acyl-CoA Binding Domain Containing 3 Research Articles

An A-kinase anchoring protein (ACBD3) coordinates traffic-induced PKA activation at the Golgi

KDEL receptor (KDELR) is a key protein that recycles escaped endoplasmic reticulum (ER) resident proteins from the Golgi apparatus back to the ER and maintains a dynamic balance between these two organelles in the early secretory pathway. Studies have shown that this retrograde transport pathway is partly regulated by two KDELR-interacting proteins, acyl-CoA-binding domain-containing 3 (ACBD3), and cyclic AMP-dependent protein kinase A (PKA). However, whether Golgi-localized ACBD3, which was first discovered as a PKA-anchoring protein in mitochondria, directly interacts with PKA at the Golgi and coordinates its signaling in Golgi-to-ER traffic has remained unclear. In this study, we showed that the GOLD domain of ACBD3 directly interacts with the regulatory subunit II (RII) of PKA and effectively recruits PKA holoenzyme to the Golgi. Forward trafficking of proteins from the ER triggers activation of PKA by releasing the catalytic subunit from RII. Furthermore, we determined that depletion of ACBD3 reduces the Golgi fraction of RII, resulting in moderate, but constitutive activation of PKA and KDELR retrograde transport, independent of cargo influx from the ER. Taken together, these data demonstrate that ACBD3 coordinates the protein secretory pathway at the Golgi by facilitating KDELR/PKA-containing protein complex formation.

Mitochondrial A-kinase anchoring proteins in cardiac ventricular myocytes.

Compartmentation of cAMP signaling is a critical factor for maintaining the integrity of receptor‐specific responses in cardiac myocytes. This phenomenon relies on various factors limiting cAMP diffusion. Our previous work in adult rat ventricular myocytes (ARVMs) indicates that PKA regulatory subunits anchored to the outer membrane of mitochondria play a key role in buffering the movement of cytosolic cAMP. PKA can be targeted to discrete subcellular locations through the interaction of both type I and type II regulatory subunits with A‐kinase anchoring proteins (AKAPs). The purpose of this study is to identify which AKAPs and PKA regulatory subunit isoforms are associated with mitochondria in ARVMs. Quantitative PCR data demonstrate that mRNA for dual specific AKAP1 and 2 (D‐AKAP1 & D‐AKAP2), acyl‐CoA‐binding domain‐containing 3 (ACBD3), optic atrophy 1 (OPA1) are most abundant, while Rab32, WAVE‐1, and sphingosine kinase type 1 interacting protein (SPHKAP) were barely detectable. Biochemical and immunocytochemical analysis suggests that D‐AKAP1, D‐AKAP2, and ACBD3 are the predominant mitochondrial AKAPs exposed to the cytosolic compartment in these cells. Furthermore, we show that both type I and type II regulatory subunits of PKA are associated with mitochondria. Taken together, these data suggest that D‐AKAP1, D‐AKAP2, and ACBD3 may be responsible for tethering both type I and type II PKA regulatory subunits to the outer mitochondrial membrane in ARVMs. In addition to regulating PKA‐dependent mitochondrial function, these AKAPs may play an important role by buffering the movement of cAMP necessary for compartmentation.

A Crucial Role of ACBD3 Required for Coxsackievirus Infection in Animal Model Developed by AAV-Mediated CRISPR Genome Editing Technique.

Genetic screens using CRISPR/Cas9 have been exploited to discover host–virus interactions. These screens have identified viral dependencies on host proteins during their life cycle and potential antiviral strategies. The acyl-CoA binding domain containing 3 (ACBD3) was identified as an essential host factor for the Coxsackievirus B3 (CVB3) infection. Other groups have also investigated the role of ACBD3 as a host factor for diverse enteroviruses in cultured cells. However, it has not been tested if ACBD3 is required in the animal model of CVB3 infection. Owing to embryonic lethality, conventional knockout mice were not available for in vivo study. As an alternative approach, we used adeno-associated virus (AAV)-mediated CRISPR genome editing to generate mice that lacked ACBD3 within the pancreas, the major target organ for CVB3. Delivery of sgRNAs using self-complementary (sc) AAV8 efficiently induced a loss-of-function mutation in the pancreas of the Cas9 knock-in mice. Loss of ACBD3 in the pancreas resulted in a 100-fold reduction in the CVB3 titer within the pancreas and a noticeable reduction in viral protein expression. These results indicate a crucial function of ACBD3 in CVB3 infection in vivo. AAV-mediated CRISPR genome editing may be applicable to many in vivo studies on the virus–host interaction and identify a novel target for antiviral therapeutics.

