GSTK1 Human

What is GSTK1 and where is it located in human cells?

GSTK1 (Glutathione S-transferase kappa 1) is an enzyme encoded by the GSTK1 gene located on chromosome 7q34 in humans. It belongs to the glutathione S-transferase (GST) superfamily, which is primarily known for cellular detoxification processes . While originally described in rodent mitochondria, GSTK1 has been definitively identified as a peroxisomal protein due to the presence of a peroxisomal targeting signal 1 (PTS1) . Some research has also suggested potential localization in the endoplasmic reticulum (ER), though this remains under debate .

Methodological approach for subcellular localization studies: Researchers can employ fluorescent fusion proteins (e.g., GSTK1-GFP) combined with organelle-specific markers for colocalization studies using confocal microscopy. Subcellular fractionation followed by Western blot analysis with organelle-specific markers can provide biochemical confirmation of GSTK1 localization in different cellular compartments.

How does GSTK1 differ structurally from other GST family members?

GSTK1 diverges significantly from other GST family members in both sequence similarity and protein structure. While GSTK1 contains the characteristic TRX-like domain and helical domain common to all GSTs, its secondary structure organization is substantially different . The key structural differences include:

• The helical domain is positioned between the βαβ and ββα motifs of the TRX-like domain, unlike the typical arrangement in other GSTs where the TRX-like domain and C-terminal helical domain are connected by a short linker of alpha-helices .

• The GSTK1 dimer adopts a distinctive butterfly shape rather than the V-shaped crevice typically observed in other GST classes .

• Structurally, GSTK1 resembles bacterial HCCA (2-hydroxychromene-2-carboxylate) isomerases and bacterial disulfide-bond-forming DsbA oxidoreductase, which has led to its alternative designation as DsbA-L .

Methodological approach for structural analysis: X-ray crystallography remains the gold standard for determining protein structure. Researchers can also employ comparative molecular modeling techniques using known bacterial HCCA isomerases and DsbA oxidoreductase structures as templates to predict functional domains in GSTK1.

What are the known alternative names and identifiers for the GSTK1 gene and protein?

GSTK1 is referenced in scientific literature and databases under several alternative names and identifiers, which is important knowledge for comprehensive literature searches and database queries:

The gene is also known as "glutathione S-transferase kappa 1" in full form . Using these alternative identifiers in literature searches ensures comprehensive coverage of research related to this gene.

What enzymatic activities have been demonstrated for GSTK1 in vitro?

GSTK1 demonstrates multiple enzymatic activities that highlight its diverse roles in cellular metabolism:

• GSH-conjugating activity: Purified GSTK1 exhibits conjugating activity towards halogenated aromatics, including 1-chloro-2,4-dinitrobenzene and 4-nitrobenzylchloride .

• Peroxidase activity: GSTK1 shows peroxidase activity against cumene hydroperoxide, tert-butyl hydroperoxide, and 15-S-hydroperocy-5,8,11,13-eicosatetraenoic acid .

• GSH-dependent disulfide bond oxidoreductase activity: Evidence suggests that GSTK1 has GSH-dependent disulfide bond oxidoreductase activity, as demonstrated by rescue experiments with GSTK1-deficient cells .

• Detoxification of xenobiotics: GSTK1 may be important for conjugating GSH to cisplatin and arsenic-based compounds, suggesting a role in detoxification of these substances .

Methodological approach for enzymatic characterization: Spectrophotometric assays using specific substrates can measure conjugation and peroxidase activities. For oxidoreductase activity, researchers can employ redox-sensitive fluorescent probes like roGFP2 to monitor changes in cellular redox state in GSTK1-expressing versus GSTK1-deficient cells.

How does GSTK1 contribute to peroxisomal redox homeostasis?

GSTK1 plays a crucial role in maintaining peroxisomal redox balance through several mechanisms:

• As the only known peroxisomal glutathione-consuming enzyme identified in humans, GSTK1 is strategically positioned to regulate the glutathione antioxidant system within peroxisomes .

• Experimental evidence shows that GSTK1 ablation significantly extends the recovery period of peroxisomal glutathione redox sensors (po-roGFP2) after treatment with thiol-specific oxidants, suggesting a role in redox recovery mechanisms .

