Recombinant Mouse Transmembrane protein 85 (Tmem85)

What is Transmembrane Protein 85 (Tmem85) and what are its primary functions?

Tmem85 is a previously uncharacterized transmembrane domain protein that has been identified as a novel anti-apoptotic sequence. Research demonstrates that Tmem85 plays a critical role in preventing cell death in response to oxidative stress. The human TMEM85 gene undergoes alternative splicing to produce multiple transcripts and proteins, making it a complex gene with conserved anti-apoptotic properties .

Key functions include:

• Protection against oxidative stress-induced cell death

• Promotion of cellular growth under stress conditions

• Potential interaction with other membrane proteins in signaling pathways

The protein's anti-apoptotic activity has been demonstrated through heterologous expression studies in yeast, where both human TMEM85 and its yeast ortholog (YGL231c) showed protective effects against hydrogen peroxide-mediated cell death .

How is Tmem85 structurally characterized?

Tmem85 is a membrane-spanning protein with multiple transmembrane domains. Based on available research data, the protein contains:

• Multiple transmembrane helices that anchor the protein in cellular membranes

• Conserved domains between species (human and yeast orthologs show functional similarity)

• Specific amino acid sequences that are crucial for its anti-apoptotic function

The specific topological arrangement of the protein is still under investigation, with current research focusing on determining which domains are exposed to the cytoplasm versus the extracellular/luminal space.

How is Tmem85 expression regulated in different tissues?

While comprehensive tissue-specific expression profiles for Tmem85 are still being developed, current research indicates variable expression across tissues. Unlike some other TMEM family proteins that show tissue-specific patterns, Tmem85 appears to have broader expression, consistent with its fundamental role in cellular survival mechanisms .

Future investigations using quantitative PCR and immunohistochemistry are needed to fully characterize tissue-specific expression patterns and regulatory mechanisms controlling Tmem85 gene expression.

What are the most effective methods for studying Tmem85 function in vitro?

Several experimental approaches have proven effective for studying Tmem85 function:

Cell-based assays:

• Oxidative stress resistance assays using hydrogen peroxide exposure

• Cell viability measurements following Tmem85 overexpression or knockdown

• Growth and proliferation assays under various stress conditions

Protein interaction studies:

• Dual-membrane yeast two-hybrid system (as used to identify TMEM85 as a GLUT9-interacting protein)

• Co-immunoprecipitation assays to confirm physical interactions with other proteins

• FRET or BiFC approaches to study protein-protein interactions in living cells

Gene expression manipulation:

• Overexpression systems using appropriate expression vectors

• RNA interference or CRISPR-Cas9 for knockdown/knockout studies

• Inducible expression systems for temporal control of Tmem85 expression

When designing experiments, researchers should consider potential confounding variables such as cell type-specific effects and the impact of oxidative stress levels on experimental outcomes .

How should researchers design experiments to investigate Tmem85's role in oxidative stress response?

A comprehensive experimental design should include:

• Treatment variables:

  • Range of oxidative stress conditions (varying H₂O₂ concentrations)

  • Time course experiments (acute vs. chronic exposure)

  • Combined stressors to assess specificity of Tmem85 protection

• Control considerations:

  • Appropriate vector-only controls for overexpression studies

  • Non-targeting controls for knockdown experiments

  • Wild-type vs. mutant Tmem85 variants to identify functional domains

• Measurement endpoints:

  • Cell viability (MTT, alamarBlue, or trypan blue exclusion)

  • Markers of apoptosis (caspase activation, PARP cleavage, Annexin V staining)

  • ROS detection methods (DCFDA, MitoSOX)

  • Mitochondrial function assessment (membrane potential, respiration)

• Experimental design structure:

  Design element	Implementation	Purpose
  Randomization	Random assignment of treatment conditions	Minimize systematic bias
  Blinding	Coded samples for analysis	Prevent observer bias
  Replication	Minimum 3 independent biological replicates	Ensure reproducibility
  Positive controls	Known anti-apoptotic proteins (Bcl-2)	Validate assay sensitivity
  Negative controls	Pro-apoptotic factors	Demonstrate response range

When analyzing results, researchers should employ appropriate statistical methods to account for variability in biological systems and potential confounding effects .

