Recombinant Mouse Thioredoxin-like protein 1 (Txnl1)

What is Mouse Thioredoxin-like Protein 1 (Txnl1) and what are its key structural features?

Txnl1, also known as TRP32 (thioredoxin-related protein of 32 kDa), is a widely expressed protein with dual functionality in cellular processes. Structurally, Txnl1 contains an N-terminal thioredoxin (TRX) domain and a C-terminal domain formerly known as DUF1000, now recommended to be called the PITH domain (proteasome-interacting thioredoxin) . The full-length protein has a molecular weight of approximately 32 kDa.

When working with recombinant mouse Txnl1, researchers should note that the protein has a distinctive domain organization that influences its functionality:

• N-terminal thioredoxin domain: Contains the redox-active C-G-P-C motif

• C-terminal PITH domain: Mediates interaction with the 26S proteasome

The protein exists predominantly in a reduced form in cells, similar to thioredoxin-1 (Trx1) .

How does Txnl1 differ from Thioredoxin-1 (Trx1) in function and cellular distribution?

While both Txnl1 and Trx1 belong to the thioredoxin family, they differ significantly in several aspects:

Unlike Trx1, Txnl1 is not secreted from cells, and its subcellular localization is not regulated by protein kinase C activity . Additionally, Txnl1 displays specific activity of about 30% compared to E. coli thioredoxin and has a reduction potential of approximately -250 mV .

What are the optimal conditions for measuring Txnl1 enzymatic activity in vitro?

To measure Txnl1 enzymatic activity, researchers can use a standard insulin reduction assay coupled to NADPH and thioredoxin reductase (TrxR1). Based on established protocols:

• Buffer composition: Use activity assay buffer containing 50 mM Tris-HCl, 2 mM EDTA, pH 7.5 .

• Reaction components:

  • NADPH: 1 mM

  • Insulin: 160-180 μM

  • TrxR1: 200 nM (containing selenium)

  • Txnl1: 10-20 μM

• Measurement conditions:

  • Temperature: 25°C

  • Use a microplate reader with 1-minute time interval readings

  • Include reaction mixtures without Txnl1 as background reference

• Kinetic analysis:

  • Plot turnover versus substrate concentration

  • Use Michaelis-Menten fit with nonlinear regression (e.g., using GraphPad Prism)

  • Correct turnover numbers for the selenium content of TrxR1 preparation

For determining free thiols upon reduction of insulin, use DTNB under denaturing conditions (1 mM DTNB, 6 M guanidine-HCl in 50 mM Tris-HCl, pH 8.0) and measure absorbance at 412 nm .

How can I effectively validate the purity and activity of recombinant mouse Txnl1 protein preparations?

A comprehensive validation approach for recombinant mouse Txnl1 should include:

• Purity assessment:

  • SDS-PAGE under reducing conditions (expected 3 μg of protein should show a single band)

  • Visualize with Coomassie blue stain

  • Purity should be >90% by SDS-PAGE

• Molecular weight confirmation:

  • Predicted MW: ~32 kDa (full-length Txnl1)

  • Confirm by MALDI-TOF or similar technique

• Activity validation:

  • Measure specific activity using insulin precipitation assay

  • Monitor increase in absorbance at 650 nm resulting from insulin reduction

  • Specific activity should be measurable as A650/cm/min/mg

• Endotoxin testing:

  • Use LAL method to ensure preparation contains <1 EU per 1μg of protein

• Western blot validation:

  • Use specific antibodies against Txnl1

  • Compare against known positive controls

  • Verify expected molecular weight (32 kDa band)

For optimal storage, recombinant Txnl1 can be stored at 2-8°C for 1 week, and for long-term storage, aliquot and store at -20°C to -80°C, avoiding repeated freeze-thaw cycles .

How can I investigate the dual functions of Txnl1 as both a redox enzyme and a chaperone in cellular systems?

To investigate the dual functions of Txnl1, employ distinct methodological approaches for each function:

For redox enzyme function:

• Use the insulin reduction assay to measure thioredoxin activity with TrxR1 and NADPH

• Create Cys-to-Ser substituted variants (particularly at the active site cysteines) to disable redox activity while maintaining protein structure

• Monitor disulfide reduction in the presence vs. absence of TrxR1/NADPH

• Use a substrate trap approach (C35S mutation in human Txnl1 equivalent) to stabilize intermolecular disulfides with protein substrates

For chaperone function:

• Test chaperone activity by measuring prevention of insulin aggregation in solution without requiring ATP

• Assess chaperone function towards whole cell lysate proteins by monitoring prevention of their aggregation during heating

• Compare wild-type Txnl1 with redox-inactive mutants to demonstrate that chaperone activity does not require redox function

• Examine chaperone function in the absence of TrxR1 and NADPH to confirm ATP-independence

Cellular analysis:

• Use siRNA knockdown of Txnl1 to assess effects on ubiquitin-protein conjugates

• Analyze protein aggregation under stress conditions in cells with and without Txnl1

• Investigate interactions between Txnl1 and the 26S proteasome using co-immunoprecipitation or nondenaturing electrophoresis followed by immunoblotting

What experimental approaches can be used to study the interaction between Txnl1 and the 26S proteasome?

