipolysis is attenuated. Collectively, these data suggest that physical contacts between peroxisomes and LDs are required for spatiotemporal translocation of ATGL, which is escorted by PEX5 upon fasting, to maintain energy homeostasis. Lipid droplets are organelles that regulate lipid metabolism but if organellar contacts play a role during lipolysis is unclear. Here, the authors show that peroxisomes and peroxisomal protein PEX5 play pivotal roles in the spatial and temporal regulation of fasting-induced lipolysis by translocating ATGL onto lipid droplets

Demyelination associated microglia (DMAM) orchestrate the regenerative response to demyelination by clearing myelin debris and promoting oligodendrocyte maturation. Peroxisomal metabolism has emerged as a candidate regulator of DMAMs, though the cell-intrinsic contribution in microglia remains undefined. Here we elucidate the role of peroxisome integrity in DMAMs, using cuprizone-mediated demyelination coupled with conditional KO of peroxisome biogenesis factor 5 (PEX5) in microglia. Absent demyelination, PEX5 conditional KO (PEX5cKO) had minimal impact on homeostatic microglia. However, during cuprizone-induced demyelination, the emergence of DMAMs unmasked a critical requirement for peroxisome integrity. At peak demyelination, PEX5cKO DMAMs exhibited increased lipid droplet burden and reduced lipophagy suggestive of impaired lipid catabolism. Although lipid droplet burden declined during the remyelination phase, PEX5cKO DMAMs accumulated intralysosomal crystals and curvilinear profiles, features that were largely absent in controls. Aberrant lipid processing was accompanied by elevated numbers of lysosomal damage markers and downregulation of the lipid exporter gene Apoe, consistent with defective lipid clearance. Furthermore, the disruptions in PEX5cKO DMAMs were associated with defective myelin debris clearance and impaired remyelination. Together, these findings delineate a stage-specific role for peroxisomes in coordinating lipid processing pathways essential to DMAM function and for enabling a pro-remyelinating environment.

Spikelet is the primary reproductive structure and a critical determinant of grain yield in rice. The molecular mechanisms regulating rice spikelet development still remain largely unclear. Here, we report that mutations in OsPEX5, which encodes a peroxisomal targeting sequence 1 (PTS1) receptor protein, cause abnormal spikelet morphology. We show that OsPEX5 can physically interact with OsOPR7, an enzyme involved in jasmonic acid (JA) biosynthesis and is required for its import into peroxisome. Similar to Ospex5 mutant, the knockout mutant of OsOPR7 generated via CRISPR-Cas9 technology has reduced levels of endogenous JA and also displays an abnormal spikelet phenotype. Application of exogenous JA can partially rescue the abnormal spikelet phenotype of Ospex5 and Osopr7. Furthermore, we show that OsMYC2 directly binds to the promoters of OsMADS1, OsMADS7 and OsMADS14 to activate their expression, and subsequently regulate spikelet development. Our results suggest that OsPEX5 plays a critical role in regulating spikelet development through mediating peroxisomal import of OsOPR7, therefore providing new insights into regulation of JA biosynthesis in plants and expanding our understanding of the biological role of JA in regulating rice reproduction.

Peroxisomes are membrane enclosed organelles hosting diverse metabolic processes in eukaryotic cells. Having no protein synthetic abilities, peroxisomes import all required enzymes from the cytosol through a peroxin (Pex) import system. Peroxisome targeting sequence 1 (PTS1)-tagged cargo is recognized by cytosolic receptor, Pex5. The cargo-Pex5 complex docks at the peroxisomal membrane translocon, composed of Pex14 and Pex13, facilitating translocation into the peroxisomal lumen. Despite its significance, the structural basis of the process is only partially understood. Here, we characterize the cargo-Pex5-Pex14 NTD ternary complex from Trypanosoma cruzi . Cryo-electron microscopy maps enabled model building for Pex5 (residues 327–462 and 487–653) bound to malate dehydrogenase (MDH; residues 1–323) cargo tetramer and Pex14 NTD (residues 21–85). The model provides insight into conformational heterogeneity and identifies secondary interfaces. Specifically, we observe that orientations of Pex5 relative to MDH span a 17° angle. Additionally, PTS1- and Wxxx(F/Y)-independent contact surfaces are observed at MDH-Pex5 and Pex5-Pex14 NTD interfaces, respectively. Mutational analysis indicates that the non-PTS1 MDH-Pex5 interface does not significantly contribute to the affinity, but limits the conformational heterogeneity of MDH-Pex5 interface. The Pex5-Pex14 NTD interface constitutes an extended binding site of Pex14 NTD over Pex5. We discuss the implications of these findings for understanding peroxisomal import mechanism. Peroxisomes import matrix proteins using a specialized membrane translocation system. Here, authors reveal the structure of an import complex from Trypanosoma cruzi, uncovering the protein-protein interactions and associated heterogeneity.