What are the optimal conditions for producing recombinant mouse Tmem85 protein?

Based on established protocols for similar transmembrane proteins, the following production conditions are recommended:

Expression systems:

• Mammalian expression systems (HEK293, CHO cells) for proper post-translational modifications

• Insect cell systems (Sf9, High Five) for higher yields while maintaining eukaryotic processing

• E. coli systems with specialized modifications for membrane protein expression

Purification strategies:

• Detergent solubilization screening (start with mild detergents like DDM, LMNG)

• Affinity purification using appropriate tags (His6 tag is commonly used)

• Size exclusion chromatography for final polishing

Quality control parameters:

• Purity assessment: >85% purity by SDS-PAGE is typically desired, with visualization by silver staining

• Functional validation: Anti-apoptotic activity in cell-based assays

• Structural integrity: Circular dichroism or thermal stability assays

Recommended storage conditions include avoiding repeated freeze-thaw cycles and storing at -70°C after reconstitution under sterile conditions for up to 3 months .

How does Tmem85 interact with other proteins in apoptotic and stress response pathways?

Current research indicates that Tmem85 participates in protein interaction networks related to stress response and cell survival:

Known interactions:

• GLUT9 (SLC2A9): Tmem85 has been identified as a GLUT9-interacting protein through dual-membrane yeast two-hybrid screening, suggesting potential involvement in urate transport regulation

Hypothesized interaction mechanisms:

• Direct physical binding to pro-apoptotic factors, potentially sequestering them

• Integration into membrane complexes that regulate stress sensing or signaling

• Modulation of mitochondrial membrane permeability through protein-protein interactions

Research approaches to further characterize interactions:

• Proximity labeling techniques (BioID, APEX) to identify near-neighbors in the membrane

• Crosslinking mass spectrometry to capture direct binding partners

• Genetic interaction screens to identify functional partners

Researchers should note that the interaction landscape may differ between cell types and under different stress conditions, necessitating context-specific investigation .

What is the relationship between Tmem85 and other anti-apoptotic proteins in cellular stress response networks?

The relationship between Tmem85 and established anti-apoptotic proteins remains to be fully characterized. Current understanding suggests:

• Complementary protection mechanisms:

  • Bcl-2 family proteins primarily target mitochondrial apoptotic pathways

  • Tmem85 may provide protection through distinct mechanisms, particularly against oxidative stress

• Potential pathway integration:

  • Tmem85 may function upstream or downstream of established anti-apoptotic factors

  • Cross-talk between Tmem85 and other stress response pathways (e.g., unfolded protein response)

• Evolutionary conservation:

  • The fact that Tmem85 function is conserved from yeast to humans suggests a fundamental role in cellular survival mechanisms

  • This conservation indicates it may represent an ancient anti-apoptotic mechanism distinct from more recently evolved pathways

Further research using systems biology approaches, including pathway analysis and network modeling, would help position Tmem85 within the broader context of cellular stress response networks.

How does post-translational modification affect Tmem85 function and localization?

While specific post-translational modifications (PTMs) of Tmem85 have not been extensively characterized, research on related transmembrane proteins suggests several potential modifications:

Potential PTMs affecting Tmem85:

• Phosphorylation: May regulate protein-protein interactions or activation state

• Ubiquitination: Could control protein turnover and stability

• Glycosylation: May affect protein folding, trafficking, and cell surface expression

Methodological approaches to study PTMs:

• Mass spectrometry-based proteomics to identify modification sites

• Site-directed mutagenesis of potential modification sites

• Pharmacological inhibitors of specific modification enzymes

• Subcellular fractionation and immunofluorescence to track localization changes

Researchers should consider that recombinant expression systems may not fully recapitulate the native PTM profile, potentially affecting functional studies with recombinant protein .