To investigate Txnl1-proteasome interactions, researchers can employ multiple complementary approaches:

• Co-immunoprecipitation studies:

  • Use specific antibodies against Txnl1 or proteasome subunits (particularly Rpn11)

  • Follow established protocols for coupling antibodies to beads:
    a) Couple antibody to beads (use protein A/G beads)
    b) Incubate with cell lysates
    c) Wash and elute bound proteins
    d) Analyze by SDS-PAGE and immunoblotting

• Nondenaturing gel electrophoresis:

  • Run cell lysates on nondenaturing gels

  • Perform immunoblotting with antibodies against Txnl1 and proteasome subunits

  • Observe co-migration of Txnl1 with 26S proteasomes (not with 20S proteasomes)

• Structural analysis:

  • Employ cryo-EM for structure determination of TXNL1-bound proteasome (recently achieved at 3.0-3.3 Å resolution)

  • Use AlphaFold prediction for full-length TXNL1 to analyze structural features

  • Perform surface charge analyses using PyMol to visualize hydrophobic features of the PITH domain

• Domain mapping:

  • Create truncation mutants to identify interaction domains

  • Previous studies have demonstrated that the DUF1000/PITH domain is necessary and sufficient to mediate interaction with the 26S proteasome

• Quantitative analysis:

  • Use quantitative immunoblotting to determine the proportion of Txnl1 associated with proteasomes (at least 85% of cellular Txnl1 has been shown to co-precipitate with 26S proteasomes)

How can Txnl1 substrate interactions be trapped and identified in vivo?

To identify physiological substrates of Txnl1 in vivo, researchers can adapt the substrate trap approach that has been successfully used for thioredoxins:

• Generate a substrate trap mutant:

  • Create a C35S mutation (or equivalent in mouse Txnl1) that replaces the highly reactive resolving thiol with a hydroxyl group

  • This mutation interrupts the oxidoreductase reaction and stabilizes the intermolecular disulfide between Txnl1 and its substrates

• In vivo application strategies:

  • Generate a transgenic mouse with inducible expression of the mutant Txnl1 transgene

  • Use a system similar to the flag-hTrx1 C35S transgenic mouse model described for Trx1

  • This can involve:
    a) Creating a founder line with attP sites in a suitable locus
    b) Inserting the flag-tagged mutant Txnl1 transgene
    c) Using ϕC31 enzyme to ensure correct integration

• Substrate identification:

  • Isolate Txnl1-substrate complexes from tissues using immunoprecipitation

  • Analyze using mass spectrometry to identify bound proteins

  • Verify with Western blot analysis under non-reducing conditions to preserve disulfide linkages

• Verification of physiological relevance:

  • Compare identified substrates with those from in vitro approaches

  • Validate specific interactions using co-immunoprecipitation studies with wild-type Txnl1

  • Confirm redox sensitivity of interactions under different oxidative stress conditions

Previous studies using this approach have identified eEF1A1 (a substrate-recruiting factor of the 26S proteasome) as a likely physiological substrate of Txnl1 .

What is the role of Txnl1 in proteostasis networks and how does this function change under cellular stress conditions?

Txnl1 represents a unique connection between protein reduction and proteolysis, two major intracellular protein quality control mechanisms. To investigate its role in proteostasis:

• Proteasome function analysis:

  • Measure proteasome activity in cells with normal vs. reduced Txnl1 levels

  • Monitor degradation of ubiquitinated proteins (knockdown of Txnl1 has been shown to moderately stabilize ubiquitin-protein conjugates)

  • Use HALO UBAUBQLN1 (TUBE) coupling to HALO LINK resin for pull-down of ubiquitinated proteins

• Stress response studies:

  • Investigate changes in Txnl1 expression under different stress conditions:

    • Oxidative stress (interaction with the proteasome may be modulated)

    • Unfolded protein response (Txnl1 is upregulated in C. elegans during UPR)

    • Proteasome inhibition (Txnl1 is induced upon proteasome inhibition)

• Redox state analysis under stress:

