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Orphanet Journal of Rare Diseases Nov 2023Zellweger spectrum disorders (ZSD) and X-linked adrenoleukodystrophy (X-ALD) are inherited metabolic diseases characterized by dysfunction of peroxisomes, that are...
BACKGROUND
Zellweger spectrum disorders (ZSD) and X-linked adrenoleukodystrophy (X-ALD) are inherited metabolic diseases characterized by dysfunction of peroxisomes, that are essential for lipid metabolism and redox balance. Oxidative stress has been reported to have a significant role in the pathogenesis of neurodegenerative diseases such as peroxisomal disorders, but little is known on the intracellular activation of Mitogen-activated protein kinases (MAPKs). Strictly related to oxidative stress, a correct autophagic machinery is essential to eliminated oxidized proteins and damaged organelles. The aims of the current study are to investigate a possible implication of MAPK pathways and autophagy impairment as markers and putative therapeutic targets in X-ALD and ZSDs.
METHODS
Three patients with ZSD (2 M, 1 F; age range 8-17 years) and five patients with X-ALD (5 M; age range 5- 22 years) were enrolled. A control group included 6 healthy volunteers. To evaluate MAPKs pathway, p-p38 and p-JNK were assessed by western blot analysis on peripheral blood mononuclear cells. LC3II/LC3I ratio was evaluated ad marker of autophagy.
RESULTS
X-ALD and ZSD patients showed elevated p-p38 values on average 2- fold (range 1.21- 2.84) and 3.30-fold (range 1.56- 4.26) higher when compared with controls, respectively. p-JNK expression was on average 12-fold (range 2.20-19.92) and 2.90-fold (range 1.43-4.24) higher in ZSD and X-ALD patients than in controls. All patients had altered autophagic flux as concluded from the reduced LC3II/I ratio.
CONCLUSIONS
In our study X-ALD and ZSD patients present an overactivation of MAPK pathways and an inhibition of autophagy. Considering the absence of successful therapies and the growing interest towards new therapies with antioxidants and autophagy inducers, the identification and validation of biomarkers to monitor optimal dosing and biological efficacy of the treatments is of prime interest.
Topics: Humans; Child; Adolescent; Child, Preschool; Young Adult; Adult; Adrenoleukodystrophy; Zellweger Syndrome; Leukocytes, Mononuclear; Peroxisomes; Oxidation-Reduction
PubMed: 37974207
DOI: 10.1186/s13023-023-02940-x -
Nature Communications Nov 2023Ubiquitination is a post-translational modification initiated by the E1 enzyme UBA1, which transfers ubiquitin to ~35 E2 ubiquitin-conjugating enzymes. While UBA1 loss...
Ubiquitination is a post-translational modification initiated by the E1 enzyme UBA1, which transfers ubiquitin to ~35 E2 ubiquitin-conjugating enzymes. While UBA1 loss is cell lethal, it remains unknown how partial reduction in UBA1 activity is endured. Here, we utilize deep-coverage mass spectrometry to define the E1-E2 interactome and to determine the proteins that are modulated by knockdown of UBA1 and of each E2 in human cells. These analyses define the UBA1/E2-sensitive proteome and the E2 specificity in protein modulation. Interestingly, profound adaptations in peroxisomes and other organelles are triggered by decreased ubiquitination. While the cargo receptor PEX5 depends on its mono-ubiquitination for binding to peroxisomal proteins and importing them into peroxisomes, we find that UBA1/E2 knockdown induces the compensatory upregulation of other PEX proteins necessary for PEX5 docking to the peroxisomal membrane. Altogether, this study defines a homeostatic mechanism that sustains peroxisomal protein import in cells with decreased ubiquitination capacity.
Topics: Humans; Ubiquitination; Ubiquitin; Protein Transport; Peroxisomes; Intracellular Membranes
PubMed: 37963875
DOI: 10.1038/s41467-023-43262-7 -
BioRxiv : the Preprint Server For... Oct 2023Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro or in cells that overexpress protein, the...