• The GSH-dependent disulfide bond oxidoreductase activity of GSTK1 likely contributes to the restoration of redox equilibrium following oxidative insults .

• GSTK1 may serve as a crucial link between glutathione metabolism and NAD(P)H levels in peroxisomes, as these redox couples often correlate .

Methodological approach for studying peroxisomal redox homeostasis: Researchers can employ genetically encoded, compartment-specific redox sensors such as po-roGFP2 (targeted to peroxisomes) to monitor real-time changes in the glutathione redox state in live cells. CRISPR-Cas9 gene editing can be used to generate GSTK1-deficient cell lines to assess the impact of GSTK1 loss on peroxisomal redox homeostasis.

What is the current understanding of GSTK1's role in adiponectin regulation?

The relationship between GSTK1 and adiponectin regulation remains controversial:

This conflicting data highlights the need for further research to clarify the precise relationship between GSTK1 and adiponectin regulation.

Methodological approach for studying adiponectin regulation: Co-immunoprecipitation experiments can assess direct interactions between GSTK1 and adiponectin. Analysis of adiponectin multimer formation using non-reducing SDS-PAGE in GSTK1-knockdown or overexpression models can further elucidate this relationship. ER stress markers should be monitored to determine whether GSTK1 effects on adiponectin are mediated through ER stress pathways.

What phenotypes have been observed in GSTK1-deficient animal models?

GSTK1 deficiency in animal models has been associated with multiple pathological phenotypes:

• Zebrafish: GSTK1 deficiency leads to hypertrophic cardiomyopathy .

• Mice: Multiple phenotypes have been documented including:

  • Diabetic renal tubular injury

  • Subclinical glomerular disease

  • Obesity-induced inflammation

  • Insulin resistance

  • Glomerular nephropathy

• Cellular models: GSTK1-deficient HEK-293 cells show impaired intraperoxisomal redox recovery after oxidative insult .

These findings collectively suggest that GSTK1 plays important roles in cardiovascular health, metabolic homeostasis, and kidney function.

Methodological approach for phenotypic analysis: Researchers can generate GSTK1 knockout models using CRISPR-Cas9 technology and characterize phenotypes through histopathological analysis, metabolic profiling, and functional testing specific to the organ systems affected (renal function tests, glucose tolerance tests, etc.).

How is GSTK1 implicated in human kidney diseases?

GSTK1 has been associated with several kidney-related conditions in humans:

• GSTK1 gene variations have been linked to familial idiopathic steroid-resistant nephrotic syndrome .

• In patients, decreased GSTK1 expression correlates with accelerated chronic kidney disease progression .

• The GSTK1 gene has been associated with several renal conditions including:

  • Nephronophthisis

  • Renal dysplasia

  • C3 glomerulonephritis

  • Frasier syndrome

  • Focal segmental glomerulosclerosis

  • Dent disease

This diverse range of kidney pathologies suggests that GSTK1 may play fundamental roles in maintaining renal function and protecting against kidney injury.

Methodological approach for human studies: Researchers can employ genome-wide association studies (GWAS) and targeted sequencing to identify GSTK1 variants in kidney disease cohorts. Expression studies using kidney biopsy samples can assess GSTK1 levels in various renal pathologies. Functional characterization of disease-associated variants can be performed in cell culture and animal models.

What is the evidence for GSTK1's role in cancer and metabolic diseases?

Research has revealed complex and sometimes contradictory relationships between GSTK1 and both cancer and metabolic diseases:

Cancer associations:

• Variations in the GSTK1 gene have been linked to certain types of cancer .

• Increased GSTK1 levels have been associated with colon cancer progression .

• Interestingly, increased GSTK1 expression correlates with improved survival in patients with luminal B breast cancer .

Metabolic disease associations:

• GSTK1 deficiency is associated with obesity-induced inflammation and insulin resistance in mice .

• Fat-specific GSTK1 overexpression protects mice from diet-induced obesity, insulin resistance, and hepatic steatosis .

• Decreased GSTK1 is linked to aging-related declines in respiratory performance .

These findings suggest context-dependent roles for GSTK1 in different tissues and disease states, highlighting the importance of tissue-specific studies.

