TLDc Domain Research Articles

Interaction between the TBC1D24 TLDc domain and the KIBRA C2 domain is disrupted by two epilepsy-associated TBC1D24 missense variants

Mutations of human TBC1D24 are associated with either deafness, epilepsy or DOORS syndrome (deafness, onychodystrophy, osteodystrophy, cognitive disability, seizures). The causal relationships between TBC1D24 variants and the different clinical phenotypes are not understood. Our hypothesis is that phenotypic heterogeneity of missense mutations of TBC1D24 results, in part, from perturbed binding of different protein partners. To discover novel protein partners of TBC1D24, we conducted a yeast two-hybrid (Y2H) screen using mouse full-length (FL) TBC1D24 as bait. KIBRA, a scaffold protein encoded by Wwc1, was identified as a partner of TBC1D24. KIBRA functions in the Hippo signaling pathway and is important for human cognition and memory. The TBC1D24 TLDc domain binds to KIBRA FL and to its C2 domain, confirmed by Y2H assays. No interaction was detected with Y2H assays between the KIBRA C2 domain and TLDc domains of NCOA7, MEAK7 and OXR1. Moreover, the C2 domains of other WWC family proteins do not interact with the TLDc domain of TBC1D24, demonstrating specificity. The mRNAs encoding TBC1D24 and KIBRA proteins in mouse are coexpressed at least in a subset of hippocampal cells indicating availability to interact in vivo. As two epilepsy-associated recessive variants (Gly511Arg and Ala515Val) in the TLDc domain of human TBC1D24 disrupt the interaction with human KIBRA C2 domain, this study reveals a pathogenic mechanism of TBC1D24-associated epilepsy, linking the TBC1D24 and KIBRA pathways. The interaction of TBC1D24-KIBRA is physiologically meaningful and necessary to reduce the risk of epilepsy.

The merger of two major families of proteins that regulate cellular processes

In this issue of Structure, Oot and Wilkens1 present new mechanistic insights to finally merge the function of V-ATPase and TLDc domain proteins. They show that TLDc proteins directly affect V-ATPase activity and assembly, expanding our understanding of how V-ATPase and TLDc proteins exert a plethora of biological functions.

Knockout of the V-ATPase Interacting Protein Tldc2 Inhibits Renal B-Intercalated Cell Function and Urine Alkalinization

The V-ATPase, or proton pump, generates electrochemical gradients across membranes driving cell biological processes. The kidney-specific V-ATPase is expressed in intercalated cells (ICs), which regulate acid-base homeostasis by adjusting plasma membrane V-ATPase via regulated traffcking. In the kidney, A-ICs transport protons into the tubule lumen while B-ICs move protons into the peritubular space and, ultimately, the blood. The V-ATPase is distributed between intracellular vesicles and the apical plasma membrane in A-ICs, whereas B-ICs express V-ATPase not only in intracellular vesicles, but also basolaterally. In general, V-ATPase dysfunction in ICs leads to distal renal tubular acidosis (dRTA) implying a functional dominance of A-IC in acid/base physiology. B-ICs have, however, been implicated in blood pressure regulation due to their role in chloride reabsorption and, indirectly, Na transport. We recently identified a new class of V-ATPase interacting proteins defined by their TLDc domain (Ncoa7, Oxr1, Tbc1d24, Tldc1, Tldc2), all of which interact with the IC-specific B1 subunit of the V-ATPase. Of importance, Ncoa7 knockout mice had decreased V-ATPase expression in ICs and developed dRTA, which led us to hypothesize that TLDc proteins regulate the kidney V-ATPase. While Ncoa7 is specifically expressed in A-ICs, Tldc2 is one of the most differentially expressed proteins in B-ICs, prompting us to ask whether Tldc2 deletion affected V-ATPase function in these cells. Using global Tldc2 knockout mice (KO), we found that urine pH is significantly more acidic in KO mice than in WT mice in both males and females (pH 5.8 in male KOs versus pH 6.4 in male WTs and pH 5.8 in female KOs versus pH 6.2 in female WTs). After an acid challenge with NH4Cl, the urine pH of WT mice was significantly decreased, but urine pH was unchanged in KO mice, since it was already low under baseline conditions (pH 5.8 for male and female WT and KO mice). After base challenging mice with NaHCO3−, urine pH was greatly increased in WT mice (pH 7.8 in male WTs and pH 7.5 in female WTs), while urine pH increased to a lesser, but still significant amount, in KO mice (pH 6.3 in male KOs and 7.2 in female KOs). Interestingly, after the bicarbonate challenge urine pH was significantly lower in KO males versus WT males (pH 6.3 in male KOs versus pH 7.8 in male WTs). In females, urine pH was also lower in KO mice versus WT mice after the base challenge but did not reach statistical significance due to high variability between mice (pH 7.2 in female KOs versus pH 7.5 in female WTs). There was no significant difference in blood pH between Tldc2 KO and Tldc2 WT mice of either sex, with or without treatment, suggesting possible respiratory compensation. The inability of KO mice to alkalinize their urine points to a defect in bicarbonate-secreting B-ICs, in which Tldc2 is specifically expressed. To examine this, localization of the B1 subunit was visualized in B-ICs by immunofluorescence, and membrane accumulation was quantified by line intensity scanning using FIJI. Our preliminary results show that basolateral membrane accumulation of B1 was reduced in KO mice relative to WT mice. In summary, our data suggest that loss of Tldc2 negatively affects B-IC function most likely by impairing basolateral accumulation of the V-ATPase. We conclude that Tldc2 is part of a regulatory mechanism that participates in the accumulation of the V-ATPase holoenzyme at the basolateral surface of B-ICs. Whether this is due to changes in assembly or traffcking of the holoenzyme remains to be determined. NIH/NIDDK R01DK121848. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Human V-ATPase function is positively and negatively regulated by TLDc proteins

