TXNL1 Human

What is TXNL1 and what are its primary functions in human cells?

TXNL1, also known as thioredoxin-related protein of 32 kDa (TRP32), is a cytosolic thioredoxin-fold protein that is expressed in all cell types and highly conserved from yeast to mammals. TXNL1 has been identified as having dual critical functions in cellular physiology. First, it supports thioredoxin reductase 1 (TrxR1)-driven redox activities in disulfide reduction reactions, similar to thioredoxin (Trx1) but with lower catalytic efficacy. Second, it functions as an ATP-independent molecular chaperone that prevents protein aggregation during cellular stress conditions . This combination of redox activity and chaperone function makes TXNL1 unique among thioredoxin family proteins, as it can both participate in redox homeostasis and protect protein structure independently of its redox function .

How does TXNL1 differ from other thioredoxin family proteins?

TXNL1 distinguishes itself from other thioredoxin family members, particularly Trx1, in several important ways. While both TXNL1 and Trx1 can reduce disulfides in substrates like insulin, cystine, and glutathione disulfide (GSSG) using NADPH and TrxR1, TXNL1 demonstrates lower catalytic efficacy due to at least one order of magnitude higher Km of TrxR1 for TXNL1 compared to Trx1 . The most striking difference is TXNL1's ATP-independent chaperone activity, which is absent in Trx1. When Trx1 reduces insulin, the reduced insulin precipitates, which is typically used as a measure of Trx1 activity. In contrast, TXNL1-reduced insulin remains in solution because TXNL1 forms non-covalent complexes with the reduced insulin . Furthermore, TXNL1 possesses a unique domain structure consisting of an N-terminal Trx domain and a C-terminal domain with the domain of unknown function 1000 (DUF1000), which interacts with the 26S proteasome .

What is the molecular structure of human TXNL1?

Human TXNL1 is a 289 amino acid protein with a molecular weight of approximately 32 kDa. The protein consists of two distinct domains: an N-terminal thioredoxin (Trx) domain that contains the redox-active site responsible for disulfide reduction reactions, and a C-terminal domain containing the domain of unknown function 1000 (DUF1000) . The C-terminal domain has been shown to interact with the 26S proteasome, suggesting a potential role in protein degradation pathways . The complete amino acid sequence of human TXNL1 has been determined, and recombinant versions of the protein can be expressed in systems such as Escherichia coli for research purposes . The redox activity of TXNL1 depends on specific cysteine residues within its thioredoxin domain, similar to other members of the thioredoxin family, while its chaperone function appears to be independent of these cysteine residues as demonstrated by studies using Cys-to-Ser variants .

How can researchers effectively express and purify human TXNL1 for in vitro studies?

For effective expression and purification of human TXNL1, researchers can employ bacterial expression systems using Escherichia coli. Recombinant human TXNL1 can be produced as a full-length protein spanning amino acids 1 to 289 . A common approach involves cloning the human TXNL1 gene into an expression vector containing a histidine tag, which facilitates purification using nickel affinity chromatography. The expression vector is then transformed into E. coli, followed by induction of protein expression, typically using IPTG (Isopropyl β-D-1-thiogalactopyranoside) . After cell lysis, the His-tagged TXNL1 protein can be purified to >95% purity using affinity chromatography methods . For functional studies, researchers often create Cys-to-Ser variants through site-directed mutagenesis to examine the role of specific cysteine residues in TXNL1's redox activity. The purified protein's integrity can be verified using SDS-PAGE and mass spectrometry techniques . This approach enables subsequent enzymatic characterization and functional studies of TXNL1 under controlled laboratory conditions.

What assays can be used to measure the dual functions of TXNL1?

To comprehensively assess the dual functions of TXNL1, researchers should employ distinct assays for its redox activity and chaperone function:

For redox activity measurement:

• Insulin reduction assay: This classic assay measures the ability of TXNL1 to reduce disulfides in insulin when coupled with TrxR1 and NADPH. Unlike with Trx1, researchers should note that TXNL1-reduced insulin remains in solution rather than precipitating .

• DTNB-based free thiol determination: After the insulin reduction reaction, free thiols can be quantified using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) under denaturing conditions, measuring absorbance at 412 nm .

