Recombinant Chicken Transmembrane protein 11, mitochondrial (TMEM11)

What is chicken TMEM11 and what are its primary functions?

TMEM11 (Transmembrane Protein 11) is an outer mitochondrial membrane protein that plays a significant role in regulating mitophagy, the selective degradation of mitochondria. Research indicates that TMEM11 forms a complex with BNIP3 and BNIP3L, co-enriching at sites of mitophagosome formation. The protein appears to function as a spatial regulator that restricts mitophagosome formation, as studies have demonstrated that mitophagy becomes hyperactive in the absence of TMEM11 during both normoxia and hypoxia-mimetic conditions . This regulation occurs through an increase in BNIP3/BNIP3L mitophagy sites when TMEM11 is absent, suggesting that it serves as a critical checkpoint in mitochondrial quality control pathways.

How does chicken TMEM11 differ from TMEM11 in other species?

While the search results don't provide direct comparative data between chicken TMEM11 and its homologs in other species, genomic analyses suggest conservation of function across species. The protein has been identified in studies of chicken genetic control of disease resistance, particularly in relation to Campylobacter colonization resistance . Researchers investigating TMEM11 should consider cross-species analysis as significant cis-eQTLs (expression quantitative trait loci) have been detected within TMEM11 in chicken studies, suggesting species-specific regulatory mechanisms may be at play .

What molecular characteristics define chicken TMEM11?

Chicken TMEM11 is characterized as a transmembrane protein localized to the outer mitochondrial membrane. While specific molecular weight and structure information for chicken TMEM11 is not directly provided in the search results, research on mitochondrial proteins suggests that it likely maintains the conserved transmembrane domains characteristic of this protein family. The protein appears to have functional domains that enable interaction with mitophagy regulators BNIP3 and BNIP3L, allowing it to form protein complexes involved in mitochondrial quality control .

What expression systems are most effective for producing recombinant chicken TMEM11?

Based on successful approaches with other chicken mitochondrial proteins, E. coli expression systems using inducible phage-T7 promoters have proven effective for recombinant protein production. Drawing from similar mitochondrial protein expression studies, researchers should consider the following protocol:

• Vector selection: pET32b(+) vector systems have demonstrated high-level expression of chicken recombinant proteins

• Expression strain: E. coli T7 Express lysY strains are recommended for optimal expression

• Induction conditions: 1.0 mM isopropyl-β-d-thiogalactopyranoside (IPTG) induction for 4 hours at 37°C

• Purification strategy: Affinity chromatography under denaturing conditions followed by slow dialysis

For improved yield, researchers should note that selection of expression plasmids on media containing kanamycin rather than ampicillin has been shown to extend the time period of maximal protein expression, as demonstrated with other mitochondrial proteins .

How does TMEM11 interact with BNIP3/BNIP3L to regulate mitophagy, and what experimental approaches can elucidate these mechanisms?

TMEM11 forms a complex with BNIP3 and BNIP3L and co-enriches at sites of mitophagosome formation. Research indicates that mitophagy becomes hyperactive when TMEM11 is absent, suggesting it functions as a negative regulator of BNIP3/BNIP3L-dependent mitophagy .

To investigate these interactions, researchers should consider the following experimental approaches:

• Co-immunoprecipitation assays to confirm physical interactions between recombinant TMEM11 and BNIP3/BNIP3L

• Fluorescence microscopy with labeled proteins to visualize co-localization at mitophagosome formation sites

• CRISPR/Cas9-mediated TMEM11 knockout studies combined with quantitative mitophagy assays under both normoxia and hypoxia-mimetic conditions

• Structure-function analyses using truncated TMEM11 constructs to identify interaction domains

The current model suggests that TMEM11 spatially restricts mitophagosome formation by regulating BNIP3/BNIP3L mitophagy sites, though the precise molecular mechanism remains to be fully characterized .

What are the challenges in producing bioactive recombinant chicken TMEM11 and how can they be addressed?

While the search results don't directly address challenges specific to TMEM11 expression, similar challenges faced with other chicken mitochondrial membrane proteins can be anticipated:

Researchers should validate bioactivity through functional assays specific to TMEM11's role in mitophagy regulation, potentially using fluorescence-based assays to monitor mitophagosome formation in the presence of the recombinant protein.

What purification protocols yield the highest purity and activity for recombinant chicken TMEM11?

Based on successful purification strategies for other chicken recombinant proteins, a multi-step approach is recommended:

• Initial capture: Affinity chromatography using His-tag or thioredoxin-tag fusion systems under denaturing conditions

• Refolding: Slow dialysis to gradually remove denaturants and allow proper protein folding

• Secondary purification: Size-exclusion chromatography to separate monomeric from oligomeric forms

• Quality control: Validation of purity using SDS-PAGE and Western blot analysis with antibodies specific to TMEM11

For membrane proteins like TMEM11, incorporating detergents such as n-dodecyl β-D-maltoside during purification may help maintain native conformation and activity. Activity assessment should include functional assays related to TMEM11's role in mitophagy regulation, potentially using cell-free systems that reconstitute aspects of mitophagosome formation .

How can researchers verify the functional activity of recombinant chicken TMEM11?

To verify functional activity of recombinant chicken TMEM11, researchers should develop assays that assess its ability to regulate mitophagy:

• In vitro binding assays with BNIP3 and BNIP3L to confirm complex formation capacity

• Cell-based mitophagy assays comparing mitophagy rates in TMEM11-knockout cells with and without recombinant TMEM11 supplementation

• Localization studies using fluorescently-tagged recombinant TMEM11 to confirm proper mitochondrial outer membrane targeting

• Co-localization experiments with mitophagosome markers to verify enrichment at formation sites

Activity validation should demonstrate that the recombinant protein recapitulates the native function of restricting BNIP3/BNIP3L-dependent mitophagy sites under both normoxic and hypoxia-mimetic conditions .

