New insights into the functions of ACBD4/5-like proteins using a combined phylogenetic and experimental approach across model organisms.

Acyl-CoA binding domain-containing proteins (ACBDs) perform diverse but often uncharacterised functions linked to cellular lipid metabolism. Human ACBD4 and ACBD5 are closely related peroxisomal membrane proteins, involved in tethering of peroxisomes to the ER and capturing fatty acids for peroxisomal ß-oxidation. ACBD5 deficiency causes neurological abnormalities including ataxia and white matter disease. Peroxisome-ER contacts depend on an ACBD4/5-FFAT motif, which interacts with ER-resident VAP proteins. As ACBD4/5-like proteins are present in most fungi and all animals, we combined phylogenetic analyses with experimental approaches to improve understanding of their evolution and functions. Notably, all vertebrates exhibit gene sequences for both ACBD4 and ACBD5, while invertebrates and fungi possess only a single ACBD4/5-like protein. Our analyses revealed alterations in domain structure and FFAT sequences, which help understanding functional diversification of ACBD4/5-like proteins. We show that the Drosophila melanogaster ACBD4/5-like protein possesses a functional FFAT motif to tether peroxisomes to the ER via Dm_Vap33. Depletion of Dm_Acbd4/5 caused peroxisome redistribution in wing neurons and reduced life expectancy. In contrast, the ACBD4/5-like protein of the filamentous fungus Ustilago maydis lacks a FFAT motif and does not interact with Um_Vap33. Loss of Um_Acbd4/5 resulted in an accumulation of peroxisomes and early endosomes at the hyphal tip. Moreover, lipid droplet numbers increased, and mitochondrial membrane potential declined, implying altered lipid homeostasis. Our findings reveal differences between tethering and metabolic functions of ACBD4/5-like proteins across evolution, improving our understanding of ACBD4/5 function in health and disease. The need for a unifying nomenclature for ACBD proteins is discussed.

A Novel Homozygous ACBD5 Variant in an Emerging Peroxisomal Disorder Presenting with Retinal Dystrophy and a Review of the Literature.

Introduction: Acyl-CoA binding domain containing 5 (ACBD5) deficiency is a newly defined inborn peroxisomal disorder with only 7 patients reported to date. Herein, we report a patient with ACBD5 deficiency who was diagnosed after a complicated diagnostic process. Case Presentation: A 6-year-old male patient was admitted with complaints of neuromotor regression and visual disturbances. He had spastic paraparesis dominated with axial hypotonic posturing and horizontal nystagmus. His very-long-chain fatty acid levels were within normal ranges with a slightly elevated C26:0/C22:0 ratio. Brain magnetic resonance imaging revealed white matter involvement. Clinical exome sequencing displayed a novel homozygous intronic splice site variant (c.936 + 2T>G) in the ACBD5 (NM_145698.5) gene. Conclusion: With this report, a novel variant in ACBD5 deficiency was described. Macular dystrophy was demonstrated with optical coherence tomography imaging for the first time in the literature in ACBD5 deficiency. In order to contribute to the knowledge about the clinical, biochemical, and genetic spectrum of ACBD5 deficiency, new patients need to be defined.

ACBD5-related retinal dystrophy with leukodystrophy due to novel mutations in ACBD5 and with additional features including ovarian insufficiency.

Acyl-CoA-binding domain-containing protein 5-related retinal dystrophy with leukodystrophy (ACBD5) is a peroxisomal disorder due to deficiency of ACBD5. Presenting features include retinal dystrophy, progressive leukodystrophy, and ataxia. Only seven cases of ACBD5-related retinal dystrophy have been reported in the literature to date, including one other case diagnosed in adulthood. Here we report a case with novel compound heterozygous ACBD5 mutations, presenting with the common features of rod monochromatism and progressive leukodystrophy with spasticity and ataxia. Additional novel clinical features included head and neck tremor and ovarian insufficiency. The patient's symptoms were present since infancy, but a diagnosis was only reached in adulthood when whole exome sequencing was performed. This case, which reports two novel mutations and additional clinical manifestations, contributes to the emerging phenotype of ACBD5-related retinal dystrophy with leukodystrophy, and delineation of the natural history and disease progression.

Differential roles for ACBD4 and ACBD5 in peroxisome-ER interactions and lipid metabolism.