Proteins that contain a highly conserved TLDc domain (Tre2/Bub2/Cdc16 LysM domain catalytic) offer protection against oxidative stress and are widely implicated in neurological health and disease. How this family of proteins exerts their function, however, is poorly understood. We have recently found that the yeast TLDc protein, Oxr1p, inhibits the proton pumping vacuolar ATPase (V-ATPase) by inducing disassembly of the pump. While loss of TLDc protein function in mammals shares disease phenotypes with V-ATPase defects, whether TLDc proteins impact human V-ATPase activity directly is unclear. Here we examine the effects of five human TLDc proteins, TLDC2, NCOA7, OXR1, TBC1D24, and mEAK7 on the activity of the human V-ATPase. We find that while TLDC2, TBC1D24, and the TLDc domains of OXR1 and NCOA7 inhibit V-ATPase by inducing enzyme disassembly, mEAK7 activates the pump. The data thus shed new light both on mammalian TLDc protein function and V-ATPase regulation.

Yeast TLDc domain proteins regulate assembly state and subcellular localization of the V-ATPase

Yeast vacuoles perform crucial cellular functions as acidic degradative organelles, storage compartments, and signaling hubs. These functions are mediated by important protein complexes, including the vacuolar-type H+-ATPase (V-ATPase), responsible for organelle acidification. To gain a more detailed understanding of vacuole function, we performed cross-linking mass spectrometry on isolated vacuoles, detecting many known as well as novel protein-protein interactions. Among these, we identified the uncharacterized TLDc-domain-containing protein Rtc5 as a novel interactor of the V-ATPase. We further analyzed the influence of Rtc5 and of Oxr1, the only other yeast TLDc-domain-containing protein, on V-ATPase function. We find that both Rtc5 and Oxr1 promote the disassembly of the vacuolar V-ATPase in vivo, counteracting the role of the RAVE complex, a V-ATPase assembly chaperone. Furthermore, Oxr1 is necessary for the retention of a Golgi-specific subunit of the V-ATPase in this compartment. Collectively, our results shed light on the in vivo roles of yeast TLDc-domain proteins as regulators of the V-ATPase, highlighting the multifaceted regulation of this crucial protein complex.

Investigation of a novel TBC1D24 variation causing autosomal dominant non-syndromic hearing loss.