What are the best approaches for detecting endogenous Tmem85 in tissue samples?

Detection of endogenous Tmem85 in tissue samples requires careful consideration of protein abundance and localization:

Immunohistochemistry/Immunofluorescence:

• Use validated antibodies specific to mouse Tmem85

• Consider antigen retrieval methods optimized for membrane proteins

• Include appropriate positive and negative controls

Western blotting:

• Optimize membrane protein extraction protocols

• Use appropriate detergents for solubilization

• Consider enrichment methods for membrane fractions

RNA detection methods:

• RT-qPCR for transcript levels

• RNA-seq for comprehensive expression analysis

• RNA in situ hybridization for spatial expression patterns

How can researchers effectively knock down or knock out Tmem85 in experimental models?

Several genetic manipulation approaches can be employed:

RNA interference:

• siRNA for transient knockdown (72-96 hours)

• shRNA for stable knockdown via lentiviral delivery

• Validated siRNA sequences should target conserved regions of Tmem85 mRNA

CRISPR-Cas9 gene editing:

• Single guide RNA design targeting early exons

• Screening for indels that cause frameshift mutations

• Verification of knockout by sequencing and protein detection methods

Conditional knockout strategies:

• Cre-loxP system for tissue-specific or inducible deletion

• Tetracycline-controlled transcriptional activation systems

Verification methods:

• RT-qPCR to confirm transcript reduction

• Western blotting to verify protein loss

• Functional assays to demonstrate phenotypic consequences

Researchers should be aware that complete loss of Tmem85 might affect cell viability, potentially necessitating inducible or partial knockdown approaches for some experimental designs .

What functional assays best demonstrate Tmem85's anti-apoptotic activity?

Based on published research on Tmem85 and related proteins, the following assays effectively demonstrate anti-apoptotic activity:

Cell viability assays:

• MTT or MTS reduction assays

• Resazurin-based metabolic assays (alamarBlue)

• ATP content measurement

• Trypan blue exclusion for membrane integrity

Apoptosis-specific assays:

• Annexin V/PI staining for phosphatidylserine externalization

• TUNEL assay for DNA fragmentation

• Caspase activity assays (especially caspase-3/7)

• Mitochondrial membrane potential using JC-1 or TMRE dyes

Oxidative stress challenge models:

• Hydrogen peroxide exposure (varying concentrations and durations)

• Paraquat or menadione treatment

• Glutathione depletion models

• Hypoxia/reoxygenation challenges

Experimental design considerations:

Researchers should employ multiple complementary assays to establish a comprehensive profile of Tmem85's anti-apoptotic functions .

How is Tmem85 function altered in disease states such as neurodegenerative disorders?

While direct evidence linking Tmem85 to neurodegenerative disorders is limited, several theoretical connections warrant investigation:

• Oxidative stress connection:

  • Neurodegenerative diseases involve increased oxidative stress

  • Tmem85's protective role against oxidative damage suggests potential relevance

  • Altered Tmem85 function might contribute to neuronal vulnerability

• Protein interaction networks:

  • Tmem85 interacts with GLUT9, which has connections to urate transport

  • Urate levels have been implicated in neurodegenerative diseases, particularly Parkinson's disease

  • This suggests a potential pathway by which Tmem85 might influence neurodegeneration

• Research approaches:

  • Analysis of Tmem85 expression in disease-affected tissues

  • Genetic association studies for Tmem85 variants in patient populations

  • Functional studies in neuronal models with disease-relevant stressors

Researchers investigating these connections should consider both cell-autonomous effects in neurons and potential influences on neuroinflammatory processes through interactions with microglial cells .

What is the potential role of Tmem85 in immune system regulation and inflammation?

