Recombinant Mouse Transmembrane and coiled-coil domain-containing protein 6 (Tmco6)

What is Transmembrane and Coiled-Coil Domain-Containing Protein 6 (Tmco6)?

Transmembrane and coiled-coil domain-containing protein 6 (Tmco6) is a protein encoded by the Tmco6 gene, which contains structural features including two reserved ARM superfamily domains and an arginine-rich region within the coiled-coil structure. The protein is considered a multi-pass membrane protein with evidence of localization to multiple cellular compartments including the nucleus, cytosol, endoplasmic reticulum, mitochondria, and plasma membrane . In humans, the TMCO6 gene is located on chromosome 5 (position 5q31.3), spanning 5568 base pairs on the positive strand, and can be alternatively spliced into different variants with variant 1 being the longest . The longest isoform (isoform 1) consists of 499 amino acids with a predicted molecular weight of approximately 55 kDa, while shorter isoforms exist due to alternative splicing events . Unlike many other proteins, TMCO6 orthologs are not found in invertebrates, fungi, plants, or bacteria, suggesting it may have evolved specifically in vertebrates .

How is Tmco6 structurally organized and what are its key domains?

The Tmco6 protein contains several key structural elements that likely contribute to its functional properties. Two reserved ARM (Armadillo/beta-catenin-like-repeat) superfamily domains form a superhelix of helices, each spanning approximately 40 amino acids in length . The protein features an arginine-rich region located within its coiled-coil domain, suggesting this positively charged area might be functionally significant for protein-protein interactions or structural stability . Additionally, Tmco6 contains 17 regions for protein binding, two transmembrane domains that anchor it to cellular membranes, and an SRP1 domain (from amino acids 23-399) which corresponds to Karyopherin/importin alpha functionality . A notable di-leucine motif, commonly associated with lysosomal targeting, is abundant in the protein sequence, while a nuclear localization sequence consisting of 5 positively charged amino acids near the 5' end suggests nuclear transport capability . The protein topology indicates that both the N-terminus (5') and C-terminus (3') are predicted to be located on the cytoplasmic side of the membrane, with only a small portion extending into the non-cytoplasmic region .

What is known about Tmco6 gene expression patterns?

The expression patterns of Tmco6 have been studied across various tissues and developmental stages, providing insights into its potential biological roles. In humans, TMCO6 expression has been detected in liver tissue and during the fetal stage of development, suggesting potential roles in liver function and embryonic development . The regulatory mechanisms controlling Tmco6 expression appear complex, with evidence that various environmental compounds can modulate its expression levels. For instance, tetrabromobisphenol A has been shown to increase TMCO6 mRNA expression, while acrylamide, aristolochic acid A, arsenous acid, and gamma-hexachlorocyclohexane have been reported to decrease its expression . Interestingly, the retinoid-related orphan receptor alpha (RORA) protein has been found to bind to the TMCO6 gene, suggesting potential transcriptional regulation through this nuclear receptor . Expression changes have also been documented in response to pharmaceutical compounds such as finasteride (increased expression) and cisplatin, cyclosporin A, doxorubicin, and paracetamol (decreased expression) . These diverse regulatory influences indicate that Tmco6 expression is dynamically controlled and may be responsive to various physiological and toxicological stimuli.

What mouse models are available for studying Tmco6 function?

Several mouse models have been developed to investigate the function of Tmco6, with the most extensively characterized being the Tmco6-knockout model. The knockout model referenced in the research literature exhibited significant neurological, physiological, and motor debilities, providing valuable insights into the in vivo functions of Tmco6 . This model demonstrated isolated Complex I deficiency specifically in heart and skeletal muscle tissues, along with abnormal cardiac electrophysiology, establishing a clear link between Tmco6 and mitochondrial respiratory chain function . The official designation for one such knockout model is Tmco6 tm1.1(KOMP)Vlcg, which is available through mouse genome informatics resources and repository systems . Additionally, researchers have employed recombinant AAV-mediated expression systems to deliver wild-type human TMCO6 to knockout mice, successfully rescuing the Complex I deficiency and electrophysiological function in the hearts of 3-month-old animals . This rescue experiment provides compelling evidence for the causal relationship between Tmco6 function and the observed phenotypes. To complement these in vivo approaches, cellular models involving TMCO6 gene silencing and overexpression have also been characterized, offering more controlled systems for mechanistic studies .