Methodological approach for disease association studies: Tissue microarray analysis can assess GSTK1 expression across different cancer types and stages. Metabolic phenotyping of tissue-specific GSTK1 knockout or overexpression models can clarify its role in metabolism. Cell-based assays measuring proliferation, migration, and response to oxidative stress can elucidate mechanisms in disease contexts.

What are the recommended approaches for studying GSTK1 enzymatic activities in cellular contexts?

To effectively characterize GSTK1 enzymatic activities in cellular contexts, researchers should consider these methodological approaches:

• Genetically encoded redox sensors: Using targeted fluorescent redox sensors like po-roGFP2 or po-GRX1-roGFP2 to monitor real-time changes in glutathione redox state within peroxisomes .

• CRISPR-Cas9 gene editing: Creating GSTK1-deficient cell lines using guide RNAs targeting specific exons (e.g., the guide sequence 5′-gtgcggcccagcctcataaca-3′ has been successfully used ).

• Rescue experiments: Reintroducing wild-type or mutant GSTK1 (e.g., S16A active site mutant) to GSTK1-deficient cells to confirm functional relationships .

• Compartment-specific analysis: Using organelle-specific targeting sequences to direct redox sensors to specific cellular compartments for selective monitoring of redox changes in peroxisomes versus cytosol .

• Oxidative challenge assays: Employing thiol-specific oxidants to assess the role of GSTK1 in redox recovery mechanisms in different cellular compartments .

Technical considerations: Ensure proper subcellular targeting of sensors by confirming colocalization with organelle markers. Include appropriate controls for each experiment, such as non-targeted sensors and catalytically inactive GSTK1 mutants.

How can researchers effectively generate and validate GSTK1-deficient cell models?

Creating reliable GSTK1-deficient cell models requires careful consideration of the following steps:

• CRISPR-Cas9 targeting strategy:

  • Design guide RNAs targeting early exons of GSTK1 for complete loss of function

  • The sequence 5′-gtgcggcccagcctcataaca-3′ has been successfully used for GSTK1 targeting

  • Clone annealed guide oligonucleotides into a Cas9-expressing vector (e.g., pSpCas9(BB)-2A-GFP)

• Validation of GSTK1 knockout:

  • Genomic validation: PCR amplification and sequencing of targeted loci

  • Protein validation: Western blot analysis to confirm absence of GSTK1 protein

  • Functional validation: Assess GSH-dependent enzymatic activities

• Potential compensatory mechanisms:

  • Assess expression of other GST family members that might compensate for GSTK1 loss

  • Monitor changes in GSH levels and redox state

• Rescue experiments:

  • Reintroduce wild-type GSTK1 to confirm phenotypes are due to GSTK1 loss

  • Use catalytically inactive GSTK1 (S16A mutant) as a control to demonstrate enzyme activity dependence

Methodological refinement: Single-cell cloning following transfection with CRISPR constructs is recommended to ensure homogeneous cell populations. Researchers should consider generating multiple independent knockout clones to control for off-target effects.

What experimental designs are optimal for investigating GSTK1's role in peroxisomal redox signaling?

Investigating GSTK1's role in peroxisomal redox signaling requires sophisticated experimental designs:

• Real-time redox imaging:

  • Express organelle-targeted redox sensors (po-roGFP2, po-GRX1-roGFP2) in wild-type and GSTK1-deficient cells

  • Measure baseline redox state and dynamic changes following oxidative challenge

  • Compare recovery kinetics between wild-type, GSTK1-deficient, and rescue models

• Metabolite profiling:

  • Quantify levels of GSH, GSSG, NAD(P)H, and NAD(P)+ in peroxisomal fractions

  • Compare metabolite ratios between wild-type and GSTK1-deficient cells under basal and stress conditions

• Peroxisomal function assessment:

  • Measure peroxisomal metabolic activities (β-oxidation, catalase activity)

  • Assess peroxisomal membrane integrity and protein import efficiency

• Interactome analysis:

  • Identify GSTK1 interaction partners in peroxisomes using proximity labeling techniques (BioID, APEX)

  • Validate interactions using co-immunoprecipitation and functional assays

• Oxidative challenge panel:

  • Expose cells to various oxidants (H₂O₂, diamide, cumene hydroperoxide)

  • Monitor compartment-specific redox changes and recovery kinetics

Experimental controls: Include cytosolic redox sensors to compare peroxisomal and cytosolic redox responses. Use peroxisome-deficient cell models (e.g., PEX gene knockouts) as additional controls to confirm peroxisome-specific effects.