Acyl-CoA-Binding Domain-Containing 3 (ACBD3; PAP7; GCP60): A Multi-Functional Membrane Domain Organizer.

Acyl-CoA-binding domain-containing 3 (ACBD3) is a multi-functional scaffolding protein, which has been associated with a diverse array of cellular functions, including steroidogenesis, embryogenesis, neurogenesis, Huntington’s disease (HD), membrane trafficking, and viral/bacterial proliferation in infected host cells. In this review, we aim to give a timely overview of recent findings on this protein, including its emerging role in membrane domain organization at the Golgi and the mitochondria. We hope that this review provides readers with useful insights on how ACBD3 may contribute to membrane domain organization along the secretory pathway and on the cytoplasmic surface of intracellular organelles, which influence many important physiological and pathophysiological processes in mammalian cells.

Gradual Phenotype Development in Huntington Disease Transgenic Minipig Model at 24 Months of Age

Background: Huntington disease (HD) is an incurable neurodegenerative disease caused by the expansion of a polyglutamine sequence in a gene encoding the huntingtin (Htt) protein, which is expressed in almost all cells of the body. In addition to small animal models, new therapeutic approaches (including gene therapy) require large animal models as their large brains are a more realistic model for translational research. Objective: In this study, we describe phenotype development in transgenic minipigs (TgHD) expressing the N-terminal part of mutated human Htt at the age of 24 months. Methods: TgHD and wild-type littermates were compared. Western blot analysis and subcellular fractionation of different tissues was used to determine the fragmentation of Htt. Immunohistochemistry and optical analysis of coronal sections measuring aggregates, Htt expression, neuroinflammation, and myelination was applied. Furthermore, the expression of Golgi protein acyl-CoA binding domain containing 3 (ACBD3) was analyzed. Results: We found age-correlated Htt fragmentation in the brain. Among various tissues studied, the testes displayed the highest fragmentation, with Htt fragments detectable even in cell nuclei. Also, Golgi protein ACBD3 was upregulated in testes, which is in agreement with previously reported testicular degeneration in TgHD minipigs. Nevertheless, the TgHD-specific mutated Htt fragments were also present in the cytoplasm of striatum and cortex cells. Moreover, microglial cells were activated and myelination was slightly decreased, suggesting the development of a premanifest stage of neurodegeneration in TgHD minipigs. Conclusions: The gradual development of a neurodegenerative phenotype, ac­companied with testicular degeneration, is observed in 24- month-old TgHD minipigs.

Overexpressed ACBD3 has prognostic value in human breast cancer and promotes the self-renewal potential of breast cancer cells by activating the Wnt/beta-catenin signaling pathway

Acyl-CoA binding domain containing 3 (ACBD3) is involved in the maintenance of Golgi structure and function through its interaction with the integral membrane protein. However, the clinical significance and biological role of ACBD3 in breast cancer remain unclear. Herein, we found that the mRNA and protein levels of ACBD3 were markedly up-regulated in breast cancer cells and tissues. Immunohistochemical analysis of breast cancer tissues demonstrated that ACBD3 overexpression was significantly associated with advanced clinicopathological features. Univariate and multivariate analysis indicated that ACBD3 overexpression correlates with poor prognosis in breast cancer. Furthermore, overexpressing ACBD3 promoted, while silencing ACBD3 inhibited, self-renewal and tumorigenesis in breast cancer cells in vitro and in vivo respectively. Importantly, upregulating ACBD3 promoted the self-renewal and tumorigenesis of breast cancer cells via activating the Wnt/beta-catenin signaling, and the pro-self-renewal effect of ACBD3 in breast cancer was antagonized by the Wnt signaling inhibitor TCF4-siRNA and Lef1-siRNA.These findings indicate that ACBD3 may represent candidate therapeutic targets to enable the elimination of breast cancer stem cells, providing the preclinical proof-of-concept for the prevention and treatment of breast cancer.