• Cystine and GSSG reduction assays: TXNL1's ability to reduce these substrates can be measured in reactions coupled to TrxR1 using NADPH, with activity monitored by the decrease in NADPH absorbance at 340 nm .

For chaperone activity assessment:

• Protein aggregation prevention assay: Measure TXNL1's ability to prevent aggregation of whole cell lysate proteins during heating, using turbidity measurements .

• Insulin solubility assay: Evaluate TXNL1's ability to form non-covalent complexes with reduced insulin, keeping it in solution instead of precipitating .

• Size exclusion chromatography: To detect complex formation between TXNL1 and client proteins .

To distinguish between redox-dependent and redox-independent functions, researchers should include Cys-to-Ser TXNL1 variants in these assays and perform experiments both with and without TrxR1/NADPH .

How can the protective effects of TXNL1 against oxidative stress be evaluated in cellular models?

To evaluate TXNL1's protective effects against oxidative stress in cellular models, researchers can utilize a systematic approach combining multiple complementary methods:

• Cell viability assays following oxidative challenge: Expose cells (such as hippocampal neuronal HT-22 cells) to H₂O₂ or glucose deprivation with and without TXNL1 overexpression or Tat-TXNL1 treatment. Measure viability using MTT, CCK-8, or LDH release assays to quantify TXNL1's protective effects .

• ROS detection assays: Employ fluorescent probes such as DCFH-DA to measure intracellular ROS levels in control versus TXNL1-overexpressing or Tat-TXNL1-treated cells following oxidative challenge. Flow cytometry or fluorescence microscopy can be used for quantification .

• Western blot analysis of stress signaling pathways: Examine the effect of TXNL1 on MAPKs (p38, JNK, ERK) phosphorylation and Akt activation in response to oxidative stress. Additionally, assess changes in pro-apoptotic and anti-apoptotic protein expression (Bax, Bcl-2) to understand the mechanism of protection .

• Gene knockdown experiments: Use siRNA or CRISPR-Cas9 to knock down endogenous TXNL1 and observe the heightened sensitivity to oxidative stress, providing evidence for TXNL1's protective role through loss-of-function studies .

• Cell-permeable fusion protein delivery: For cells that are difficult to transfect, utilize cell-permeable versions of TXNL1, such as Tat-TXNL1 fusion protein, which can be directly added to culture media to enhance cellular uptake .

These methodologies allow for comprehensive assessment of TXNL1's cytoprotective function and underlying mechanisms in various cellular models of oxidative stress.

How does TXNL1 interact with the 26S proteasome and what are the functional implications?

TXNL1 interacts with the 26S proteasome through its C-terminal domain, which contains the domain of unknown function 1000 (DUF1000) . This interaction has significant functional implications for cellular protein homeostasis. The 26S proteasome is the primary machinery for controlled protein degradation in eukaryotic cells, and TXNL1's association with this complex suggests a role in coordinating redox regulation with protein quality control mechanisms. TXNL1 is involved in the function of the regulatory particle non-ATPase 11 (Rpn11), a subunit of the 26S proteasome, as well as translation elongation factor 1A (EF1A) . Through these interactions, TXNL1 appears to facilitate the transfer of misfolded proteins to the degradation machinery, serving as a bridge between its chaperone function and protein clearance mechanisms . This dual capacity to both protect proteins from aggregation as a chaperone and guide terminally misfolded proteins to degradation makes TXNL1 an important component of cellular protein quality control systems. The proteasomal interaction may also explain why TXNL1 overexpression can affect cell proliferation and act as a transcriptional repressor in certain cellular contexts , potentially through the degradation of specific regulatory proteins.

What is the kinetic difference between TXNL1 and Trx1 in disulfide reduction reactions?