Recent research on TMEM family proteins suggests potential immune regulatory functions for Tmem85:

Possible mechanisms of immune regulation:

• Modulation of immune cell apoptosis during inflammation resolution

• Potential regulation of immune cell activation thresholds

• Protection against oxidative burst damage in inflammatory environments

Research evidence from TMEM family members:

• Some TMEM proteins influence macrophage polarization (M1/M2 balance)

• TMEM proteins can affect T cell infiltration and activity in tissue microenvironments

• Several TMEM proteins modulate inflammatory cytokine production

While direct evidence for Tmem85's role in immunity is still emerging, its anti-apoptotic function suggests it may protect immune cells from activation-induced cell death or oxidative damage during inflammatory responses. Further research using immune cell-specific Tmem85 manipulation would help clarify these potential functions .

How does the function of mouse Tmem85 compare with human TMEM85 in experimental models?

Comparative studies between mouse and human TMEM85 reveal important insights:

Functional conservation:

• Both human TMEM85 and mouse Tmem85 exhibit anti-apoptotic properties

• The heterologous expression of human TMEM85 in yeast promotes growth and prevents cell death in response to oxidative stress, similar to the function of the yeast ortholog

Structural similarities:

• Sequence homology suggests conserved transmembrane topology

• Key functional domains appear to be maintained across species

Experimental considerations when comparing species:

• Expression level differences may affect functional outcomes

• Cellular context (including interacting proteins) may vary between species

• Post-translational modification patterns might differ

Research approach for cross-species comparison:

• Rescue experiments in knockout models

• Direct side-by-side functional assays

• Domain swapping between species variants

The high degree of functional conservation suggests mouse models can provide valuable insights into human TMEM85 biology, though species-specific differences should be considered when translating findings .

What are the critical quality control parameters for recombinant mouse Tmem85 protein preparations?

Ensuring high-quality recombinant Tmem85 preparations requires rigorous quality control:

Purity assessment:

• SDS-PAGE analysis with silver staining (target: >85% purity)

• Mass spectrometry for identity confirmation

• Endotoxin testing for preparations intended for immune cell studies

Functional validation:

• Anti-apoptotic activity in cellular assays

• Protein interaction verification (e.g., GLUT9 binding)

• Proper folding assessment via circular dichroism

Stability parameters:

• Thermal stability testing

• Freeze-thaw tolerance evaluation

• Long-term storage stability monitoring

Documentation requirements:

For collaborative research, standardized quality control reporting facilitates reproducibility across laboratories .

How should researchers optimize transfection and expression systems for studying recombinant Tmem85?

Optimization strategies for Tmem85 expression include:

Mammalian expression systems:

• Cell line selection: HEK293T cells often provide good expression of membrane proteins

• Vector considerations: CMV promoter for high expression; inducible systems for temporal control

• Transfection method: Lipid-based methods typically work well; electroporation for difficult-to-transfect cells

• Timing: 24-72 hour expression period depending on experimental needs

Expression optimization:

• Codon optimization for improved translation efficiency

• Signal sequence modifications for enhanced membrane targeting

• Addition of stabilizing fusion tags (e.g., EGFP) for visualization and stability

• Temperature modulation (30-37°C) to balance expression and proper folding

Validation approaches:

• Western blotting to confirm expression level and molecular weight

• Fluorescence microscopy to verify membrane localization (if fluorescently tagged)

• Functional assays to confirm biological activity

Researchers should note that overexpression of membrane proteins can sometimes lead to mislocalization or aggregation, necessitating careful optimization of expression levels for functional studies .

What are the most effective methods for preserving activity of purified recombinant Tmem85 protein?