ACBD3 functions as a scaffold to organize the Golgi stacking proteins and a Rab33b‐GAP

Golgin45 plays important roles in Golgi stack assembly and is known to bind both the Golgi stacking protein GRASP55 and Rab2 in the medial-Golgi cisternae. In this study, we sought to further characterize the cisternal adhesion complex using a proteomics approach. We report here that Acyl-CoA binding domain containing 3 (ACBD3) is likely to be a novel binding partner of Golgin45. ACBD3 interacts with Golgin45 via its GOLD domain, while its co-expression significantly increases Golgin45 targeting to the Golgi. Furthermore, ACBD3 recruits TBC1D22, a Rab33b GTPase activating protein (GAP), to a large multi-protein complex containing Golgin45 and GRASP55. These results suggest that ACBD3 may provide a scaffolding to organize the Golgi stacking proteins and a Rab33b-GAP at the medial-Golgi.

A role for TSPO in mitochondrial Ca2+ homeostasis and redox stress signaling

The 18 kDa translocator protein TSPO localizes on the outer mitochondrial membrane (OMM). Systematically overexpressed at sites of neuroinflammation it is adopted as a biomarker of brain conditions. TSPO inhibits the autophagic removal of mitochondria by limiting PARK2-mediated mitochondrial ubiquitination via a peri-organelle accumulation of reactive oxygen species (ROS). Here we describe that TSPO deregulates mitochondrial Ca2+ signaling leading to a parallel increase in the cytosolic Ca2+ pools that activate the Ca2+-dependent NADPH oxidase (NOX) thereby increasing ROS. The inhibition of mitochondrial Ca2+ uptake by TSPO is a consequence of the phosphorylation of the voltage-dependent anion channel (VDAC1) by the protein kinase A (PKA), which is recruited to the mitochondria, in complex with the Acyl-CoA binding domain containing 3 (ACBD3). Notably, the neurotransmitter glutamate, which contributes neuronal toxicity in age-dependent conditions, triggers this TSPO-dependent mechanism of cell signaling leading to cellular demise. TSPO is therefore proposed as a novel OMM-based pathway to control intracellular Ca2+ dynamics and redox transients in neuronal cytotoxicity.

CircRNA_0046367 Prevents Hepatoxicity of Lipid Peroxidation: An Inhibitory Role against Hepatic Steatosis.

Hepatic steatosis reflects the miRNA-related pathological disorder with triglyceride accumulation and lipid peroxidation, which leads to nonalcoholic steatohepatitis, liver fibrosis/cirrhosis, and even hepatocellular carcinoma. Circular RNA (circRNA)/miRNA interaction reveals a novel layer of epigenetic regulation, yet the miRNA-targeting circRNA remains uncertain in hepatic steatosis. Here, we uncover circRNA_0046367 to be endogenous modulator of miR-34a that underlies hepatic steatosis. In contrast to its expression loss during the hepatocellular steatosis in vivo and in vitro, circRNA_0046367 normalization abolished miR-34a's inhibitory effect on peroxisome proliferator-activated receptor α (PPARα) via blocking the miRNA/mRNA interaction with miRNA response elements (MREs). PPARα restoration led to the transcriptional activation of genes associated with lipid metabolism, including carnitine palmitoyltransferase 2 (CPT2) and acyl-CoA binding domain containing 3 (ACBD3), and then resulted in the steatosis resolution. Hepatotoxicity of steatosis-related lipid peroxidation, being characterized by mitochondrial dysfunction, growth arrest, and apoptosis, is resultantly prevented after the circRNA_0046367 administration. These findings indicate a circRNA_0046367/miR-34a/PPARα regulatory system underlying hepatic steatosis. Normalized expression of circRNA_0046367 may ameliorate the lipoxidative stress on the basis of steatosis attenuation. circRNA_0046367, therefore, is suggested to be potential approach to the therapy of lipid peroxidative damage.

Regulation of Mitochondrial Signaling and Quality Control by the 18KDA Translocator Protein (TSPO)

