GSTZ1 Human

What is GSTZ1 and what is its primary function in human metabolism?

GSTZ1 is a member of the zeta-1 family isoform of Glutathione S-Transferases that plays crucial roles in both endogenous metabolism and xenobiotic biotransformation. It primarily catalyzes the oxygenation of dichloroacetic acid (DCA) to glyoxylic acid and functions in xenobiotic α-haloacid metabolism . GSTZ1 is also involved in the tyrosine degradation pathway, as evidenced by the accumulation of fumarylacetoacetate (FAA) and succinylacetone (SA) in GSTZ1-deficient models .

Methodologically, researchers studying GSTZ1 function typically measure its activity through conversion of 14C-DCA to 14C-glyoxylate in the presence of glutathione, using saturating concentrations of DCA (0.2 mM) and glutathione (1 mM) to ensure maximal rates of biotransformation .

What are the common haplotypes of GSTZ1 identified in human populations?

GSTZ1 haplotypes are defined by three key non-synonymous SNPs and one promoter SNP:

• G94>A (rs7975): Results in Glu→Lys substitution at amino acid position 32 (E32K)

• G124>A (rs7972): Results in Gly→Arg at position 42 (G42R)

• C245>T (rs1046428): Results in Thr→Met at position 82 (T82M)

• Promoter SNP: -1002 G>A (rs7160195)

These polymorphisms combine to form several common haplotypes including EGT, KGT, EGM, and KRT. The nomenclature reflects the amino acids at positions 32, 42, and 82 respectively .

How does GSTZ1 expression vary between normal and cancerous tissues?

GSTZ1 expression shows significant variability between normal and cancerous tissues, particularly in hepatocellular carcinoma (HCC). Analysis of 363 HCC tissues from The Cancer Genome Atlas (TCGA) database revealed significantly decreased GSTZ1 mRNA expression in tumor tissues compared with normal liver tissues (P<0.001) . This downregulation was further confirmed through qRT-PCR and protein expression analysis in paired HCC and non-tumor tissues .

The clinical significance of this expression pattern is highlighted by the association between decreased GSTZ1 expression and advanced tumor stage (stage I/II versus stage III/IV, P=0.005) . Methodologically, researchers typically quantify GSTZ1 protein levels using Western blotting with rabbit anti-human GSTZ1 polyclonal antibodies and standardization against purified hGSTZ1C protein .

What experimental models are available for studying GSTZ1 function?

Several experimental models have been developed to study GSTZ1 function:

• GSTZ1 knockout (GSTZ1-KO) cell lines: The search results describe GSTZ1 knockout HepG2 cell lines established using the CRISPR/Cas9 system .

• GSTZ1 overexpression (GSTZ1-OE) models: Adenoviral vectors expressing GSTZ1 (AdGSTZ1) have been used to overexpress GSTZ1, with adenovirus expressing green fluorescent protein (AdGFP) serving as a control .

• Animal models: Gstz1−/− mice have been used to study GSTZ1 deficiency, with these mice showing accumulation of FAA and succinylacetone in urine. When stressed with a high-phenylalanine diet, these mice exhibited rapid weight loss, renal and hepatic damage, necrosis, and lethality .

• Xenograft tumor models: AdGSTZ1-, AdGFP-, or mock-infected MHCC-97H cells injected subcutaneously into nude mice have been used to study the effects of GSTZ1 expression on tumor growth in vivo .

How do GSTZ1 haplotype variations affect protein expression and enzymatic activity?

GSTZ1 haplotype variations significantly influence both protein expression and enzymatic activity in a population-specific manner:

In Caucasians, GSTZ1 protein expression differs significantly between K carrier and non-K carrier haplotypes (p=0.001). Specifically, individuals with K carrier haplotypes (KGT, KRT) show lower GSTZ1 protein expression than those with non-K carrier haplotypes (EGT, EGM) .

This expression difference translates directly to enzymatic activity. Liver cytosol from Caucasian donors without lysine at position 32 (non-K carriers) showed mean DCA metabolism activity of 0.63 ± 0.36 nmol glyoxylate/min/mg protein, which was significantly higher than K carriers .

Interestingly, this haplotype-dependent expression pattern is not observed in African-Americans (p=0.277), highlighting the importance of population genetics in GSTZ1 research .

What is the linkage disequilibrium pattern of GSTZ1 SNPs across different populations?

