Recombinant Human Transmembrane protein C9orf123 (C9orf123)

What is C9orf123 and what are its alternative nomenclatures?

C9orf123 (Chromosome 9 Open Reading Frame 123) is now officially designated as TMEM261 (Transmembrane Protein 261) or DMAC1 (Distal Membrane Arm Assembly Component 1). This protein is encoded by a gene located on chromosome 9p24.1 in humans . The nomenclature evolution reflects improved understanding of the protein's function, particularly its role in mitochondrial respiratory chain complex I assembly .

What are the basic structural characteristics of TMEM261/C9orf123?

TMEM261 is a relatively small protein with the following structural features:

• Length: 111-129 amino acids, depending on the isoform

• Size: Approximately 13.5 kDa for the largest isoform

• Domains: Contains two potential transmembrane domains that are evolutionarily conserved

• DUF4536: Contains a domain of unknown function (DUF4536) that is highly conserved in the C-terminal region across species

• The protein's primary sequence shows conservation particularly in mammals, with the most essential regions maintained even in distant homologues

What is the genomic organization of the TMEM261 gene?

The TMEM261 gene has the following genomic features:

• Location: Chromosome 9p24.1 on the reverse strand

• Length: 91,891 base pairs

• Structure: 2 exons and 1 intron

• Transcript variants: 6 primary transcript variants

• Neighboring gene: PTPRD (protein tyrosine phosphatase receptor type delta) also on the reverse strand

What is the current understanding of TMEM261/DMAC1 function?

DMAC1 is required for the assembly of the mitochondrial NADH:ubiquinone oxidoreductase complex (complex I). Specifically, it is involved in the assembly of the distal region of complex I . This function places it in the critical pathway of mitochondrial energy production, which is essential for cellular metabolism. The protein is localized to the mitochondrial inner membrane and colocalizes with respiratory chain complex I .

Where is TMEM261/C9orf123 expressed in human tissues?

TMEM261 shows ubiquitous expression across human tissues. Expression profiling reveals:

• Highest expression in the heart (94% relative expression), particularly in heart fibroblast cells

• High expression in the thymus (90% relative expression)

• High expression in the thyroid (93% relative expression), particularly in thyroid glandular cells

• Cancer cells showing intermediate to high expression in breast, colorectal, ovarian, skin, urothelial, head, and neck cells

What expression systems are optimal for recombinant TMEM261/C9orf123 production?

For optimal recombinant TMEM261 production, insect cell expression systems have proven effective:

• Human TMEM261 protein has been successfully raised in insect cells using multi-step, protein-specific purification processes to ensure crystallization grade quality

• State-of-the-art algorithms for plasmid design (gene synthesis) improve expression efficiency

• The protein requires careful folding consideration due to its transmembrane domains

How can researchers verify the correct folding of recombinant TMEM261?

To ensure correct folding of recombinant TMEM261:

• Use expert laboratory assessment before delivery to verify correct folding

• Measure protein concentration using absorbance at 280nm against specific reference buffers

• Calculate protein concentration using specific absorption coefficients

• Utilize tools such as Expasy's protparam to determine the absorption coefficient of the protein

• Consider the transmembrane nature of the protein when assessing folding quality

What are the advantages of made-to-order recombinant TMEM261 proteins?

Made-to-order recombinant TMEM261 proteins offer several advantages:

• No financial obligation if the protein cannot be expressed or purified

• Avoidance of fees for expression plasmid development, first expression experiments, or purification optimization that might occur with other companies

• Payment only upon receipt of correctly folded protein

• Reduced financial risk while ensuring expert handling throughout the production process

What is known about TMEM261/C9orf123's involvement in cancer?

TMEM261/C9orf123 has been implicated in cancer through several studies:

• Identified in a genome-wide association study for pancreatic neoplasms

• Shows elevated copy number and over-expression in pancreatic tumor patients

• Copy number variations (CNV) and over-expression have been documented in the COSMIC database

• Its locus has been associated with gene amplification and rearrangements in colorectal cancer, breast cancer, and lymphomas

What evidence links TMEM261/C9orf123 to neurodegenerative diseases?

The connection between TMEM261/C9orf123 and neurodegenerative diseases is emerging:

• C9orf123 was identified in computational analysis of pathogenetic pathways in Alzheimer's disease with a significant false discovery rate (fdr) p-value of 0.0029 and a z-value of 4.7

• Since DMAC1 is involved in mitochondrial function, and mitochondrial dysfunction is a hallmark of many neurodegenerative diseases, this connection merits further investigation

• Diseases associated with DMAC1 include Leigh Syndrome, a severe neurological disorder caused by mitochondrial dysfunction

How does TMEM261/DMAC1 dysfunction potentially contribute to disease mechanisms?