Hearing loss is considered one of the most common sensory neurological defects, with approximately 60% of cases attributed to genetic factors. Human pathogenic variants in the TBC1D24 gene are associated with various clinical phenotypes, including dominant nonsyndromic hearing loss DFNA65, characterized by progressive hearing loss after the development of language. This study provides an in-depth analysis of the causative gene and mutations in a family with hereditary deafness. We recruited a three-generation family with autosomal dominant nonsyndromic hearing loss (ADNSHL) and conducted detailed medical histories and relevant examinations. Next-generation sequencing (NGS) was used to identify genetic variants in the proband, which were then validated using Sanger sequencing. Multiple computational software tools were employed to predict the impact of the variant on the function and structure of the TBC1D24 protein. A series of bioinformatics tools were applied to determine the conservation characteristics of the sequence, establish a three-dimensional structural model, and investigate changes in molecular dynamics. A detailed genotype and phenotype analysis were carried out. The family exhibited autosomal dominant, progressive, postlingual, and nonsyndromic sensorineural hearing loss. A novel heterozygous variant, c.1459C>T (p.His487Tyr), in the TBC1D24 gene was identified and confirmed to be associated with the hearing loss phenotype in this family. Conservation analysis revealed high conservation of the amino acid affected by this variant across different species. The mutant protein showed alterations in thermodynamic stability, elasticity, and conformational dynamics. Molecular dynamics simulations indicated changes in RMSD, RMSF, Rg, and SASA of the mutant structure. We computed the onset age of non-syndromic hearing loss associated with mutations in the TBC1D24 gene and identified variations in the hearing progression time and annual threshold deterioration across different frequencies. The identification of a new variant associated with rare autosomal dominant nonsyndromic hereditary hearing loss in this family broadens the range of mutations in the TBC1D24 gene. This variant has the potential to influence the interaction between the TLDc domain and TBC domain, thereby affecting the protein's biological function.

A loss-of-function mutation in human Oxidation Resistance 1 disrupts the spatial–temporal regulation of histone arginine methylation in neurodevelopment

BackgroundOxidation Resistance 1 (OXR1) gene is a highly conserved gene of the TLDc domain-containing family. OXR1 is involved in fundamental biological and cellular processes, including DNA damage response, antioxidant pathways, cell cycle, neuronal protection, and arginine methylation. In 2019, five patients from three families carrying four biallelic loss-of-function variants in OXR1 were reported to be associated with cerebellar atrophy. However, the impact of OXR1 on cellular functions and molecular mechanisms in the human brain is largely unknown. Notably, no human disease models are available to explore the pathological impact of OXR1 deficiency.ResultsWe report a novel loss-of-function mutation in the TLDc domain of the human OXR1 gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generate patient-derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identify that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1-dependent mechanisms regulating gene expression during neurodevelopment. We model the function of OXR1 in early human brain development using patient-derived brain organoids revealing that OXR1 contributes to the spatial–temporal regulation of histone arginine methylation in specific brain regions.ConclusionsThis study provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency in patients.

Tender love and disassembly: How a TLDc domain protein breaks the V-ATPase.

Vacuolar ATPases (V-ATPases, V1 Vo -ATPases) are rotary motor proton pumps that acidify intracellular compartments, and, when localized to the plasma membrane, the extracellular space. V-ATPase is regulated by a unique process referred to as reversible disassembly, wherein V1 -ATPase disengages from Vo proton channel in response to diverse environmental signals. Whereas the disassembly step of this process is ATP dependent, the (re)assembly step is not, but requires the action of a heterotrimeric chaperone referred to as the RAVE complex. Recently, an alternative pathway of holoenzyme disassembly was discovered that involves binding of Oxidation Resistance 1 (Oxr1p), a poorly characterized protein implicated in oxidative stress response. Unlike conventional reversible disassembly, which depends on enzyme activity, Oxr1p induced dissociation can occur in absence of ATP. Yeast Oxr1p belongs to the family of TLDc domain containing proteins that are conserved from yeast to mammals, and have been implicated in V-ATPase function in a variety of tissues. This brief perspective summarizes what we know about the molecular mechanisms governing both reversible (ATP dependent) and Oxr1p driven (ATP independent) V-ATPase dissociation into autoinhibited V1 and Vo subcomplexes.