Linkage disequilibrium (LD) patterns of GSTZ1 SNPs vary significantly between populations, explaining the differential effects of haplotypes on protein expression:

In Caucasians, there is strong LD between the K allele (A allele from G>A SNP rs7975) and the promoter -1002 G>A SNP (rs7160195) A allele. This linkage explains why K carrier haplotypes show lower expression in Caucasians - the K allele is linked to a promoter variant that reduces transcription .

In contrast, African-Americans show no LD between these two polymorphisms, explaining why K carrier status does not affect GSTZ1 expression in this population .

These population differences were identified using genotype data from the 1,000 Genomes Project for European and African populations, with pairwise LD values (D' and r²) calculated and visualized using Haploview software .

How can allele-specific mRNA quantification be applied to study GSTZ1 expression?

Allele-specific mRNA quantification provides valuable insights into the transcriptional mechanisms underlying haplotype-dependent GSTZ1 expression. The methodology involves:

• Identifying individuals heterozygous for both GSTZ1 promoter SNP (-1002 G>A) and coding SNP G94>A (E32K)

• Using pyrosequencing-based methodology to quantify the relative expression of each allele in mRNA transcripts from liver samples

• Comparing the proportion of major allele in DNA (expected to be 50% for heterozygotes) versus the proportion in cDNA (which may deviate from 50% if one allele is preferentially expressed)

• Statistical analysis using Wilcoxon Signed Ranks Test to determine significance of allelic imbalance

This approach allows researchers to determine whether certain GSTZ1 haplotypes are associated with differential expression at the transcriptional level, providing mechanistic insights into how genetic variations influence GSTZ1 function.

What mechanisms underlie the downregulation of GSTZ1 in hepatocellular carcinoma?

GSTZ1 is significantly downregulated in HCC compared to normal liver tissues, as confirmed by multiple methodologies:

• Analysis of TCGA data from 363 HCC tissues showed reduced GSTZ1 mRNA expression in tumors (P<0.001)

• qRT-PCR, immunoblot, and immunohistochemistry assays confirmed lower GSTZ1 mRNA and protein levels in HCC versus paired non-tumor tissues

While the precise mechanisms driving this downregulation weren't fully detailed in the search results, the consistent pattern across multiple patient cohorts suggests a fundamental role for GSTZ1 loss in HCC pathogenesis rather than a secondary effect.

Methodologically, researchers typically use paired tumor/normal samples to control for inter-individual variability and employ multiple techniques (mRNA quantification, protein expression analysis) to confirm GSTZ1 downregulation at different molecular levels.

How does GSTZ1 deficiency influence the NRF2/IGF1R signaling axis in cancer cells?

GSTZ1 deficiency activates the NRF2/IGF1R axis in HCC through a mechanism that results in upregulation of IGF1R:

• Correlation analysis of 363 HCC patients in the TCGA database showed a significant negative correlation between GSTZ1 and IGF1R mRNA expression (r=-0.31, P<0.0001)

• This negative correlation was confirmed at both mRNA and protein levels in HCC tissues using qRT-PCR, immunoblot, and IHC assays (r=-0.68, P<0.0001, n=30)

• Mechanistically, experimental manipulation of GSTZ1 levels directly affected IGF1R expression: GSTZ1 overexpression downregulated IGF1R in Huh7 cells, while GSTZ1 knockout upregulated IGF1R in HepG2 cells

The NRF2 connection is evidenced by observations that GSTZ1 deficiency in mice causes constitutive oxidative stress and suggests activation of the NRF2 antioxidant response pathway . The research concludes that "GSTZ1-1 functions as an important tumor suppressor by inhibiting NRF2/IGF1R axis in HCC" .

What are the implications of GSTZ1 expression levels on patient prognosis in HCC?

GSTZ1 expression levels have significant prognostic implications in HCC:

These findings establish GSTZ1 as an independent prognostic biomarker in HCC, with potential applications in clinical risk stratification. The methodology involved dividing patients into high and low GSTZ1 expression groups based on median expression values and using Kaplan-Meier analysis with log-rank tests to compare survival outcomes .

What approaches are optimal for quantifying GSTZ1 genetic variations in clinical samples?