Although both TXNL1 and Trx1 can reduce disulfides in substrates like insulin, cystine, and glutathione disulfide (GSSG) using NADPH and TrxR1, significant kinetic differences exist between these two thioredoxin family proteins. The most notable difference is that TXNL1 demonstrates lower catalytic efficacy compared to Trx1, primarily due to TXNL1 having at least one order of magnitude higher Km (Michaelis constant) when interacting with TrxR1 . This higher Km value indicates a lower affinity between TXNL1 and TrxR1, resulting in less efficient coupling in enzymatic reactions. The practical consequence of this kinetic difference is that at equivalent concentrations, TXNL1 would reduce disulfides more slowly than Trx1 in cellular environments where TrxR1 is the electron donor. This difference suggests that while TXNL1 contributes to cellular redox balance, it likely plays a complementary rather than redundant role to Trx1 in maintaining redox homeostasis. The distinct kinetic properties, combined with TXNL1's additional chaperone function, indicate evolutionary specialization within the thioredoxin family to address multiple cellular needs simultaneously .

How does TXNL1 protect against ischemic brain injury and what are the molecular mechanisms involved?

TXNL1 demonstrates significant neuroprotective effects against ischemic brain injury through multiple molecular mechanisms targeting oxidative stress and apoptotic pathways. Studies utilizing cell-permeable Tat-TXNL1 fusion protein have shown that TXNL1 markedly reduces neuronal cell death following ischemic damage both in vitro and in vivo . The primary protective mechanisms include:

• Inhibition of ROS production: Tat-TXNL1 significantly reduces reactive oxygen species in H₂O₂-exposed hippocampal neuronal (HT-22) cells, thereby preventing oxidative damage to cellular components .

• Modulation of MAPK signaling pathways: Excessive ROS increases phosphorylation of MAPKs (p38, JNK, ERK), leading to neuronal cell death. TXNL1 inhibits the phosphorylation of these MAPKs, interrupting cell death signaling cascades in oxidative stress conditions .

• Regulation of apoptotic protein expression: TXNL1 favorably modulates the expression of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins, increasing the Bcl-2/Bax ratio and thereby preventing activation of caspase-dependent apoptotic pathways .

• Suppression of glial activation: In ischemic animal models, Tat-TXNL1 significantly reduces the activation of astrocytes (GFAP-positive cells) and microglia (Iba-1-positive cells), which are known to exacerbate neuroinflammation and subsequent neuronal damage following ischemic injury .

• Activation of survival pathways: TXNL1 modulates Akt activation, a key component of pro-survival signaling in neurons under stress conditions .

These mechanisms collectively contribute to TXNL1's neuroprotective effects, suggesting its potential as a therapeutic protein for ischemic brain injury .

What is the potential application of cell-permeable Tat-TXNL1 fusion protein in treating oxidative stress-related disorders?

Cell-permeable Tat-TXNL1 fusion protein represents a promising therapeutic approach for treating oxidative stress-related disorders due to its dual functionality and efficient cellular uptake capabilities. The Tat peptide, derived from the HIV trans-activator of transcription (Tat) protein, serves as a protein transduction domain (PTD) that enables efficient delivery of TXNL1 across cellular membranes, overcoming one of the major challenges in protein therapy . This fusion protein demonstrates several therapeutic advantages:

• Size-independent protein delivery: Unlike other delivery methods, Tat PTD fusion proteins are not limited by the protein size, making TXNL1 delivery feasible regardless of its 32 kDa molecular weight .

• Broad neuroprotective effects: Tat-TXNL1 has shown protective effects against H₂O₂-induced oxidative stress in vitro and ischemic brain injury in vivo, suggesting applications in stroke, traumatic brain injury, and neurodegenerative disorders where oxidative stress plays a central role .

• Multi-targeted mechanism: By simultaneously reducing ROS production, modulating MAPK signaling pathways, regulating apoptotic protein expression, and suppressing neuroinflammation, Tat-TXNL1 addresses multiple pathological mechanisms involved in oxidative stress-related disorders .

• Potential for combination therapy: The direct antioxidant and anti-apoptotic properties of Tat-TXNL1 could complement existing therapeutic approaches, potentially enhancing their efficacy when used in combination .

What is the relationship between TXNL1 expression and cancer progression or chemotherapy resistance?

TXNL1 exhibits complex relationships with cancer progression and chemotherapy resistance that appear to be context-dependent across different cancer types. Research has revealed several important mechanisms:

• Transcriptional regulation: TXNL1 overexpression can inhibit mammalian cell proliferation and act as a transcriptional repressor through direct binding to the transcription factor B-Myb in certain cancer cell lines such as SNU-1 . This suggests a potential tumor-suppressive role in some contexts.