Liquid-liquid phase separation (LLPS) underlies diverse biological processes. Because most LLPS studies were performed in vitro or in cells that overexpress protein, the physiological relevance of LLPS is unclear. PERIOD proteins are central mammalian circadian clock components and interact with other clock proteins in the core circadian negative feedback loop. Different core clock proteins were previously shown to form large complexes. Here we show that when transgene was stably expressed, PER2 formed nuclear phosphorylation-dependent LLPS condensates that recruited other clock proteins. Super-resolution microscopy of endogenous PER2, however, revealed formation of circadian-controlled, rapidly diffusing microbodies that were resistant to protein concentration changes, hexanediol treatment, and loss of phosphorylation, indicating that they are distinct from the LLPS condensates caused by overexpression. Surprisingly, only a small fraction of endogenous PER2 microbodies transiently interact with endogenous BMAL1 and CRY1, a conclusion that was confirmed in cells and in mice tissues, suggesting an enzyme-like mechanism in the circadian negative feedback process. Together, these results demonstrate that the dynamic interactions of core clock proteins is a key feature of mammalian circadian clock mechanism and the importance of examining endogenous proteins in LLPS and circadian studies.
PubMed: 37961341
DOI: 10.1101/2023.10.19.563153 -
Scientific Reports Nov 2023Diffusion and mobility are essential for cellular functions, as molecules are usually distributed throughout the cell and have to meet to interact and perform their...
Diffusion and mobility are essential for cellular functions, as molecules are usually distributed throughout the cell and have to meet to interact and perform their function. This also involves the cytosolic migration of cellular organelles. However, observing such diffusion and interaction dynamics is challenging due to the high spatial and temporal resolution required and the accurate analysis of the diffusional tracks. The latter is especially important when identifying anomalous diffusion events, such as directed motions, which are often rare. Here, we investigate the migration modes of peroxisome organelles in the cytosol of living cells. Peroxisomes predominantly migrate randomly, but occasionally they bind to the cell's microtubular network and perform directed migration, which is difficult to quantify, and so far, accurate analysis of switching between these migration modes is missing. We set out to solve this limitation by experiments and analysis with high statistical accuracy. Specifically, we collect temporal diffusion tracks of thousands of individual peroxisomes in the HEK 293 cell line using two-dimensional spinning disc fluorescence microscopy at a high acquisition rate of 10 frames/s. We use a Hidden Markov Model with two hidden states to (1) automatically identify directed migration segments of the tracks and (2) quantify the migration properties for comparison between states and between different experimental conditions. Comparing different cellular conditions, we show that the knockout of the peroxisomal membrane protein PEX14 leads to a decrease in the directed movement due to a lowered binding probability to the microtubule. However, it does not eradicate binding, highlighting further microtubule-binding mechanisms of peroxisomes than via PEX14. In contrast, structural changes of the microtubular network explain perceived eradication of directed movement by disassembly of microtubules by Nocodazole-treatment.
Topics: Humans; Peroxisomes; HEK293 Cells; Microtubules; Intracellular Membranes; Microscopy, Fluorescence
PubMed: 37951993
DOI: 10.1038/s41598-023-46812-7 -
Redox Biology Nov 2023Despite intensive research on peroxisome biochemistry, the role of glutathione in peroxisomal redox homeostasis has remained a matter of speculation for many years, and... (Review)
Review
Despite intensive research on peroxisome biochemistry, the role of glutathione in peroxisomal redox homeostasis has remained a matter of speculation for many years, and only recently has this issue started to be experimentally addressed. Here, we summarize and compare data from several organisms on the peroxisome-glutathione topic. It is clear from this comparison that the repertoire of glutathione-utilizing enzymes in peroxisomes of different organisms varies widely. In addition, the available data suggest that the kinetic connectivity between the cytosolic and peroxisomal pools of glutathione may also be different in different organisms, with some possessing a peroxisomal membrane that is promptly permeable to glutathione whereas in others this may not be the case. However, regardless of the differences, the picture that emerges from all these data is that glutathione is a crucial component of the antioxidative system that operates inside peroxisomes in all organisms.