The 18-kDa translocator protein (TSPO) is localized to the outer mitochondrial membrane (OMM) where it is found in complex with the voltage dependent anion channel (VDAC). TSPO expression is elevated in cancer and inflammatory conditions and it is well known for its ligand binding properties and application in PET imaging as a marker of neuroinflammation. TSPO is implicated in multiple mitochondrial functions including steroidogenesis, apoptosis and ATP production although precise molecular mechanisms remain to be elucidated. We recently demonstrated that increased TSPO expression limits mitochondrial ubiquitination and mitophagy through a reactive oxygen species (ROS) -dependent process, which leads to changes in mitochondrial morphology and biomass.Here we have corroborated this pathway by establishing an interplay with the protein kinase A (PKA) signaling complex, which is recruited to mitochondria by TSPO via acyl-CoA binding domain containing 3 (ACBD3). Using targeted luminescent protein chimeras, we show that the combined accumulation of TSPO and PKA alters subcellular Ca2+ dynamics and metabolism, which we propose arises from increased VDAC phosphorylation and subsequent changes in channel activity. Of note, this pathway is activated under glutamate-induced cell stress in the neuronal cell line, SH-SY5Y.This work highlights an important role for the OMM in fine-tuning mitochondrial signaling and mitophagy. As an inducible factor, TSPO represents a critical physiological element that facilitates mitochondrial adaptation to altered cellular metabolic requirements under stress conditions.

Acyl-CoA-binding domain containing 3 modulates NAD+ metabolism through activating poly(ADP-ribose) polymerase 1.

NAD(+) plays essential roles in cellular energy homoeostasis and redox state, functioning as a cofactor along the glycolysis and citric acid cycle pathways. Recent discoveries indicated that, through the NAD(+)-consuming enzymes, this molecule may also be involved in many other cellular and biological outcomes such as chromatin remodelling, gene transcription, genomic integrity, cell division, calcium signalling, circadian clock and pluripotency. Poly(ADP-ribose) polymerase 1 (PARP1) is such an enzyme and dysfunctional PARP1 has been linked with the onset and development of various human diseases, including cancer, aging, traumatic brain injury, atherosclerosis, diabetes and inflammation. In the present study, we showed that overexpressed acyl-CoA-binding domain containing 3 (ACBD3), a Golgi-bound protein, significantly reduced cellular NAD(+) content via enhancing PARP1's polymerase activity and enhancing auto-modification of the enzyme in a DNA damage-independent manner. We identified that extracellular signal-regulated kinase (ERK)1/2 as well as de novo fatty acid biosynthesis pathways are involved in ACBD3-mediated activation of PARP1. Importantly, oxidative stress-induced PARP1 activation is greatly attenuated by knocking down the ACBD3 gene. Taken together, these findings suggest that ACBD3 has prominent impacts on cellular NAD(+) metabolism via regulating PARP1 activation-dependent auto-modification and thus cell metabolism and function.

New route for the activation of poly(ADP-ribose) polymerase-1: a passage that links poly(ADP-ribose) polymerase-1 to lipotoxicity?

In this issue of Biochemical Journal, Chen and colleagues characterize an interaction between ACBD3 (acyl-CoA-binding domain-containing 3) protein and PARP [poly(ADP-ribose) polymerase]-1 through the activation of ERKs (extracellular-signal-regulated kinases). This study envisages a pathway through which ABCD3 translates enhanced fatty acid levels to ERK and consequently PARP-1 activation. The consequences of PARP-1 activation lead to cellular and tissue damage, implying that the ACBD3/PARP-1 pathway is an important pathway in lipotoxicity events.

Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment

Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD(+)-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake.

C11 Transgenic Minipigs For N-terminal Part Of Human Mutated Huntingtin: Following Disease Development

Background In order to facilitate studies of pathogenesis and therapy of Huntington’s disease (HD), we have generated transgenic minipigs using microinjection of a lentiviral vector encoding N-terminal (548 aa) part of human huntingtin (Htt) containing 126 CAG/CAA repeats under the control of the human Htt promoter. The progression of HD in human patients is predominantly linked with formation of aggregates, oligomers, and fragments of Htt. Among other factors corresponding to HD is lowered BDNF (brain-derived neurotrophic factor) expression in brain, accumulation of ACBD3 (acyl-CoA binding domain containing 3), and formation of toxic Rhes/mtHtt/ACBD3 complex. Aim To follow disease development in transgenic (TgHD) minipigs using specific biomarkers of HD. Methods At present, TgHD and WT (wild-type) minipigs of F0, F1, F2 and F3 generations are being used for invasive (24 and 36 months old animals) and non-invasive approaches (up to five years old animals). We perform biochemical assays on tissues (with main focus on brain) and cells (fibroblasts, and buccal swabs) in order to follow HD development in our model. Results To date, we have not been successful in the aggregate detection, which could be due to the young age of TgHD animals. However, we have detected Htt oligomers, several Htt fragments of lower molecular weight in samples from TgHD minipigs and not from WT siblings. In addition, we monitor altered levels of proteins previously reported as markers of HD development, namely BDNF and Rhes/mtHtt/ACBD3 complex Conclusion We provide complex information about HD phenotype development in transgenic minipig model between 24–60 months of age. Support CHDI Foundation (A-5378), TACR (TA01011466), European Regional Development FundCZ.1.05/2.1.00/03.0124