What analytical techniques are most appropriate for structural characterization of recombinant chicken TMEM11?

For comprehensive structural characterization of recombinant chicken TMEM11, researchers should employ multiple complementary techniques:

These approaches have proven successful for other recombinant chicken proteins, as demonstrated by the crystallization of recombinant chicken mitochondrial creatine kinase, which formed crystals of space group P42(1)2 with identical structure to the native protein .

How is TMEM11 implicated in disease resistance in chickens, particularly regarding Campylobacter colonization?

Genomic and transcriptomic analyses have identified TMEM11 as a gene of interest in the context of Campylobacter colonization resistance in broiler chickens. Significant cis-eQTLs have been detected within TMEM11, suggesting it may play a role in immune response pathways related to bacterial colonization resistance .

The genetic control of Campylobacter colonization in broilers shows a modest but significant heritability (h²=0.11 ± 0.03), with genome-wide association studies identifying quantitative trait loci (QTLs) on chromosomes 14, 16, 19, and 26 . TMEM11 appears to be associated with these resistance mechanisms, potentially through involvement in immune signaling pathways. Pathway and network analysis has implicated cooperative functional pathways in colonization resistance, including those related to antigen presentation, innate and adaptive immune responses, calcium, and renin-angiotensin signaling .

What potential does recombinant chicken TMEM11 have for developing immunotherapy approaches against tumors?

While the search results don't directly address TMEM11's role in tumor immunity, the research on other recombinant chicken proteins provides insights into potential applications. Matrix metalloproteinase-11 (MMP-11), for example, is selectively expressed in tumor tissues and has been identified as a promising target for immunotherapy .

If TMEM11 demonstrates similar selective expression patterns in tumor versus normal tissues, researchers could explore:

• Development of TMEM11-targeted immunotherapies using recombinant proteins to generate antibodies

• Investigation of TMEM11's role in mitophagy regulation in tumor cells, potentially exploiting cancer cells' altered metabolic dependencies

• Examination of TMEM11 expression profiles in various tumor types to identify cancer-specific patterns

Further research is needed to characterize TMEM11 expression in normal versus neoplastic tissues and to determine whether it plays a role in tumor progression or metastasis.

How can researchers use TMEM11 to better understand mitochondrial quality control in avian species?

TMEM11's role in regulating mitophagy through interaction with BNIP3/BNIP3L provides an excellent model system for studying mitochondrial quality control in avian species . Researchers can leverage recombinant chicken TMEM11 to:

• Compare mitophagy regulation mechanisms between avian and mammalian systems

• Investigate tissue-specific TMEM11 expression patterns and potential specialized functions in different chicken tissues

• Examine how TMEM11-regulated mitophagy responds to various stressors relevant to poultry health, including hypoxia, oxidative stress, and bacterial challenges

• Develop cell-based assays using primary chicken cells to study mitochondrial quality control under physiologically relevant conditions

Understanding TMEM11's role in mitochondrial quality control could provide insights into metabolic adaptation, stress responses, and cellular homeostasis mechanisms specific to avian biology, with potential applications in poultry health and production.

What are the key unanswered questions regarding chicken TMEM11 structure and function?

Despite emerging research on TMEM11, several critical questions remain unanswered:

• What is the high-resolution structure of chicken TMEM11, and how does it compare to mammalian homologs?

• What are the precise molecular mechanisms by which TMEM11 restricts BNIP3/BNIP3L-dependent mitophagy sites?

• Does TMEM11 interact with other mitochondrial proteins beyond BNIP3 and BNIP3L?

• How is TMEM11 expression regulated under different physiological and pathological conditions?

• What are the tissue-specific functions of TMEM11 in different chicken organs?

Addressing these questions will require multidisciplinary approaches combining structural biology, biochemistry, cell biology, and genomics. Development of specific antibodies against chicken TMEM11 would significantly facilitate these investigations.

How might TMEM11 function differ between chicken breeds with varying disease resistance profiles?

Given the identification of TMEM11 in studies of genetic control of Campylobacter colonization resistance, exploring breed-specific variations in TMEM11 structure and function represents an important research direction . Researchers should consider:

• Comparative genomic analysis of TMEM11 sequences across chicken breeds with different disease resistance phenotypes

• Transcriptomic profiling to identify breed-specific TMEM11 expression patterns

• Functional analysis of variant TMEM11 proteins to determine impact on mitophagy regulation

• Association studies linking TMEM11 variants to specific disease resistance phenotypes

Understanding breed-specific TMEM11 variants could provide insights into the genetic basis of disease resistance in chickens and inform breeding programs aimed at enhancing natural resistance mechanisms.

What novel experimental systems could advance our understanding of TMEM11 in mitochondrial biology?

To accelerate research on chicken TMEM11, development of the following experimental systems is recommended:

• CRISPR/Cas9-engineered chicken cell lines with TMEM11 knockout or targeted mutations

• Fluorescent reporter systems for real-time monitoring of TMEM11-regulated mitophagy

• Organoid cultures derived from chicken tissues to study TMEM11 function in more physiologically relevant contexts

• In vitro reconstitution systems combining purified recombinant TMEM11 with artificial membrane systems and mitophagy components

These experimental approaches would enable more detailed investigation of TMEM11's molecular mechanisms and potentially reveal new functions beyond its established role in mitophagy regulation.