Peroxisomes and the endoplasmic reticulum (ER) are intimately linked subcellular organelles, physically connected at membrane contact sites. While collaborating in lipid metabolism, for example, of very long-chain fatty acids (VLCFAs) and plasmalogens, the ER also plays a role in peroxisome biogenesis. Recent work identified tethering complexes on the ER and peroxisome membranes that connect the organelles. These include membrane contacts formed via interactions between the ER protein VAPB (vesicle-associated membrane protein-associated protein B) and the peroxisomal proteins ACBD4 and ACBD5 (acyl-coenzyme A-binding domain protein). Loss of ACBD5 has been shown to cause a significant reduction in peroxisome-ER contacts and accumulation of VLCFAs. However, the role of ACBD4 and the relative contribution these two proteins make to contact site formation and recruitment of VLCFAs to peroxisomes remain unclear. Here, we address these questions using a combination of molecular cell biology, biochemical, and lipidomics analyses following loss of ACBD4 or ACBD5 in HEK293 cells. We show that the tethering function of ACBD5 is not absolutely required for efficient peroxisomal ß-oxidation of VLCFAs. We demonstrate that loss of ACBD4 does not reduce peroxisome-ER connections or result in the accumulation of VLCFAs. Instead, the loss of ACBD4 resulted in an increase in the rate of ß-oxidation of VLCFAs. Finally, we observe an interaction between ACBD5 and ACBD4, independent of VAPB binding. Overall, our findings suggest that ACBD5 may act as a primary tether and VLCFA recruitment factor, whereas ACBD4 may have regulatory functions in peroxisomal lipid metabolism at the peroxisome-ER interface.

Assessing Peroxisomal Protein Interaction by Immunoprecipitation.

Organelles physically interact with each other via protein tethering complexes that bridge the opposing membranes. Organelle membrane contacts are highly dynamic, implying dynamism of the tethering complexes. Alterations in the binding of the tethering proteins can be assessed by immunoprecipitation. Antibody-conjugated beads allow for purification of the target protein with its binding partners, which can subsequently be examined by western blot analysis. We present immunoprecipitation methods and strategies to examine protein interaction domains, and for the identification of residues important for the regulation of the interaction, here focusing on phosphorylation. We use the peroxisomal membrane protein ACBD5 and its paralog ACBD4, which both bind ER membrane protein VAPB to mediate peroxisome-ER contacts, as example. However, this method can be applied to other peroxisomal and non-peroxisomal (membrane) proteins.

Fission Impossible (?)-New Insights into Disorders of Peroxisome Dynamics.

Peroxisomes are highly dynamic and responsive organelles, which can adjust their morphology, number, intracellular position, and metabolic functions according to cellular needs. Peroxisome multiplication in mammalian cells involves the concerted action of the membrane-shaping protein PEX11ß and division proteins, such as the membrane adaptors FIS1 and MFF, which recruit the fission GTPase DRP1 to the peroxisomal membrane. The latter proteins are also involved in mitochondrial division. Patients with loss of DRP1, MFF or PEX11ß function have been identified, showing abnormalities in peroxisomal (and, for the shared proteins, mitochondrial) dynamics as well as developmental and neurological defects, whereas the metabolic functions of the organelles are often unaffected. Here, we provide a timely update on peroxisomal membrane dynamics with a particular focus on peroxisome formation by membrane growth and division. We address the function of PEX11ß in these processes, as well as the role of peroxisome-ER contacts in lipid transfer for peroxisomal membrane expansion. Furthermore, we summarize the clinical phenotypes and pathophysiology of patients with defects in the key division proteins DRP1, MFF, and PEX11ß as well as in the peroxisome-ER tether ACBD5. Potential therapeutic strategies for these rare disorders with limited treatment options are discussed.

Multiple Ways to Keep FFAT Under Control!

Peroxisomes and the ER are closely inter-connected organelles, which collaborate in the metabolism of lipids. In a recent research paper in the Journal of Cell Biology, we describe a novel mechanism by which peroxisome-ER membrane contact sites are regulated, via phosphorylation of the peroxisomal protein ACBD5. We found that the interaction between ACBD5 and the ER protein VAPB, which we have previously shown to form a tether complex at peroxisome-ER contacts, is controlled by phosphorylation of ACBD5 at two different sites of its FFAT motif - the VAPB binding site. We also identify the kinase GSK3-ß as being responsible for direct phosphorylation of ACBD5 to negatively regulate interaction with VAPB, leading to reduced peroxisome-ER contacts. In this article we provide additional insights into how this work, in combination with other studies on phosphorylation of VAP interactors, suggests a complex system of both positive and negative regulation of the FFAT motif via phosphorylation.

Variables in the ACBD5 Gene Leading to Distinct Phenotypes: A Case Report.