Maintaining the functional integrity of purified Tmem85 requires careful handling:

Buffer optimization:

• pH stability range determination (typically pH 7.0-7.4 for membrane proteins)

• Salt concentration optimization (typically 150-300 mM NaCl)

• Addition of stabilizing agents (glycerol 10-20%, specific lipids)

• Consideration of detergent types and concentrations for solubilization

Storage recommendations:

• Aliquoting to avoid repeated freeze-thaw cycles

• Flash freezing in liquid nitrogen

• Storage at -70°C for long-term stability

• Short-term storage at 4°C with appropriate preservatives for active use

Reconstitution guidelines:

• Reconstitute lyophilized protein at 200-500 μg/mL in appropriate buffer

• Allow complete solubilization before use

• Centrifuge to remove any insoluble material

• Validate activity after reconstitution

Stability monitoring:

• Regular activity testing of stored samples

• SDS-PAGE analysis to assess degradation

• Dynamic light scattering to monitor aggregation

For functional studies, freshly prepared or minimally manipulated protein preparations typically provide optimal results .

What emerging technologies could advance our understanding of Tmem85 structure and function?

Several cutting-edge technologies hold promise for Tmem85 research:

Structural biology approaches:

• Cryo-electron microscopy for membrane protein structure determination

• Advanced NMR techniques for dynamic structural studies

• In silico molecular dynamics simulations based on homology models

Functional genomics:

• CRISPR screens to identify genetic interactions with Tmem85

• Single-cell transcriptomics to characterize cellular responses to Tmem85 manipulation

• Spatial transcriptomics to map Tmem85 expression in complex tissues

Protein interaction technologies:

• Proximity labeling methods (TurboID, APEX2) in live cells

• Advanced mass spectrometry for complex membrane protein interactions

• Optical techniques for tracking protein interactions in real-time

Therapeutic exploration:

• Small molecule screening for Tmem85 modulators

• Peptide-based approaches targeting specific Tmem85 domains

• RNA-based therapeutics to modulate Tmem85 expression

These emerging approaches could reveal new aspects of Tmem85 biology and potentially lead to therapeutic applications in stress-related pathologies .

How might artificial intelligence and computational methods contribute to Tmem85 research?

AI and computational approaches offer powerful tools for advancing Tmem85 research:

Structure prediction:

• AlphaFold2 and RoseTTAFold for accurate protein structure prediction

• Molecular dynamics simulations to study conformational changes

• Binding site prediction for potential ligand or protein interactions

Systems biology integration:

• Network analysis to position Tmem85 in cellular pathways

• Multi-omics data integration to understand context-dependent function

• Predictive modeling of cellular responses to Tmem85 manipulation

Drug discovery applications:

• Virtual screening for Tmem85-targeting compounds

• Structure-based drug design for modulators of Tmem85 function

• Prediction of off-target effects and optimization of specificity

Experimental design optimization:

• Machine learning for optimal experimental parameter selection

• Automated image analysis for high-content screening data

• Predictive models to prioritize hypotheses for experimental testing

Researchers should leverage these computational approaches while validating predictions with rigorous experimental testing .

What are the potential therapeutic applications of modulating Tmem85 activity in disease models?

Based on Tmem85's anti-apoptotic and stress-protective functions, several therapeutic directions warrant exploration:

Neurodegenerative disorders:

• Enhancement of Tmem85 function could protect neurons from oxidative stress

• Particularly relevant in conditions with known oxidative components (Parkinson's, ALS)

• Could be targeted to specific neuronal populations through advanced delivery systems

Inflammatory conditions:

• Modulation of Tmem85 in immune cells might influence inflammation resolution

• Potential applications in chronic inflammatory disorders

• May help balance protective immunity with tissue damage control

Ischemia-reperfusion injuries:

• Cardiac, cerebral, and renal ischemic events involve oxidative damage

• Tmem85 enhancement could protect cells during reperfusion phase

• Temporary modulation could be achieved through targeted delivery approaches

Drug development considerations:

• Small molecule modulators of Tmem85 activity or stability

• Gene therapy approaches for Tmem85 delivery

• Peptide-based drugs targeting specific Tmem85 interactions

Researchers should consider tissue-specific effects and potential unintended consequences in proliferative disorders when developing Tmem85-targeted therapeutics .