  • Monitor the redox state of Txnl1 using redox Western blotting techniques

  • Examine how different stressors affect the Txnl1 redox state and interactions

• Specific stress-related interactions:

  • Recent research has identified key binding interfaces between TXNL1 and proteasomal subunits required for ubiquitin-independent degradation of TXNL1 upon cellular exposure to compounds causing oxidative stress

  • Investigate this stress-induced degradation pathway to understand how cellular stress modulates Txnl1 levels and function

• Comparative analysis with other proteostasis components:

  • Examine interplay between Txnl1 and other systems involved in protein quality control

  • Analyze potential redundancy or compensation mechanisms when Txnl1 function is compromised

The recently determined structure of TXNL1-bound proteasome provides insights into molecular interactions required for stress-induced degradation, suggesting that Txnl1 may regulate proteasomal activity under stress conditions .

How can I optimize Western blot detection of endogenous mouse Txnl1 in tissue samples?

For optimal detection of endogenous mouse Txnl1 in tissue samples, follow these methodological recommendations:

• Sample preparation:

  • Homogenize snap-frozen tissue in buffer containing:

    • 1 mM NaHPO₄ anhydrous

    • 5 mM EDTA (pH 8.0)

    • 1% protease inhibitor cocktail

    • 1% phosphatase inhibitor cocktails

    • 0.1 mM PMSF

  • Incubate on ice for 30 minutes

  • Centrifuge at 10,000 rpm for 15 minutes at 4°C

• Protein quantification:

  • Determine protein concentration using BCA assay

  • Standardize loading amounts (typically 20-50 μg total protein per lane)

• Gel electrophoresis conditions:

  • Use 10-12% SDS-PAGE gels

  • Include reducing agent (e.g., β-mercaptoethanol or DTT)

  • Run at 100-120V until adequate separation

• Transfer parameters:

  • Transfer to PVDF membrane at 100V for 60-90 minutes or 30V overnight at 4°C

  • Alternatively, use nitrocellulose membranes

• Antibody selection and dilution:

  • Primary antibody: Anti-TXNL1 antibody [EPR16061(B)] (targeting N-terminal) has shown good specificity in mouse samples at 1:1000 dilution

  • Secondary antibody: HRP-conjugated or IRDye-labeled secondary antibodies at manufacturer-recommended dilutions (typically 1:5000-1:20000)

• Signal detection optimization:

  • Expected molecular weight: ~32 kDa

  • Use appropriate controls (knockout or siRNA-treated samples if available)

  • For weak signals, consider extended exposure times or enhanced chemiluminescence substrates

• Troubleshooting steps:

  • For high background: Increase blocking time/concentration or add 0.05-0.1% Tween-20 to wash buffers

  • For weak signals: Increase antibody concentration, protein loading, or use signal enhancement systems

  • For multiple bands: Verify antibody specificity against knockout controls or preincubate antibody with recombinant Txnl1

What are the key considerations when designing experiments to study cross-talk between Txnl1 and other redox systems?

When investigating the cross-talk between Txnl1 and other redox systems, consider these methodological approaches:

• Experimental design principles:

  • Include appropriate controls for each redox system (e.g., Trx1, glutathione system)

  • Design experiments to distinguish direct from indirect effects

  • Consider both acute and chronic modulation of redox systems

• Modulation approaches:

  • Genetic: Use siRNA, CRISPR-Cas9, or inducible expression systems for targeted manipulation

  • Pharmacological: Employ specific inhibitors:

    • For TrxR1: Auranofin (1-2 μM) can inhibit thioredoxin reductase activity

    • For glutathione system: Buthionine sulfoximine (BSO) depletes GSH

• Readout systems:

  • Monitor multiple parameters simultaneously:

    • Protein thiol status (using redox Western blotting)

    • General cellular redox state (with redox-sensitive fluorescent proteins)

    • Functional outcomes (e.g., protein aggregation, proteasome activity)

• Multi-omics integration:

  • Combine targeted approaches with broader analyses:

    • Proteomics: PISA (Protein Integral Stability Alteration) assay can track protein stability

    • Redox proteomics: Identify changes in thiol oxidation states globally

    • REXpression: Use non-Cys containing peptides for protein abundance information

• Data analysis considerations:

  • Calculate correlation coefficients between different parameters

  • Examples from literature show good correlation (R=0.91) between conventional protein expression and REXpression measures

  • Compare coefficient of variation (CV) values (median values of 8% vs. 7% for conventional vs. REXpression methods)

• Validation in multiple systems:

  • Use both recombinant proteins and cellular systems

  • Compare results across different cell types and tissue contexts

  • Consider the impact of cellular compartmentalization on redox systems