We report the cases of a father and his daughter, the former diagnosed with retinitis pigmentosa (RP) and the latter with early foveal atrophy; while both shared a novel variant of uncertain significance (VUS) in the ACBD5 gene (variant c.431G>A), they exhibited different clinical profiles and disease manifestations. The father was a 48-year-old man who presented with nyctalopia that had persisted since age seven. He had mild disk pallor, vessel attenuation, retinal pigment epithelium (RPE) changes nasal to the fovea, and few mid-peripheral bone spicules. Sequencing analysis showed that he carried seven VUS in five genes: ACBD5 c.431G>A (p.Gly144Asp), CYP4V2 c.296T>C (p.Met99Thr), EYS c.1852G>A (p.Gly618Ser), HMCN1 c.280G>A (p.Val94Met), HMCN1 c.8939A>C (p.Asn2980Thr), RP1L1 c.575C>A (p.Pro192His), and RP1L1 c.1375A>C (p.Thr459Pro). He shared only the ACBD5 gene with his 18-year-old daughter. The daughter had 20/20 visual acuity, but further testing showed foveal atrophy and hyperautofluorescence. Intrafamilial phenotypic heterogeneity was detected in our patients. Studies on the role of hormonal factors leading to phenotypic variability are warranted.

Newly defined peroxisomal disease with novel ACBD5 mutation.

Peroxisomal disorders are a heterogeneous group of diseases caused by mutations in a large number of genes. One of the genetic disorders known to cause this situation is ACBD5 (Acyl-CoA binding-domain-containing-5) gene mutations that have been described in recent years. Here, we report two siblings with a novel homozygous nonsense variation (c.1297C>T, p.Arg433*) in ACBD5 (NM_145698.4) gene using Clinical Exome Sequencing (Sophia Genetics).

First reported adult patient with retinal dystrophy and leukodystrophy caused by a novel ACBD5 variant: A case report and review of literature.

Peroxisomes play an essential role in lipid metabolism via interaction with other intracellular organelles. The information about the role of the Acyl-CoA-binding domain containing-protein 5 (ACBD5) in these interactions in human cells is emerging. Moreover, a few patients with retinal dystrophy and leukodystrophy caused by pathogenic variants in ACBD5 have been recently introduced. Here, we present a 36-year-old female with retinal dystrophy, leukodystrophy, and psychomotor regression due to a novel homozygous variant in ACBD5. Our study adds to the growing knowledge of this peroxisomal disorder by providing phenotypic details of the first adult patient.

Organelle interplay-peroxisome interactions in health and disease.

Peroxisomes are multifunctional, dynamic, membrane-bound organelles with important functions in cellular lipid metabolism, rendering them essential for human health and development. Important roles for peroxisomes in signaling and the fine-tuning of cellular processes are emerging, which integrate them in a complex network of interacting cellular compartments. Like many other organelles, peroxisomes communicate through membrane contact sites. For example, peroxisomal growth, positioning, and lipid metabolism involves contacts with the endoplasmic reticulum (ER). Here, we discuss the most recent findings on peroxisome-organelle interactions including peroxisome-ER interplay at membrane contacts sites, and functional interplay with mitochondria, lysosomes, and lipid droplets in mammalian cells. We address tether proteins, metabolic cooperation, and the impact of peroxisome interactions on human health and disease.

Fluorescent tools to analyse peroxisome-ER interactions in mammalian cells.

Peroxisomes and the endoplasmic reticulum (ER) cooperate extensively in lipid-related metabolic pathways, and the ER also provides phospholipids to enable the peroxisomal membrane to expand prior to division. Recently, we identified peroxisomal proteins ACBD5 and ACBD4, and the ER protein VAPB as tethering components which physically interact to foster peroxisome-ER associations at membrane contact sites. Overexpression or loss of these tether proteins alters the extent of peroxisome-ER interactions, impacting on lipid exchange between these two compartments. To facilitate further studies into peroxisome-ER associations at the level of membrane contact sites, their role, composition and regulation, we have developed two fluorescence-based systems to monitor peroxisome-ER interactions. We modified a proximity ligation assay and a split-fluorescence reporter system using split superfolder green fluorescent protein. Using the proximity ligation assay we were able to measure changes in peroxisome-ER interactions whilst the split-fluorescence reporter was more limited and only allowed us to label ER-peroxisome contacts. We show that both techniques can be useful additions to the toolkit of methods to study peroxisome-ER associations and explore the relative merits of each.

The peroxisome: an update on mysteries 2.0.