Topics: Peroxisomes; Glutathione; Antioxidants; Oxidation-Reduction; Homeostasis
PubMed: 37804696
DOI: 10.1016/j.redox.2023.102917 -
Cell Reports Oct 2023The enhanced response of glucagon and its Drosophila homolog, adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous...
The enhanced response of glucagon and its Drosophila homolog, adipokinetic hormone (Akh), leads to high-caloric-diet-induced hyperglycemia across species. While previous studies have characterized regulatory components transducing linear Akh signaling promoting carbohydrate production, the spatial elucidation of Akh action at the organelle level still remains largely unclear. In this study, we find that Akh phosphorylates extracellular signal-regulated kinase (ERK) and translocates it to peroxisome via calcium/calmodulin-dependent protein kinase II (CaMKII) cascade to increase carbohydrate production in the fat body, leading to hyperglycemia. The mechanisms include that ERK mediates fat body peroxisomal conversion of amino acids into carbohydrates for gluconeogenesis in response to Akh. Importantly, Akh receptor (AkhR) or ERK deficiency, importin-associated ERK retention from peroxisome, or peroxisome inactivation in the fat body sufficiently alleviates high-sugar-diet-induced hyperglycemia. We also observe mammalian glucagon-induced hepatic ERK peroxisomal translocation in diabetic subjects. Therefore, our results conclude that the Akh/glucagon-peroxisomal-ERK axis is a key spatial regulator of glycemic control.
Topics: Animals; Carbohydrates; Drosophila; Extracellular Signal-Regulated MAP Kinases; Glucagon; Glycemic Control; Hyperglycemia; Peroxisomes; Drosophila Proteins
PubMed: 37796662
DOI: 10.1016/j.celrep.2023.113200 -
Nature Communications Sep 2023Mitochondrial morphology, which is controlled by mitochondrial fission and fusion, is an important regulator of the thermogenic capacity of brown adipocytes....
Mitochondrial morphology, which is controlled by mitochondrial fission and fusion, is an important regulator of the thermogenic capacity of brown adipocytes. Adipose-specific peroxisome deficiency impairs thermogenesis by inhibiting cold-induced mitochondrial fission due to decreased mitochondrial membrane content of the peroxisome-derived lipids called plasmalogens. Here, we identify TMEM135 as a critical mediator of the peroxisomal regulation of mitochondrial fission and thermogenesis. Adipose-specific TMEM135 knockout in mice blocks mitochondrial fission, impairs thermogenesis, and increases diet-induced obesity and insulin resistance. Conversely, TMEM135 overexpression promotes mitochondrial division, counteracts obesity and insulin resistance, and rescues thermogenesis in peroxisome-deficient mice. Mechanistically, thermogenic stimuli promote association between peroxisomes and mitochondria and plasmalogen-dependent localization of TMEM135 in mitochondria, where it mediates PKA-dependent phosphorylation and mitochondrial retention of the fission factor Drp1. Together, these results reveal a previously unrecognized inter-organelle communication regulating mitochondrial fission and energy homeostasis and identify TMEM135 as a potential target for therapeutic activation of BAT.
Topics: Animals; Mice; Adipocytes, Brown; Adipose Tissue, Brown; Homeostasis; Insulin Resistance; Mice, Knockout; Mitochondrial Dynamics; Obesity; Peroxisomes; Thermogenesis
PubMed: 37773161
DOI: 10.1038/s41467-023-41849-8 -
Journal of the American Chemical Society Nov 2023Autophagy is responsible for the degradation of large intracellular contents, such as unwanted protein aggregates and organelles. Impaired autophagy can therefore lead...