• Oxidative stress modulation: TXNL1 prevents cell death and may influence cancer progression through inactivation of oxidative stress-induced phosphatase of regenerating liver (PRL) . Since cancer cells often exhibit elevated ROS levels, TXNL1's antioxidant functions could either support tumor cell survival or help maintain redox homeostasis to prevent malignant transformation, depending on the stage and type of cancer.

• Chemotherapy resistance mechanisms: In gastric cancer, TXNL1 expression correlates with chemotherapy response through regulation of the base excision repair protein XRCC1 (X-ray repair cross-complementing group 1). Upregulation of TXNL1 promotes the degradation of XRCC1, whereas TXNL1 downregulation leads to XRCC1 upregulation in cisplatin-resistant gastric cancer cells (BGC823/DDP) . Since XRCC1 is involved in DNA repair, its regulation by TXNL1 can influence how cancer cells respond to DNA-damaging chemotherapeutic agents.

The dual role of TXNL1 in both promoting cell survival through antioxidant mechanisms and potentially inhibiting cell proliferation through transcriptional repression suggests that its effects on cancer progression are likely dependent on the specific cellular context, cancer type, and treatment conditions . These findings indicate that TXNL1 and its interaction partners could serve as potential biomarkers for predicting chemotherapy response or as drug targets for adjuvant chemotherapy, particularly in gastric cancer.

How might the ATP-independent chaperone activity of TXNL1 be exploited to address protein misfolding diseases?

The ATP-independent chaperone activity of TXNL1 represents a unique opportunity for therapeutic intervention in protein misfolding diseases such as neurodegenerative disorders. Unlike many molecular chaperones that require ATP hydrolysis, TXNL1 can prevent protein aggregation independently of energy consumption, making it potentially more resilient under stress conditions when cellular energy resources may be compromised . To exploit this property, researchers could pursue several strategic approaches:

The unique ability of TXNL1 to maintain chaperone function even when its redox activity is compromised (as demonstrated with Cys-to-Ser variants) suggests that these functions could be separately targeted or enhanced for therapeutic benefit . This property distinguishes TXNL1 from other thioredoxin family proteins and positions it as a promising candidate for addressing protein misfolding under conditions where ATP availability or redox balance may be compromised.

What methodological approaches could resolve the structural basis of TXNL1's chaperone activity independent of its redox function?

Resolving the structural basis of TXNL1's chaperone activity independent of its redox function requires sophisticated methodological approaches that can distinguish between these dual functionalities. Several complementary techniques could be employed:

• High-resolution structural studies of TXNL1-substrate complexes:

  • Cryo-electron microscopy (cryo-EM) to visualize TXNL1 bound to client proteins during chaperone action

  • X-ray crystallography of TXNL1 variants with substrates to determine binding interfaces

  • NMR spectroscopy to examine dynamic changes in TXNL1 structure upon substrate binding

  These approaches should be applied to both wild-type TXNL1 and Cys-to-Ser variants that lack redox activity but retain chaperone function , allowing identification of structural elements specifically involved in chaperone activity.

• Targeted mutagenesis beyond redox-active cysteines:

  • Alanine-scanning mutagenesis of surface residues to identify regions critical for chaperone function

  • Domain truncation and chimeric protein construction to isolate the minimal regions required for chaperone activity

  • Site-directed photocrosslinking to map protein-substrate interaction sites

• Biophysical characterization of chaperone-substrate interactions:

  • Isothermal titration calorimetry (ITC) to determine binding energetics

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions that undergo conformational changes upon substrate binding

  • Small-angle X-ray scattering (SAXS) to determine solution structures of TXNL1-substrate complexes

• Comparative analysis with other chaperones:

  • Structural alignment with known ATP-independent chaperones to identify conserved structural features

  • Competition assays with other chaperones to determine substrate specificity

  • In silico modeling and molecular dynamics simulations to predict chaperone-active regions

By combining these methodological approaches, researchers could develop a comprehensive structural model explaining how TXNL1 performs its chaperone function independently of its redox activity. This would significantly advance our understanding of this unique dual-function protein and potentially inform the development of therapeutics targeting specific TXNL1 functions.