How can recombinant Tmco6 protein be effectively produced and purified?

The production and purification of recombinant Tmco6 protein requires careful consideration of its structural features and expression systems. For effective expression of functional recombinant mouse Tmco6, researchers should consider utilizing mammalian expression systems such as HEK293 or CHO cells to ensure proper post-translational modifications and folding of this multi-domain protein with two transmembrane regions. The expression construct should be designed to include the full-length mouse Tmco6 cDNA (encoding the 499 amino acid protein for isoform 1) with an appropriate affinity tag (such as His6, FLAG, or GST) positioned to avoid interference with the transmembrane domains or functional regions . For membrane protein purification, a two-step approach is recommended: initial solubilization using mild detergents (such as n-dodecyl-β-D-maltoside or digitonin) that maintain protein structure, followed by affinity chromatography utilizing the engineered tag. Since Tmco6 contains coiled-coil domains and multiple protein binding regions, additional purification steps such as size exclusion chromatography may be necessary to obtain homogeneous protein preparations. For functional studies, consideration should be given to reconstituting the purified protein into liposomes or nanodiscs to maintain its native membrane environment, particularly important given its localization to the mitochondrial inner membrane and involvement in Complex I function .

What techniques are most effective for studying Tmco6 interactions with Complex I?

The interaction between Tmco6 and Complex I of the mitochondrial respiratory chain can be effectively studied using a combination of biochemical, proteomic, and imaging techniques. Co-immunoprecipitation experiments have successfully demonstrated the physical association between Tmco6 and Complex I components, providing direct evidence of their interaction . Two-dimensional blue native gel electrophoresis (2D-BNGE) has proven particularly valuable, showing co-localization of TMCO6 with the Complex I holocomplex . This technique allows visualization of intact respiratory chain complexes and supercomplexes, enabling assessment of how Tmco6 deficiency affects the stability and assembly of these structures. For more detailed interaction mapping, crosslinking mass spectrometry (XL-MS) can identify specific contact points between Tmco6 and Complex I subunits. Proximity-dependent biotin identification (BioID) or APEX2 proximity labeling offer complementary approaches for identifying neighboring proteins within the native cellular environment. Super-resolution microscopy techniques have been successfully employed to confirm the mitochondrial localization of Tmco6 and could be further utilized to visualize its co-localization with Complex I components at nanometer resolution . Functional assessment of this interaction can be performed using Seahorse XF analyzers to measure oxygen consumption rates and Complex I activity in cellular models with modified Tmco6 expression, complemented by in vitro biochemical assays of Complex I enzymatic activity using isolated mitochondria.

What methods are used to assess mitochondrial function in Tmco6-deficient models?

Assessment of mitochondrial function in Tmco6-deficient models employs a multi-parametric approach focusing on respiratory chain activity, particularly Complex I. Biochemical measurement of Complex I enzymatic activity using spectrophotometric assays that monitor NADH oxidation rates provides direct quantification of functional deficits, as demonstrated in skeletal muscle biopsies from patients with TMCO6 mutations and in Tmco6-knockout mouse tissues . Blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by in-gel activity assays offers visualization of active respiratory complexes and supercomplexes, revealing the destabilization of CI-containing supercomplexes observed in patient-derived fibroblasts . Oxygen consumption measurements using instruments like the Seahorse XF Analyzer allow real-time assessment of mitochondrial respiration in intact cells, providing insights into basal respiration, ATP production, maximal respiratory capacity, and substrate utilization. Mitochondrial membrane potential can be evaluated using fluorescent indicators such as TMRM or JC-1, while reactive oxygen species production (often elevated with Complex I dysfunction) can be measured using probes like MitoSOX. For cardiac function assessment in the Tmco6-knockout mouse model, electrophysiological recordings have been particularly informative, demonstrating abnormalities that could be rescued by recombinant AAV-mediated expression of wildtype human TMCO6 . Complementary histological and ultrastructural analyses using electron microscopy can reveal morphological changes in mitochondria resulting from Tmco6 deficiency.

How does Tmco6 contribute to mitochondrial Complex I assembly and function?