Autophagy is responsible for the degradation of large intracellular contents, such as unwanted protein aggregates and organelles. Impaired autophagy can therefore lead to the accumulation of pathological aggregates, correlating with aging and neurodegenerative diseases. However, a broadly applicable methodology is not available for the targeted degradation of protein aggregates or organelles in mammalian cells. Herein, we developed a series of autophagy receptor-inspired targeting chimeras (AceTACs) that can induce the targeted degradation of aggregation-prone proteins and protein aggregates (e.g., huntingtin, TDP-43, and FUS mutants), as well as organelles (e.g., mitochondria, peroxisomes, and endoplasmic reticulum). These antibody-fusion-based AceTAC degraders were designed to mimic the function of autophagy receptors, simultaneously binding with the cellular targets and the LC3 proteins on the autophagosomal membrane, eventually transporting the target to the autophagy-lysosomal process for degradation. The AceTAC degradation system provides design principles for antibody-based degradation through autophagy, largely expanding the scope of intracellular targeted degradation technologies.
Topics: Animals; Protein Aggregates; Autophagy; Endoplasmic Reticulum; Lysosomes; Peroxisomes; Mammals
PubMed: 37748140
DOI: 10.1021/jacs.3c05199 -
Trends in Cell Biology May 2024Peroxisomes are vital metabolic organelles that import their lumenal (matrix) enzymes from the cytosol using mobile receptors. Surprisingly, the receptors can even... (Review)
Review
Peroxisomes are vital metabolic organelles that import their lumenal (matrix) enzymes from the cytosol using mobile receptors. Surprisingly, the receptors can even import folded proteins, but the underlying mechanism has been a mystery. Recent results reveal how import receptors shuttle cargo into peroxisomes. The cargo-bound receptors move from the cytosol across the peroxisomal membrane completely into the matrix by a mechanism that resembles transport through the nuclear pore. The receptors then return to the cytosol through a separate retrotranslocation channel, leaving the cargo inside the organelle. This cycle concentrates imported proteins within peroxisomes, and the energy for cargo import is supplied by receptor export. Peroxisomal protein import thus fundamentally differs from other previously known mechanisms for translocating proteins across membranes.
Topics: Peroxisomes; Protein Transport; Humans; Animals; Cytosol; Receptors, Cytoplasmic and Nuclear
PubMed: 37743160
DOI: 10.1016/j.tcb.2023.08.005 -
Nature Communications Sep 2023The double-ring AAA+ ATPase Pex1/Pex6 is required for peroxisomal receptor recycling and is essential for peroxisome formation. Pex1/Pex6 mutations cause severe...
The double-ring AAA+ ATPase Pex1/Pex6 is required for peroxisomal receptor recycling and is essential for peroxisome formation. Pex1/Pex6 mutations cause severe peroxisome associated developmental disorders. Despite its pathophysiological importance, mechanistic details of the heterohexamer are not yet available. Here, we report cryoEM structures of Pex1/Pex6 from Saccharomyces cerevisiae, with an endogenous protein substrate trapped in the central pore of the catalytically active second ring (D2). Pairs of Pex1/Pex6(D2) subdomains engage the substrate via a staircase of pore-1 loops with distinct properties. The first ring (D1) is catalytically inactive but undergoes significant conformational changes resulting in alternate widening and narrowing of its pore. These events are fueled by ATP hydrolysis in the D2 ring and disengagement of a "twin-seam" Pex1/Pex6(D2) heterodimer from the staircase. Mechanical forces are propagated in a unique manner along Pex1/Pex6 interfaces that are not available in homo-oligomeric AAA-ATPases. Our structural analysis reveals the mechanisms of how Pex1 and Pex6 coordinate to achieve substrate translocation.
Topics: ATPases Associated with Diverse Cellular Activities; Cryoelectron Microscopy; Mutation; Peroxisomes; Proton-Translocating ATPases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity
PubMed: 37741838
DOI: 10.1038/s41467-023-41640-9