Golgi Protein ACBD3 Mediates Neurotoxicity Associated with Huntington’s Disease

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disease caused by the expansion of polyglutamine repeats in the gene for huntingtin (Htt). In HD, the corpus striatum selectively degenerates despite the uniform expression of mutant huntingtin (mHtt) throughout the brain and body. Striatal selectivity reflects the binding of the striatal-selective protein Rhes to mHtt to augment cytotoxicity, but molecular mechanisms underlying the toxicity have been elusive. Here, we report that the Golgi protein acyl-CoA binding domain containing 3 (ACBD3) mediates mHtt cytotoxicity via a Rhes/mHtt/ACBD3 complex. ACBD3 levels are markedly elevated in the striatum of HD patients, in a striatal cell line harboring polyglutamine repeats, and in the brains of HD mice. Moreover, ACBD3 deletion abolishes HD neurotoxicity, which is increased by ACBD3 overexpression. Enhanced levels of ACBD3 elicited by endoplasmic reticulum, mitochondrial, and Golgi stresses may account for HD-associated augmentation of ACBD3 and neurodegeneration.

Maturation and Activity of Sterol Regulatory Element Binding Protein 1 Is Inhibited by Acyl-CoA Binding Domain Containing 3

Imbalance of lipid metabolism has been linked with pathogenesis of a variety of human pathological conditions such as diabetes, obesity, cancer and neurodegeneration. Sterol regulatory element binding proteins (SREBPs) are the master transcription factors controlling the homeostasis of fatty acids and cholesterol in the body. Transcription, expression, and activity of SREBPs are regulated by various nutritional, hormonal or stressful stimuli, yet the molecular and cellular mechanisms involved in these adaptative responses remains elusive. In the present study, we found that overexpressed acyl-CoA binding domain containing 3 (ACBD3), a Golgi-associated protein, dramatically inhibited SREBP1-sensitive promoter activity of fatty acid synthase (FASN). Moreover, lipid deprivation-stimulated SREBP1 maturation was significantly attenuated by ACBD3. With cell fractionation, gene knockdown and immunoprecipitation assays, it was showed that ACBD3 blocked intracellular maturation of SREBP1 probably through directly binding with the lipid regulator rather than disrupted SREBP1-SCAP-Insig1 interaction. Further investigation revealed that acyl-CoA domain-containing N-terminal sequence of ACBD3 contributed to its inhibitory effects on the production of nuclear SREBP1. In addition, mRNA and protein levels of FASN and de novo palmitate biosynthesis were remarkably reduced in cells overexpressed with ACBD3. These findings suggest that ACBD3 plays an essential role in maintaining lipid homeostasis via regulating SREBP1's processing pathway and thus impacting cellular lipogenesis.

Acyl-CoA binding domain containing 3 (ACBD3) recruits the protein phosphatase PPM1L to ER–Golgi membrane contact sites

The metal-dependent protein phosphatase family (PPM) governs a number of signaling pathways. PPM1L, originally identified as a negative regulator of stress-activated protein kinase signaling, was recently shown to be involved in the regulation of ceramide trafficking at ER–Golgi membrane contact sites. Here, we identified acyl-CoA binding domain containing 3 (ACBD3) as an interacting partner of PPM1L. We showed that this association, which recruits PPM1L to ER–Golgi membrane contact sites, is mediated by a GOLD (Golgi dynamics) domain in ACBD3. These results suggested that ACBD3 plays a pivotal role in ceramide transport regulation at the ER–Golgi interface. Structured summary of protein interactionsACBD3 and PPM1Lcolocalize by fluorescence microscopy (View interaction)FYCO1physically interacts with PPM1L by pull down (View interaction)SEC14L2physically interacts with PPM1L by pull down (View interaction)ACBD3physically interacts with PPM1L by pull down (View interaction)SEC14L1physically interacts with PPM1L by pull down (View interaction)PPM1Lphysically interacts with ACBD3 by two hybrid (View interaction)