Loss of Ncoa7 leads to oxidative stress in kidney intercalated cells

Oxidative stress (OS) results from excessive production of reactive oxygen species, relative to endogenous antioxidant defenses, leading to cellular damage. The metabolically active kidney is vulnerable to OS, which is a key step in the progression of many renal pathologies. Furthermore, many non-renal pathologies are characterized by OS. Therefore, although our work focuses on kidney epithelial cells, it has significant implications for understanding how OS is regulated at a molecular level in many organs and cell types.The V-ATPase, or proton pump, is required for the acidification of intracellular compartments. Recently, we identified a family of V-ATPase interacting proteins defined by their TLDc domain (Ncoa7, Oxr1, Tbc1d24, Tldc1, and Tldc2). TLDc proteins are protective against OS in the nervous system and mutations cause human neurological pathologies. However, little is known about the molecular mechanisms that provide these protective properties. The V-ATPase, like TLDc proteins, also protects against OS. Therefore, we hypothesize that TLDc proteins play a V-ATPase-dependent protective role against OS. This is supported by our previous findings that mutating an amino acid in Ncoa7 that is protective against OS abolishes the interaction with the V-ATPase, and Ncoa7 knockout (KO) decreases V-ATPase-dependent urinary acidification in mice.To test this, we focused on Ncoa7 because it showed the most robust protective effect against OS in previous studies and is highly expressed in the kidney where it is enriched in epithelial intercalated cells (ICs). RNA sequencing of isolated ICs from Ncoa7 KO mice revealed significant downregulation of antioxidant enzymes including glutathione peroxidase-3 (Gpx3) and carbonyl reductase-2 (Cbr2). Immunofluorescence (IF) showed Cbr2 enrichment in medullary ICs from control mouse kidneys and a decrease in Cbr2 fluorescent signal intensity in ICs from Ncoa7 KO mice. Furthermore, Cbr2 localizes to the mitochondria of ICs, a significant source of cellular OS.To investigate whether the loss of Ncoa7 promotes OS in ICs we examined changes in OS markers by IF. We monitored the expression of Advanced Glycation End products (AGEs) and malondialdehyde (MDA) in fixed kidney sections using specific antibodies. AGEs are generated by glycation events accompanying OS and their formation can be suppressed by Gpx3. MDA is a product of lipid oxidation due to OS and can be reduced by Cbr2. ICs in Ncoa7 KO mice exhibited a striking accumulation of both AGEs and MDA, often found in large V-ATPase-bound intracellular compartments. Furthermore, knockdown of Ncoa7 in cultured kidney cells increased expression of AGEs, supporting loss of Ncoa7 as the mechanism behind the increased accumulation of intracellular AGEs.Based on these data we conclude that loss of Ncoa7 promotes OS in renal intercalated cells potentially through a regulatory interaction with the V-ATPase, which leads to decreased expression of antioxidant enzymes. NIH/NIDDK T32DK007540-33 and NIH/NIDDK R01DK121848 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The involvement of an interferon-induced protein 44-like (CgIFI44L) in the antiviral immune response of Crassostrea gigas

Interferon-stimulated genes (ISGs) encoding proteins are the essential executors of interferon (IFN) mediated antiviral defense. In the present study, an ISG member, interferon-induced protein 44-like (IFI44L) gene (designed as CgIFI44L-1) was identified from the Pacific oyster Crassostrea gigas. The ORF of CgIFI44L-1 cDNA was of 1437 bp encoding a polypeptide of 479 amino acids with a TLDc domain and an MMR_HSR1 domain. The mRNA transcripts of CgIFI44L-1 were detected in all the tested tissues with highest level in haemocytes, which was 15.78-fold of that in gonad (p < 0.001). Among the haemocytes, the CgIFI44L-1 protein was detected to be highly expressed in granulocytes with dominant distribution in cytoplasm. The mRNA expression level of CgIFI44L-1 in haemocytes was significantly induced by poly (I:C) stimulation, and the expression level peaked at 24 h, which was 24.24-fold (p < 0.0001) of that in control group. After the treatment with the recombinant protein of an oyster IFN-like protein (rCgIFNLP), the mRNA expression level of CgIFI44L-1 was significantly enhanced at 6 h, 12 h and 24 h, which was 2.67-fold (p < 0.001), 5.44-fold (p < 0.001) and 5.16-fold (p < 0.001) of that in control group, respectively. When the expressions of CgSTAT and CgIFNLP were knocked down by RNA interference (RNAi), the mRNA transcripts of CgIFI44L-1 were significantly down-regulated after poly (I:C) stimulation, which was 0.09-fold (p < 0.001) and 0.06-fold (p < 0.001) of those in EGFP group, respectively. These results suggested that CgIFI44L-1 was a conserved ISG in oyster, which was regulated by CgIFNLP and CgSTAT, and involved in the oyster antiviral immune response.