Skeletal muscles, which constitute 40–50% of body mass, regulate whole-body energy expenditure and glucose and lipid metabolism. Peroxisomes are dynamic organelles that play a crucial role in lipid metabolism and clearance of reactive oxygen species, however their role in skeletal muscle remains poorly understood. To clarify this issue, we generated a muscle-specific transgenic mouse line with peroxisome import deficiency through the deletion of peroxisomal biogenesis factor 5 ( Pex5 ). Here, we show that Pex5 inhibition results in impaired lipid metabolism, reduced muscle force and exercise performance. Moreover, mitochondrial structure, content, and function are also altered, accelerating the onset of age-related structural defects, neuromuscular junction degeneration, and muscle atrophy. Consistent with these observations, we observe a decline in peroxisomal content in the muscles of control mice undergoing natural aging. Altogether, our findings show the importance of preserving peroxisomal function and their interplay with mitochondria to maintain muscle health during aging. The role of peroxisomes in skeletal muscle remains largely unexplored. Here, the authors show that peroxisomal dysfunction and disrupted crosstalk with mitochondria drive age-related muscle decline, underscoring the need to preserve peroxisomes for muscle health.

Animal models have been utilized to understand the pathogenesis of Zellweger spectrum disorders (ZSDs); however, the link between clinical manifestations and molecular pathways has not yet been clearly established. We generated peroxin 5 homozygous mutant zebrafish (pex5−/−) to gain insight into the molecular pathogenesis of peroxisome dysfunction. pex5−/− display hallmarks of ZSD in humans and die within one month after birth. Fasting rapidly depletes lipids and glycogen in pex5−/− livers and expedites their mortality. Mechanistically, deregulated mitochondria and mechanistic target of rapamycin (mTOR) signaling act together to induce metabolic alterations that deplete hepatic nutrients and accumulate damaged mitochondria. Accordingly, chemical interventions blocking either the mitochondrial function or mTOR complex 1 (mTORC1) or a combination of both improve the metabolic imbalance shown in the fasted pex5−/− livers and extend the survival of animals. In addition, the suppression of oxidative stress by N-acetyl L-cysteine (NAC) treatment rescued the apoptotic cell death and early mortality observed in pex5−/−. Furthermore, an autophagy activator effectively ameliorated the early mortality of fasted pex5−/−. These results suggest that fasting may be detrimental to patients with peroxisome dysfunction, and that modulating the mitochondria, mTORC1, autophagy activities, or oxidative stress may provide a therapeutic option to alleviate the symptoms of peroxisomal diseases associated with metabolic dysfunction.

Leishmaniasis, an infectious disease caused by pathogenic Leishmania parasites, affects millions of people in developing countries, and its re-emergence in developed countries, particularly in Europe, poses a growing public health concern. The limitations of current treatments and the absence of effective vaccines necessitate the development of novel therapeutics. In this study, we focused on identifying small molecule inhibitors which prevents the interaction between peroxin 5 (PEX5) and peroxisomal targeting signal 1 (PTS1), pivotal for kinetoplastid parasite survival. The Leishmania donovani PEX5, containing a C-terminal tetratricopeptide repeat (TPR) domain, was expressed and purified, followed by the quantification of kinetic parameters of PEX5-PTS1 interactions. A fluorescence polarization-based high-throughput screening assay was developed and small molecules inhibiting the LdPEX5-PTS1 interaction were discovered through the screening of a library of 51,406 compounds. Based on the confirmatory assay, nine compounds showed half maximal inhibitory concentration (IC50) values ranging from 3.89 to 24.50 µM. In silico docking using a homology model of LdPEX5 elucidated that the molecular interactions between LdPEX5 and the inhibitors share amino acids critical for PTS1 binding. Notably, compound P20 showed potent activity against the growth of L. donovani promastigotes, L. major promastigotes, and Trypanosoma brucei blood stream form, with IC50 values of 12.16, 19.21, and 3.06 μM, respectively. The findings underscore the potential of targeting LdPEX5-PTS1 interactions with small molecule inhibitors as a promising strategy for the discovery of new anti-parasitic compounds.

Peroxisomes are ubiquitous membrane-enclosed organelles involved in lipid processing and reactive oxygen detoxification. Mutations in human peroxisome biogenesis genes (Peroxin, PEX, or Pex) cause developmental disabilities and often early death. Pex5 and Pex7 are receptors that recognize different peroxisomal targeting signals called PTS1 and PTS2, respectively, and traffic proteins to the peroxisomal matrix. We characterized mutants of Drosophila melanogaster Pex5 and Pex7 and found that adult animals are affected in lipid processing. Pex5 mutants exhibited severe developmental defects in the embryonic nervous system and muscle, similar to what is observed in humans with PEX5 mutations, while Pex7 fly mutants were weakly affected in brain development, suggesting different roles for fly Pex7 and human PEX7. Of note, although no PTS2-containing protein has been identified in Drosophila, Pex7 from Drosophila can function as a bona fide PTS2 receptor because it can rescue targeting of the PTS2-containing protein thiolase to peroxisomes in PEX7 mutant human fibroblasts.