Given its role in mitochondrial complex I assembly, DMAC1 dysfunction may contribute to disease through:

• Impaired energy metabolism due to compromised complex I function

• Increased reactive oxygen species production from dysfunctional electron transport chain

• Disrupted mitochondrial membrane potential affecting cellular processes

• Potential triggering of cellular stress responses or apoptotic pathways

• Contribution to neurodegenerative processes through energy deficits in high-energy demanding neural tissues

What experimental approaches are most effective for studying TMEM261/DMAC1 interactions?

To study TMEM261/DMAC1 interactions with other proteins:

• Co-immunoprecipitation assays have been successful in identifying interactions with complex I assembly components

• Isolation using antibodies against Myc or FLAG tags attached to the protein can help identify binding partners

• For mitochondrial studies, consider both overexpression and knockdown approaches to observe effects on complex I assembly

• RNA interference screens can identify potential downstream effectors or pathways

How can researchers effectively analyze TMEM261/C9orf123 in genome-wide association studies?

For effective GWAS analysis of TMEM261/C9orf123:

• Utilize databases such as the International Cancer Genome Consortium (ICGC) to identify mutations in patient samples

• Verify mutations using the COSMIC database for copy number variations (CNV) and expression levels

• Employ tools like cBioPortal to identify coding sequence mutations with somatic mutation scores

• Consider both SNP-level and gene-level evidence in association studies

• Use multiple statistical approaches including meta-analysis across different study populations to improve statistical power

What mitochondrial functional assays are appropriate for studying TMEM261/DMAC1?

To assess TMEM261/DMAC1's role in mitochondrial function:

• Monitor microtubule-associated protein 1A/1B light chain 3 (LC3) flux to assess effects on autophagy

• Use EGFP-LC3 conversion on immunoblots to track autophagy induction

• Evaluate distribution of EGFP-LC3 in cells with modified DMAC1 expression

• Employ Torin1 and bafilomycin A1 treatments to study autophagy regulation

• Assess interactions with the ULK1 complex components (FIP200, ULK1, ATG13) to understand the mechanism of action

How should researchers address contradictions in TMEM261/C9orf123 functional studies?

When facing contradictory data on TMEM261/C9orf123 function:

What are the main technical challenges in studying TMEM261/DMAC1?

Key technical challenges include:

• Small protein size (13.5 kDa) making detection and visualization difficult

• Transmembrane nature complicating purification and structural studies

• Mitochondrial localization requiring specialized isolation techniques

• Limited commercial antibodies with validated specificity

• Multiple isoforms potentially serving different functions

• Ubiquitous expression making tissue-specific effects harder to identify

• Overlapping functions with other mitochondrial proteins potentially obscuring phenotypes

What future research directions might clarify TMEM261/DMAC1's role in health and disease?

Promising future research directions include:

• CRISPR-Cas9 knockout studies to definitively establish function

• Structural biology approaches to elucidate protein-protein interactions

• Patient-derived iPSCs to study disease-relevant mutations

• Tissue-specific conditional knockout animal models

• Metabolomics studies to assess impact on cellular energy pathways

• Integration of multi-omics data to place DMAC1 in broader cellular networks

• High-resolution imaging to visualize DMAC1's role in mitochondrial complex assembly

What bioinformatic resources are most valuable for TMEM261/C9orf123 research?

Key bioinformatic resources include:

• GeneCards: Provides comprehensive gene information with ID HGNC:30536

• NCBI Gene: ID 90871

• Ensembl: ENSG00000137038

• OMIM: 617261

• UniProtKB/Swiss-Prot: Q96GE9

• Open Targets Platform: ENSG00000137038

• COSMIC database: For somatic mutations and copy number variations

• ICGC database: For mutations in cancer patient samples

• Expasy's protparam: For protein parameter analysis

What experimental models have been successful in TMEM261/DMAC1 functional studies?

Successful experimental models include:

• HeLa and HEK293 cell lines for protein overexpression and knockdown studies

• Rat primary cortical neurons for studying effects on autophagy

• siRNA and miRNA approaches for gene silencing

• Co-immunoprecipitation assays with tagged proteins for interaction studies

• Insect cell expression systems for recombinant protein production

• EGFP-LC3 reporter systems for monitoring autophagy