CryoEM of endogenous mammalian V-ATPase interacting with the TLDc protein mEAK-7.

V-ATPases are rotary proton pumps that serve as signaling hubs with numerous protein binding partners. CryoEM with exhaustive focused classification allowed detection of endogenous proteins associated with porcine kidney V-ATPase. An extra C subunit was found in ∼3% of complexes, whereas ∼1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 showed that mEAK-7's TLDc domain interacts with V-ATPase's stator, whereas its C-terminal α helix binds V-ATPase's rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike the yeast TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that the mEAK-7:V-ATPase interaction is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. These results show that V-ATPase binding by TLDc domain proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzyme's activity.

Molecular basis of mEAK7-mediated human V-ATPase regulation

The activity of V-ATPase is well-known to be regulated by reversible dissociation of its V1 and Vo domains in response to growth factor stimulation, nutrient sensing, and cellular differentiation. The molecular basis of its regulation by an endogenous modulator without affecting V-ATPase assembly remains unclear. Here, we discover that a lysosome-anchored protein termed (mammalian Enhancer-of-Akt-1-7 (mEAK7)) binds to intact V-ATPase. We determine cryo-EM structure of human mEAK7 in complex with human V-ATPase in native lipid-containing nanodiscs. The structure reveals that the TLDc domain of mEAK7 engages with subunits A, B, and E, while its C-terminal domain binds to subunit D, presumably blocking V1–Vo torque transmission. Our functional studies suggest that mEAK7, which may act as a V-ATPase inhibitor, does not affect the activity of V-ATPase in vitro. However, overexpression of mEAK7 in HCT116 cells that stably express subunit a4 of V-ATPase represses the phosphorylation of ribosomal protein S6. Thus, this finding suggests that mEAK7 potentially links mTOR signaling with V-ATPase activity.

V‐ATPase Domain Assembly is Increased in Ncoa7 KO Mice

The V‐ATPase is a multisubunit, transmembrane proton pump that generates electrochemical gradients across membranes driving essential cell biological processes. Critically, for proton pumping activity, the two distinct domains of the V‐ATPase (V1 and VO) must assemble into a functional holoenzyme. The kidney‐specific V‐ATPase is expressed in intercalated cells (ICs), which regulate acid‐base homeostasis by adjusting the number of apical V‐ATPase holoenzymes via regulated trafficking. Thus, V‐ATPase dysfunction in ICs leads to distal renal tubular acidosis (dRTA), which is characterized by an accumulation of acid in the blood. Despite this, little is known about the molecular regulation of the kidney V‐ATPase.We recently identified a new class of V‐ATPase interacting proteins defined by their TLDc domain which protect against oxidative stress (Ncoa7, Oxr1, Tbc1d24, Tldc1, Tldc2). We showed that the purified TLDc domains of Ncoa7, Oxr1, and Tldc2 interact with the IC‐specific B1 subunit of the V‐ATPase. Furthermore, endogenous Ncoa7, Oxr1, and Tbc1d24 interacted with the B1 subunit. Endogenous Tldc1 or Tldc2 were expressed at low levels in the kidney, but proteins overexpressed in cell cultures interacted with the B1 V‐ATPase from kidney lysates. Of importance, Ncoa7 knockout (KO) mice had decreased V‐ATPase expression in ICs and developed dRTA, which led us to hypothesize that the TLDc proteins regulate the kidney V‐ATPase.Here, we asked whether Ncoa7 deletion affected apical trafficking of the V‐ATPase or assembly of the V1 and VO domains into a holoenzyme in renal ICs after stimulation of proton secretion. Wild type (WT) and Ncoa7 KO mice were injected with CPT‐cAMP, to stimulate proton secretion, or saline as a control. Localization of the B1 and a4 subunits was visualized in ICs by immunofluorescence using specific antibodies, and apical accumulation was quantified by line intensity scanning. Our results show that cAMP significantly increased apical accumulation of B1 and a4 in WT mice from 55% to 78% of the cellular total, as expected. However, ICs in the Ncoa7 KO mice were already highly activated at baseline, with only a slight trend towards increased apical accumulation of the V‐ATPase after cAMP treatment (79% to 82%). A Pearson’s correlation analysis of the colocalization of B1 and a4, as a measure of V‐ATPase assembly, revealed a significant increase in assembly in Ncoa7 KO mice, relative to control animals, after both saline and cAMP treatment. Furthermore, a Proximity Ligation Assay also showed a marked increase in assembly of the V‐ATPase in untreated Ncoa7 KO ICs compared to control WT mice.These data suggest that loss of Ncoa7 does not affect apical accumulation of the V‐ATPase in ICs. Ncoa7 KO mice ICs are already highly activated in untreated mice. This is probably a compensatory response to the prevailing acidosis observed in these mice, which may be due to decreased total V‐ATPase protein expression in their ICs. Our data shows, however, increased assembly of the remaining V‐ATPase in Ncoa7 KO mice. Thus, we conclude that Ncoa7 is part of a regulatory mechanism that inhibits assembly of the V‐ATPase into a holoenzyme in WT mice, while not affecting trafficking of V‐ATPase subunits to the apical surface. How this negative effect is balanced in vivo to determine the overall degree of V‐ATPase holoenzyme assembly remains to be determined.