Tmco6 plays a critical role in maintaining the integrity and function of mitochondrial Complex I, as evidenced by multiple experimental approaches. Co-localization studies using 2D-BNGE have demonstrated that TMCO6 physically associates with the Complex I holocomplex, suggesting direct involvement in its assembly or stability . This interaction has been further corroborated by immunoprecipitation experiments, confirming the physical association between Tmco6 and Complex I components . In Tmco6-deficient models, including patient-derived skin fibroblasts with TMCO6 mutations and Tmco6-knockout mice, researchers have observed destabilization of CI-containing supercomplexes and isolated CI enzymatic deficiency, particularly in heart and skeletal muscle tissues . The functional importance of this relationship is dramatically illustrated by rescue experiments, where expression of wild-type human TMCO6 in 3-month-old knockout mice hearts successfully restored Complex I deficiency and normalized electrophysiological function . In contrast, expression of patient mutant protein variants failed to recover the isolated CI deficiency, establishing a clear causal link between Tmco6 function and Complex I integrity . Given its localization to the mitochondrial inner membrane, Tmco6 likely functions as an assembly factor or structural component that facilitates the proper formation or stabilization of Complex I and its integration into respiratory supercomplexes.

What is the relationship between Tmco6 and nuclear-mitochondrial communication?

The structural and functional characteristics of Tmco6 suggest it may serve as a mediator in nuclear-mitochondrial communication pathways. The protein possesses both an SRP1 domain (Karyopherin/importin alpha) involved in nuclear transport and a nuclear localization sequence, indicating potential roles in nucleocytoplasmic trafficking . Simultaneously, its established localization to the mitochondrial inner membrane and involvement in respiratory chain function position it at a critical junction between these two organelles . This dual association may enable Tmco6 to participate in retrograde signaling from mitochondria to the nucleus, particularly in response to changes in respiratory chain function or mitochondrial stress. The protein's predicted function in enabling nuclear import signal receptor activity further supports this potential role in bidirectional communication . Evidence that various compounds and drugs modulate Tmco6 expression suggests it may respond to cellular stress conditions, potentially triggering adaptive responses that require coordination between mitochondrial and nuclear activities . Additionally, the multiple protein binding regions identified in Tmco6 provide ample opportunity for interactions with various factors involved in organelle crosstalk . Though direct experimental evidence specifically addressing Tmco6's role in mitochondrial-nuclear communication remains limited, its structural features, subcellular distribution, and involvement in mitochondrial function make it a prime candidate for future investigations into this critical cellular coordination mechanism.

What molecular pathways are affected by Tmco6 deficiency?

Tmco6 deficiency triggers a cascade of molecular disturbances primarily centered around mitochondrial respiratory function but extending to broader cellular pathways. The most directly affected pathway involves Complex I (NADH:ubiquinone oxidoreductase) of the electron transport chain, with biochemical and structural analyses revealing reduced enzymatic activity and destabilization of CI-containing supercomplexes in both patient fibroblasts and mouse models . This primary defect likely compromises oxidative phosphorylation efficiency, reducing ATP production capacity particularly in high-energy demanding tissues such as heart and skeletal muscle. Consequently, energy metabolism pathways including TCA cycle activity and substrate utilization patterns may undergo compensatory adjustments. The abnormal cardiac electrophysiology observed in Tmco6-knockout mice suggests disruption of ion homeostasis pathways, potentially involving calcium handling proteins whose function is intimately linked to mitochondrial energetics . The severe cardiac fibrosis resulting from expression of mutant Tmco6 indicates activation of tissue remodeling and fibrotic pathways, potentially involving TGF-β signaling and extracellular matrix production . Neurological deficits in the knockout model point to disruption of pathways essential for neuronal function and survival, possibly including synapse maintenance and axonal transport mechanisms dependent on mitochondrial distribution . The progressive cerebral and cerebellar atrophy observed in patients with TMCO6 mutations further suggests activation of cell death pathways in neural tissues over time .

What protein interactions have been identified for Tmco6?