Peroxisomes are key metabolic organelles, which contribute to cellular lipid metabolism, e.g. the ß-oxidation of fatty acids and the synthesis of myelin sheath lipids, as well as cellular redox balance. Peroxisomal dysfunction has been linked to severe metabolic disorders in man, but peroxisomes are now also recognized as protective organelles with a wider significance in human health and potential impact on a large number of globally important human diseases such as neurodegeneration, obesity, cancer, and age-related disorders. Therefore, the interest in peroxisomes and their physiological functions has significantly increased in recent years. In this review, we intend to highlight recent discoveries, advancements and trends in peroxisome research, and present an update as well as a continuation of two former review articles addressing the unsolved mysteries of this astonishing organelle. We summarize novel findings on the biological functions of peroxisomes, their biogenesis, formation, membrane dynamics and division, as well as on peroxisome-organelle contacts and cooperation. Furthermore, novel peroxisomal proteins and machineries at the peroxisomal membrane are discussed. Finally, we address recent findings on the role of peroxisomes in the brain, in neurological disorders, and in the development of cancer.

Peroxisome Motility Measurement and Quantification Assay.

Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation with mitochondria and the endoplasmic reticulum (ER) is essential for the ß-oxidation of fatty acids and the synthesis of myelin lipids and polyunsaturated fatty acids. A key assay to assess peroxisome motility in mammalian cells is the expression of a fluorescent fusion protein with a peroxisomal targeting signal (e.g., GFP-PTS1), which targets the peroxisomal matrix and allows live-cell imaging of peroxisomes. Here, we first present a protocol for the transfection of cultured mammalian cells with the peroxisomal marker EGFP-SKL to observe peroxisomes in living cells. This approach has revealed different motile behaviour of peroxisomes and novel insight into peroxisomal membrane dynamics (Rapp et al., 1996; Wiemer et al., 1997; Schrader et al., 2000). We then present a protocol which combines the live-cell approach with peroxisome motility measurements and quantification of peroxisome dynamics in mammalian cells. More recently, we used this approach to demonstrate that peroxisome motility and displacement is increased when a molecular tether, which associates peroxisomes with the ER, is lost (Costello et al., 2017b). Silencing of the peroxisomal acyl-CoA binding domain protein ACBD5, which interacts with ER-localised VAPB, increased peroxisome movement in skin fibroblasts, indicating that membrane contact sites can modulate organelle distribution and motility. The protocols described can be adapted to other cell types and organelles to measure and quantify organelle movement under different experimental conditions.

Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells.

Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus that anchors them to the membranes of organelles where they mediate critical cellular processes. Accordingly, mutations in genes encoding TA proteins have been identified in a number of severe inherited disorders. Despite the importance of correctly targeting a TA protein to its appropriate membrane, the mechanisms and signals involved are not fully understood. In this study, we identify additional peroxisomal TA proteins, discover more proteins that are present on multiple organelles, and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelle locations in mammals. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We show that subtle changes in those parameters can shift TA proteins between organelles, explaining why peroxisomes and mitochondria have many of the same TA proteins. This enabled us to associate characteristic physicochemical parameters in TA proteins with particular organelle groups. Using this classification allowed successful prediction of the location of uncharacterized TA proteins for the first time.

Atg37 regulates the assembly of the pexophagic receptor protein complex.

Like other selective autophagy pathways, the selective autophagy of peroxisomes, pexophagy, is controlled by receptor protein complexes (RPCs). The pexophagic RPC in Pichia pastoris consists of several proteins: Pex3 and Pex14 ligands in the peroxisomal membrane, Atg30 receptor, Atg11, and Atg17 scaffolds, and the phagophore protein Atg8. Recently, we identified a new component of the pexophagic RPC, Atg37, which is involved in the assembly of this complex. Atg37 is an integral peroxisomal membrane protein (PMP) that binds Pex3 and Atg30, but not Pex14 or Atg8. In the absence of Atg37, the recognition of Pex3 and recruitment of Atg17 by Atg30 are normal. However, the recruitment of Atg11 is severely affected suggesting that the role of Atg37 is to facilitate the Atg30-Atg11 interaction. Palmitoyl-CoA competes with Atg30 for the acyl-CoA binding domain of Atg37 in vitro and might regulate the dynamics of the pexophagic RPC in vivo. The human counterpart of Atg37, ACBD5, also localizes to peroxisomes and is specifically required for pexophagy. Therefore, it is tempting to speculate that ACBD5/ATG37 regulates the assembly of the pexophagic RPC in mammalian cells.