Age-Related Neuroprotection by Dietary Restriction Requires OXR1-Mediated Retromer Function

Dietary restriction (DR) is the most robust method to delay aging and the onset of neurogenerative disorders across multiple species, though the mechanisms behind this phenomenon remain unknown. To elucidate how DR mediates lifespan extension, we analyzed natural genetic variants that associate with increased longevity under DR conditions in the Drosophila Genetic Reference Panel. We found that neuronal expression of the fly homolog of human Oxidation Resistance 1 (OXR1) is necessary for DR-mediated lifespan extension. Neuronal knockdown of OXR1 also accelerated visual decline but not physical decline, arguing for a specific role of OXR1 in neuronal signaling. Further, we find that overexpression of the TLDc domain from human OXR1 is sufficient for lifespan extension in a diet-dependent manner. Studies from the Accelerating Medicines Partnership - Alzheimer's Disease network show that patients with reduced OXR1 protein levels are more prone to Alzheimer's disease diagnosis, and we find that overexpression of human OXR1 is protective in animal and cell Alzheimer's models. In seeking the mechanism by which OXR1 protects against age-related neuronal decline, we discovered that it provides a necessary function in regulating the neuronal retromer complex, which is essential for the recycling of transmembrane receptors and for maintenance of autophagy. We further discovered that OXR1 deficiency can be rescued by genetic or pharmacological enhancement of retromer function, and that this enhancement extends lifespan and healthspan. Understanding how OXR1 operates could help uncover novel mechanisms to slow neurodegeneration including Alzheimer's disease.

The evolutionary conserved TLDc domain defines a new class of (H+)V-ATPase interacting proteins

We recently found that nuclear receptor coactivator 7 (Ncoa7) and Oxr1 interact with the proton-pumping V-ATPase. Ncoa7 and Oxr1 belong to a group of proteins playing a role in the oxidative stress response, that contain the conserved “TLDc” domain. Here we asked if the three other proteins in this family, i.e., Tbc1d24, Tldc1 and Tldc2 also interact with the V-ATPase and if the TLDc domains are involved in all these interactions. By co-immunoprecipitation, endogenous kidney Tbc1d24 (and Ncoa7 and Oxr1) and overexpressed Tldc1 and Tldc2, all interacted with the V-ATPase. In addition, purified TLDc domains of Ncoa7, Oxr1 and Tldc2 (but not Tbc1d24 or Tldc1) interacted with V-ATPase in GST pull-downs. At the amino acid level, point mutations G815A, G845A and G896A in conserved regions of the Ncoa7 TLDc domain abolished interaction with the V-ATPase, and S817A, L926A and E938A mutations resulted in decreased interaction. Furthermore, poly-E motifs upstream of the TLDc domain in Ncoa7 and Tldc2 show a (nonsignificant) trend towards enhancing the interaction with V-ATPase. Our principal finding is that all five members of the TLDc family of proteins interact with the V-ATPase. We conclude that the TLDc motif defines a new class of V-ATPase interacting regulatory proteins.