Investigation of Tmco6's interactome has revealed several significant protein associations that provide insights into its functional roles. Two-hybrid experimental evidence has identified UBQLN1 (ubiquilin 1) as having high potential to interact with TMCO6, suggesting possible involvement in protein quality control and degradation pathways . Co-immunoprecipitation studies have demonstrated physical association between Tmco6 and components of the mitochondrial Complex I, establishing a direct link to the respiratory chain machinery . Additionally, there is evidence that the retinoid-related orphan receptor alpha (RORA) protein binds to the TMCO6 gene, indicating a potential regulatory relationship at the transcriptional level . The presence of 17 predicted protein binding regions within the Tmco6 sequence suggests capacity for numerous additional interactions that remain to be characterized experimentally . The protein's structural features, including ARM domains (known to mediate protein-protein interactions, particularly in beta-catenin signaling), coiled-coil domains (common in protein complexes), and an arginine-rich region, provide multiple interfaces for potential binding partners . The SRP1/Karyopherin domain suggests interactions with nuclear pore components and cargo proteins involved in nuclear import . While the full complement of Tmco6 binding partners awaits comprehensive interactome analysis using techniques such as affinity purification-mass spectrometry or proximity labeling approaches, the identified interactions support roles in mitochondrial function, protein homeostasis, and possibly transcriptional regulation.

What is the evidence linking Tmco6 mutations to mitochondrial disease?

The causal relationship between Tmco6 mutations and mitochondrial disease has been established through convergent lines of evidence from clinical, genetic, biochemical, and experimental model studies. A homozygous recessive variant in TMCO6 (NM_018502.5: c.271C>T) was identified in a pediatric patient presenting with the classical clinical manifestations of mitochondrial disease, including severe developmental delay, generalized hypotonia, and progressive cerebral and cerebellar atrophy . Biochemical analysis of skeletal muscle biopsy from this patient revealed Complex I enzymatic deficiency, a hallmark feature of mitochondrial respiratory chain disorders . Patient-derived skin fibroblasts demonstrated destabilization of CI-containing supercomplexes, providing cellular evidence of mitochondrial dysfunction . The pathogenicity of TMCO6 mutations was further confirmed through functional complementation studies, where stable expression of wildtype human TMCO6 in a Tmco6-knockout mouse model rescued the Complex I deficiency and electrophysiological abnormalities . Critically, expression of the patient mutant protein variant failed to recover the isolated CI deficiency and additionally resulted in severe cardiac fibrosis, demonstrating the specific pathogenicity of this mutation . The Tmco6-knockout mouse model recapitulated key aspects of human mitochondrial disease, exhibiting neurological and physiological abnormalities along with isolated Complex I deficiency in heart and skeletal muscle . Together, these findings establish a clear genetic and mechanistic link between TMCO6 mutations and mitochondrial respiratory chain disorders.

What therapeutic approaches show promise for Tmco6-related disorders?

Therapeutic strategies for Tmco6-related disorders are emerging from experimental evidence and understanding of the protein's function. Gene therapy approaches show particular promise, as demonstrated by successful rescue experiments using recombinant AAV-mediated expression of wildtype human TMCO6 in Tmco6-knockout mice . This intervention restored Complex I deficiency and normalized cardiac electrophysiological function in 3-month-old knockout mice hearts, providing proof-of-principle for gene replacement therapy . The specificity of TMCO6's role in Complex I function suggests that targeted approaches to boost Complex I activity, such as compounds that stabilize or enhance respiratory chain supercomplexes, might provide functional benefits. Given the mitochondrial inner membrane localization of Tmco6, delivery strategies that efficiently target therapeutic agents to this compartment will be crucial for success . For addressing the progressive neurological manifestations observed in patients with TMCO6 mutations, neuroprotective agents that prevent or slow neuronal loss might be beneficial as adjunctive therapy. The cardiac fibrosis that results from expression of mutant Tmco6 suggests that anti-fibrotic agents could help manage cardiac complications . Since energy metabolism is compromised due to respiratory chain dysfunction, metabolic support therapies including specific vitamins, cofactors, and substrate-level phosphorylation enhancers might improve energy availability in affected tissues. Early intervention appears critical, as the successful rescue in 3-month-old mice suggests therapeutic windows may exist before irreversible damage occurs.

How can patient-derived cells be utilized to study Tmco6-related pathologies?