How can researchers reconcile conflicting data regarding GSTK1's role in adiponectin regulation?

The contradictory findings regarding GSTK1's role in adiponectin regulation require systematic investigation:

• Tissue and cell-type specific analyses:

  • Compare GSTK1-adiponectin relationships across different adipose tissue depots

  • Investigate potential species-specific differences in GSTK1 function

• Mechanistic dissection:

  • Distinguish between direct effects on adiponectin multimerization and indirect effects via ER stress pathways

  • Identify the specific molecular interactions between GSTK1 and adiponectin using structural biology approaches

• Temporal dynamics:

  • Examine adiponectin regulation by GSTK1 under different metabolic states (fasting, feeding, insulin resistance)

  • Use inducible knockout systems to determine acute versus chronic effects of GSTK1 loss

• Integrative approaches:

  • Combine transcriptomics, proteomics, and metabolomics to build comprehensive models of GSTK1-adiponectin relationships

  • Validate molecular mechanisms in human adipose tissue samples from subjects with varying metabolic health profiles

Methodological approach: Time-course experiments with conditional GSTK1 knockout models can help differentiate primary from secondary effects on adiponectin regulation. Cell-free reconstitution systems can test direct effects on adiponectin multimerization.

What are the key knowledge gaps in understanding GSTK1's unique structural features and their functional implications?

Despite significant progress, several knowledge gaps remain regarding GSTK1's structure-function relationships:

• Structural basis for substrate specificity:

  • The butterfly-shaped dimer structure of GSTK1 differs from the V-shaped crevice in other GSTs, but the functional implications remain unclear

  • The positioning of the helical domain between βαβ and ββα motifs likely affects substrate binding, but detailed structural analyses are needed

• Catalytic mechanism:

  • The role of specific residues, particularly C27 and C176 (reported glutathionylation targets), in GSTK1's catalytic mechanism requires clarification

  • The structural basis for GSTK1's GSH-dependent disulfide bond oxidoreductase activity remains to be fully elucidated

• Post-translational regulations:

  • How post-translational modifications affect GSTK1 activity in different cellular compartments

  • Whether GSTK1 activity is regulated by redox-sensitive modifications

• Evolutionary significance:

  • The functional significance of GSTK1's structural similarity to bacterial HCCA isomerases and DsbA oxidoreductase

  • How GSTK1's unique structural features evolved and their adaptative significance

Methodological approach: Structure-guided mutagenesis coupled with activity assays can identify critical residues for different enzymatic functions. Crystallography of GSTK1 with various substrates and cofactors can reveal binding determinants and catalytic mechanisms.

How might GSTK1 research inform therapeutic strategies for kidney diseases and metabolic disorders?

GSTK1 research has significant translational potential for treating kidney diseases and metabolic disorders:

• Biomarker development:

  • GSTK1 expression levels or specific variants could serve as biomarkers for kidney disease progression

  • Association with accelerated chronic kidney disease progression suggests potential prognostic value

• Therapeutic targeting strategies:

  • Enhancing GSTK1 expression or activity in adipose tissue might protect against obesity and metabolic dysregulation

  • Tissue-specific GSTK1 modulation might be necessary given its context-dependent effects in different tissues

• Drug metabolism and toxicity:

  • GSTK1's role in conjugating GSH to cisplatin and arsenic-based compounds suggests it may influence chemotherapy outcomes

  • Understanding GSTK1 polymorphisms could help predict drug sensitivity and toxicity

• Novel therapeutic modalities:

  • Cell-penetrating peptides mimicking GSTK1's functional domains

  • Small molecule activators of GSTK1 for metabolic and renal protection

  • Gene therapy approaches to restore GSTK1 function in deficiency states

Methodological approach for translational research: High-throughput screening for GSTK1 modulators, followed by validation in disease-relevant cell and animal models. Retrospective analysis of clinical outcomes in patients with different GSTK1 polymorphisms can guide personalized medicine approaches.