Crystal structure of the TLDc domain of human NCOA7-AS.

The TLDc [Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic] domain is associated with oxidation-resistance related functions and is well conserved among eukaryotes. Seven proteins possess a TLDc domain in humans, notably proteins belonging to the oxidation resistance protein (OXR), nuclear receptor coactivator 7 (NCOA7) and TBC1 domain family member 24 (TBC1D24) families. Although the mechanism is unknown, a protective role of TLDc proteins against oxidative stress, notably in the brain, has been demonstrated. Neurobiological disorders caused by mutations in the TLDc domain have also been reported. The human NCOA7 gene encodes several mRNA isoforms; among these, isoform 4, named NCOA7-AS, is up-regulated by type 1 interferon in response to viral infection. NCOA7 and NCOA7-AS both interact with several subunits of the vacuolar proton pump V-ATPase, which leads to increased acidification of the endolysosomal system and consequently impairs infection by viruses that enter their host cells through the endosomal pathway, such as influenza A virus and hepatitis C virus. Similarly to full-length NCOA7, NCOA7-AS possesses a TLDc domain in its C-terminus. Structures of TLDc domains have been reported from zebrafish and fly but not from humans. Here, the expression, purification and crystallization of the TLDc domain from NCOA7 and NCOA7-AS is reported. The crystal structure solved at 1.8 Å resolution is compared with previously solved three-dimensional structures of TLDc domains.

TBC1D24 emerges as an important contributor to progressive postlingual dominant hearing loss

Several TBC1D24 variants are causally involved in the development of profound, prelingual hearing loss (HL) and different epilepsy syndromes inherited in an autosomal recessive manner. Only two TBC1D24 pathogenic variants have been linked with postlingual progressive autosomal dominant HL (ADHL). To determine the role of TBC1D24 in the development of ADHL and to characterize the TBC1D24-related ADHL, clinical exome sequencing or targeted multigene (n = 237) panel were performed for probands (n = 102) from multigenerational ADHL families. In four families, TBC1D24-related HL was found based on the identification of three novel, likely pathogenic (c.553G>A, p.Asp185Asn; c.1460A>T, p. His487Leu or c.1461C>G, p.His487Gln) and one known (c.533C>T, p.Ser178Leu) TBC1D24 variant. Functional consequences of these variants were characterized by analyzing the proposed homology models of the human TBC1D24 protein. Variants not only in the TBC (p.Ser178Leu, p.Asp185Asn) but also in the TLDc domain (p.His487Gln, p.His487Leu) are involved in ADHL development, the latter two mutations probably affecting interactions between the domains. Clinically, progressive HL involving mainly mid and high frequencies was observed in the patients (n = 29). The progression of HL was calculated by constructing age-related typical audiograms. TBC1D24-related ADHL originates from the cochlear component of the auditory system, becomes apparent usually in the second decade of life and accounts for approximately 4% of ADHL cases. Given the high genetic heterogeneity of ADHL, TBC1D24 emerges as an important contributor to this type of HL.

The Evolutionarily Conserved TLDc Domain Defines a New Class of V‐ATPase Interacting Proteins