Patient-derived cells represent invaluable tools for investigating Tmco6-related pathologies and developing personalized therapeutic approaches. Skin fibroblasts from patients with TMCO6 mutations have already provided significant insights, demonstrating destabilization of CI-containing supercomplexes and establishing cellular phenotypes that correlate with clinical manifestations . These fibroblasts can be subjected to comprehensive mitochondrial function analysis, including respirometry, membrane potential measurements, and ROS production assessment, to characterize the cellular consequences of specific TMCO6 variants. Additionally, patient fibroblasts can be reprogrammed to induced pluripotent stem cells (iPSCs) and subsequently differentiated into disease-relevant cell types such as neurons, cardiomyocytes, or skeletal muscle cells to study tissue-specific manifestations of Tmco6 deficiency. Such cellular models enable high-throughput drug screening to identify compounds that rescue mitochondrial function or prevent secondary pathological processes. CRISPR/Cas9 gene editing can be employed to correct patient-specific mutations in these cellular models, providing isogenic controls and potential therapeutic validation. Live-cell imaging approaches can track changes in mitochondrial dynamics, morphology, and distribution in patient cells, potentially revealing additional disease mechanisms. Complementation studies involving expression of wild-type or modified TMCO6 variants can determine specific functional domains required for rescuing cellular phenotypes, as well as test the efficacy of gene therapy constructs before moving to animal models .

What phenotypic manifestations characterize Tmco6 deficiency in animal models?

Tmco6-deficient animal models display a constellation of phenotypic abnormalities that provide valuable insights into the protein's physiological functions. The Tmco6-knockout mouse model exhibits significant neurological deficits, including various motor debilities that reflect the neurological involvement seen in human patients with TMCO6 mutations . Physiological abnormalities are prominent, with particular impact on tissues with high energy demands such as heart and skeletal muscle, consistent with Tmco6's role in mitochondrial respiratory function . Cardiac abnormalities are especially noteworthy, with knockout mice demonstrating abnormal electrophysiological function that can be rescued by recombinant AAV-mediated expression of wildtype human TMCO6 . Biochemical analysis reveals isolated Complex I deficiency specifically in heart and skeletal muscle tissues, establishing a direct link between Tmco6 and respiratory chain function . Histopathological examination shows tissue-specific changes, with severe cardiac fibrosis developing when patient mutant Tmco6 variants are expressed . The phenotypic profile resembles that of other mitochondrial disease models with Complex I deficiency, featuring progressive deterioration of neurological and muscle function over time. Metabolic alterations likely include adaptive changes in substrate utilization and energy conservation pathways to compensate for reduced oxidative phosphorylation capacity. These comprehensive phenotypic manifestations in animal models closely mirror the clinical presentation in human patients, supporting the translational relevance of these models for understanding disease mechanisms and testing therapeutic interventions.

How might Tmco6 function differ across various tissues and developmental stages?

The tissue-specific and developmental aspects of Tmco6 function represent an important frontier for future research, with several lines of evidence suggesting differential roles across contexts. The isolated Complex I deficiency observed specifically in heart and skeletal muscle of Tmco6-knockout mice, despite the protein's broader expression pattern, indicates tissue-specific dependencies on Tmco6 function . This selectivity may reflect varying energy demands, different compositions of respiratory chain supercomplexes, or tissue-specific interacting partners that modulate Tmco6 activity. Developmentally, the known expression of TMCO6 during the fetal stage in humans suggests potential roles in embryonic development that may differ from its functions in mature tissues . The progressive nature of neurological manifestations in patients with TMCO6 mutations, including cerebral and cerebellar atrophy, points to age-dependent consequences of Tmco6 dysfunction that may reflect cumulative damage or changing functional requirements over time . Investigation of Tmco6 expression and function across different developmental timepoints in various tissues would illuminate these temporal aspects. The potential involvement of Tmco6 in nuclear-mitochondrial communication pathways may also vary by tissue and developmental context, depending on the specific signaling requirements of different cell types. Comparative studies of Tmco6-dependent transcriptional programs across tissues could reveal context-specific regulatory networks. Understanding these tissue-specific and developmental aspects of Tmco6 function would inform more targeted therapeutic approaches for TMCO6-related disorders.

What is the evolutionary significance of Tmco6's restricted phylogenetic distribution?