The kidney-specific V-ATPase regulates physiological acid-base homeostasis, and its dysfunction causes distal renal tubular acidosis (dRTA). We recently found that nuclear receptor coactivator 7 (NCOA7) interacts with the V-ATPase in the kidney, and its deletion in mice resulted in dRTA. NCOA7 belongs to a group of five proteins that contain the evolutionarily conserved TLDc domain, which are known to play a role in the oxidative stress response, and like the V-ATPase, mutations in TLDc proteins lead to serious human neurological diseases. In addition to NCOA7, we found that the TLDc protein, OXR1, interacts with the kidney V-ATPase. Here we asked if the other proteins that make up this family, TBC1D24, TLDC1 and TLDC2, also, interact with the kidney V-ATPase and whether this interaction is mediated by their TLDc domains. Interactions with the V-ATPase were assessed by GST pull-down using mouse kidney lysates and purified GST-tagged TLDc domains of NCOA7, OXR1, TBC1D24, TLDC1 and TLDC2 or the NCOA7 TLDc domain harboring pathogenic mutations (G802A, G815A, S817A, G845A, G896A, L926A, E938A), followed by western blotting. These experiments revealed that the purified TLDc domains of NCOA7, OXR1 and TLDC2, but not TBC1D24 or TLDC1, interact with the kidney-enriched B1 isoform of the V-ATPase. Moreover, the G815A, G845A and G896A mutants found in evolutionarily conserved regions of the NCOA7 TLDc domain did not interact with V-ATPase, L926A and E938A mutations decreased interaction, while S817A or the non-conserved G802A mutation, did not attenuate interaction at all. Since we could not identify an interaction between purified TBC1D24 or TLDC1 and the kidney V-ATPase we investigated the interactions between endogenous TBC1D24, TLDC1 and V-ATPase by co-immunoprecipitation (co-IP) and western blotting. In co-IP studies using mouse kidney lysate, we found that endogenous TBC1D24 interacts with the B1 isoform of the V-ATPase, but not with the more ubiquitous B2 isoform. On the other hand, we did not detect any interaction between endogenous TLDC1 and either the B1 or B2 isoform of the V-ATPase in kidney lysates. Because we were unable to detect TLDC1 in kidney lysates, we performed a co-IP using mouse lung lysate, which expresses high levels of TLDC1. In lung we found that TLDC1 interacts with the B2, but not the B1 subunit of the V-ATPase. However, B1 was expressed at low levels in the lung, which could explain the lack of detectable interaction with TLDC1. Of note, co-IP experiments using equal parts lung and kidney lysate showed a stronger interaction with B2 than B1, suggesting that TLDC1 may preferentially interact with the B2 subunit of the V-ATPase. In the kidney, NCOA7 and OXR1, as well as TBC1D24 and possibly TLDC2 interacted with the V-ATPase and may play a role in V-ATPase-dependent regulation of renal acid-base homeostasis. Furthermore, the evolutionary conserved amino acids within the TLDc domain of Ncoa7 are critical for its interaction with the kidney V-ATPase. Although TLDC1 was not detectable in the kidney, it interacts with the B2 isoform of the V-ATPase in the lung. We conclude, that the TLDc motif is a protein-protein interaction domain that defines a new class of V-ATPase interacting regulatory proteins.

Evolutionary Conserved TLDc Domain Defines a New Class of V-ATPase Interacting Proteins

Evolutionary Conserved TLDc Domain Defines a New Class of V-ATPase Interacting Proteins

The sunflower TLDc-containing protein HaOXR2 confers tolerance to oxidative stress and waterlogging when expressed in maize plants

The sunflower (Helianthus annuus L.) genome encodes six proteins containing a TLDc domain, typical of the eukaryotic OXidation Resistance (OXR) protein family. Expression of sunflower HaOXR2 in Arabidopsis generated plants with increased rosette diameter, higher number of leaves and increased seed production. Maize inbred lines expressing HaOXR2 also showed increased total leaf area per plant. In addition, heterologous expression of HaOXR2 induced an increase in the oxidative stress tolerance in Arabidopsis and maize. Maize transgenic plants expressing HaOXR2 experienced less oxidative damage and exhibited increased photosynthetic performance and efficiency than non-transgenic segregant plants after treatment of leaves with the reactive oxygen species generating compound Paraquat. Expression of HaOXR2 in maize also improved tolerance to waterlogging. The number of expanded leaves, aerial biomass, and stem height and cross-section area were less affected by waterlogging in HaOXR2 expressing plants, which also displayed less aerial tissue damage under these conditions. Transgenic plants also showed an increased production of roots, a typical adaptive stress response. The results show the existence of functional conservation of OXR proteins in dicot and monocot plants and indicate that HaOXR2 could be useful to improve plant performance under conditions that increase oxidative stress.