The distinctive evolutionary profile of Tmco6, with orthologs absent in invertebrates, fungi, plants, and bacteria, raises intriguing questions about its evolutionary history and functional significance. This restricted phylogenetic distribution suggests Tmco6 emerged relatively recently in evolutionary time, potentially coinciding with the evolution of vertebrate-specific traits or physiological adaptations . The protein has been characterized as a fairly fast-evolving entity, comparable to fibrinogen in its evolutionary rate, indicating potential adaptive pressure or relaxed constraint on its sequence . This rapid evolution could reflect coevolution with interacting partners or adaptation to specific vertebrate mitochondrial functions. The absence of Tmco6 in invertebrates and other lineages that possess mitochondria indicates that its role in Complex I function represents either a vertebrate-specific refinement of respiratory chain regulation or a complementary pathway that evolved specifically in the vertebrate lineage. Comparative analysis of respiratory chain assembly and function across species with and without Tmco6 orthologs could reveal alternative mechanisms employed by organisms lacking this protein. Additionally, examination of the genomic context and synteny relationships of the Tmco6 locus across vertebrate species might provide insights into its evolutionary origins. Research into whether Tmco6's dual roles in mitochondrial function and potential nuclear-cytoplasmic trafficking represent an evolutionary innovation for coordinating these compartments in vertebrate cells would illuminate its broader significance in eukaryotic cell biology.

How do post-translational modifications regulate Tmco6 function?

The regulation of Tmco6 through post-translational modifications (PTMs) represents an unexplored dimension that could significantly impact its function in different contexts. Given the protein's complex structure, including multiple protein binding regions, coiled-coil domains, and an arginine-rich region, various PTMs could modulate its interactions, localization, stability, or activity . Phosphorylation sites within Tmco6 could serve as regulatory switches controlling its association with Complex I components or other interacting partners, potentially in response to changes in cellular energy status or mitochondrial function. The arginine-rich region presents potential sites for methylation or citrullination, modifications that could alter the protein's charge distribution and thereby affect its binding properties or conformational states. SUMOylation or ubiquitination might regulate Tmco6's stability, subcellular trafficking between different compartments, or function in protein quality control pathways, particularly considering its interaction with ubiquilin 1 (UBQLN1) . Acetylation of lysine residues could influence the protein's association with membranes or other proteins. Systematic proteomic analysis of Tmco6 PTMs under different physiological and stress conditions would establish a modification profile that could be correlated with functional outcomes. Site-directed mutagenesis of identified modification sites, followed by functional assessment of mitochondrial Complex I activity, would establish causal relationships between specific PTMs and Tmco6 function. Additionally, identification of the enzymes responsible for adding or removing these modifications could reveal regulatory pathways controlling Tmco6 activity in response to cellular signaling events.

What potential roles might Tmco6 play in aging and age-related diseases?

The involvement of Tmco6 in mitochondrial function positions it as a potential contributor to aging processes and age-related pathologies, warranting dedicated investigation. Mitochondrial dysfunction, particularly Complex I deficiency, is a well-established feature of normal aging and various age-related diseases, suggesting Tmco6 activity or regulation might change during senescence . The progressive nature of symptoms in patients with TMCO6 mutations, including neurodegeneration, parallels age-related decline in mitochondrial function and neurological integrity . Investigation of Tmco6 expression and function across the lifespan in different tissues would establish whether changes occur with advancing age. The protein's potential role in nuclear-mitochondrial communication could be particularly relevant to aging, as coordination between these compartments becomes dysregulated during senescence . If Tmco6 contributes to mitochondrial quality control through its interaction with ubiquilin 1, age-related alterations in this protein might contribute to the accumulation of dysfunctional mitochondria characteristic of aging tissues . Studies in aged Tmco6-heterozygous animals could reveal whether partial deficiency sensitizes to age-related pathologies, while examination of Tmco6 function in models of accelerated aging might uncover connections to established aging pathways. The cardiovascular phenotypes observed in Tmco6-deficient models, including cardiac electrophysiological abnormalities and fibrosis, suggest potential contributions to age-related cardiac diseases . Similarly, the neurological manifestations point to possible roles in neurodegenerative conditions. Comparative analysis of Tmco6 sequence variants in long-lived versus average-lifespan individuals might identify polymorphisms associated with healthy aging or longevity.