GSTZ1 genotyping in clinical samples typically involves a multi-step approach:

• DNA isolation from liver tissue samples using commercial kits (e.g., QiaGen Tissue kit)

• PCR amplification of target regions containing the SNPs of interest:

  • G94>A (E32K)

  • G124>A (G42R)

  • C245>T (T82M)

  • Promoter SNP -1002 G>A

• Pyrosequencing for SNP genotyping using a PSQ HS 96 System (QiaGen)

• Haplotype inference from the unphased genotype data using computational methods such as PHASE software version 2.0.2

• Quality control including testing for Hardy-Weinberg equilibrium within each racial/ethnic group using chi-squared test

This comprehensive approach allows for accurate determination of both individual SNPs and the haplotypes they form, which is crucial given the functional significance of specific GSTZ1 haplotypes.

What experimental approaches are optimal for measuring GSTZ1 enzymatic activity?

GSTZ1 enzymatic activity is optimally measured using a radiometric assay that quantifies the conversion of 14C-DCA to 14C-glyoxylate in the presence of glutathione:

• Preparation of liver cytosol samples using standard methods

• Incubation with 14C-DCA (American Radiolabeled Chemicals) and glutathione (Sigma-Aldrich)

• Use of saturating concentrations: 0.2 mM DCA and 1 mM glutathione to ensure maximal rates of biotransformation

• Measurement under linear conditions of product formation

• Expression of activity as nmol glyoxylate/min/mg protein

This approach provides a reliable quantitative measure of GSTZ1's catalytic efficiency that can be compared across different samples, genotypes, and experimental conditions. The use of saturating substrate concentrations ensures that differences in activity reflect differences in enzyme amount or intrinsic catalytic properties rather than substrate availability.

How can GSTZ1 protein expression be accurately quantified in tissue samples?

Accurate quantification of GSTZ1 protein expression in tissue samples involves:

• Preparation of cytosolic protein fractions from tissue samples

• Western blotting using rabbit anti-human GSTZ1 polyclonal primary antibody

• Quantification using a standard curve constructed with expressed, purified hGSTZ1C protein

• Normalization of GSTZ1 content to total cytosolic protein (expressed as ng GSTZ1 per μg cytosolic protein)

• Control for gel-to-gel variability by loading 5 μg of a single sample of human liver cytosol on each gel as an internal standard

This methodology allows for absolute quantification of GSTZ1 protein levels rather than relative comparisons, making it possible to establish reference ranges and compare results across different studies. The use of internal standards and standardized protocols minimizes technical variability, enhancing reproducibility.

How does GSTZ1 influence cellular apoptotic pathways in the context of cancer?

GSTZ1 exerts profound effects on apoptotic pathways in HCC cells through modulation of the IGF1R signaling cascade:

• GSTZ1 overexpression in HCC cells results in:

  • Decreased phosphorylation of AKT, ERK, BAD, and CREB

  • Increased cleavage of caspase 9 and caspase 3

  • Decreased levels of the antiapoptotic protein BCL2

• Conversely, GSTZ1 knockout produces opposite effects, promoting cell survival

• The mechanism appears to be mediated through IGF1R suppression, as treatment with IGF1R inhibitor PPP or IGF1R shRNA blocked the antiapoptotic effect of GSTZ1 depletion in GSTZ1-deficient cells

These findings establish GSTZ1 as a tumor suppressor that promotes apoptosis by inhibiting the IGF1R-mediated survival pathway. The methodology involved manipulating GSTZ1 expression in cell lines and assessing effects on downstream signaling molecules and apoptotic markers by Western blotting.

What implications do GSTZ1 haplotype variations have for personalized medicine approaches?

GSTZ1 haplotype variations have significant implications for personalized medicine, particularly in the context of drugs metabolized by this enzyme:

• Caucasians with K carrier haplotypes show lower GSTZ1 expression and enzymatic activity, resulting in slower metabolism of dichloroacetic acid (DCA) compared to non-K carriers

• This genotype-dependent metabolism has direct implications for drug dosing: "These results further define safe, genetics-based dosing regimens for chronic DCA administration"

• Population differences in haplotype frequencies and linkage disequilibrium patterns (e.g., between Caucasians and African-Americans) necessitate population-specific approaches to GSTZ1-based personalized medicine

The methodological approach for implementing GSTZ1-based personalized medicine would involve genotyping patients for key GSTZ1 SNPs, determining their haplotypes, and adjusting drug dosing according to expected metabolic capacity - lower doses for slow metabolizers (K carriers in Caucasians) and potentially higher doses for rapid metabolizers.

What potential therapeutic targets emerge from the GSTZ1-regulated pathways in HCC?

Research on GSTZ1 in HCC has identified several promising therapeutic targets in GSTZ1-regulated pathways:

• IGF1R: GSTZ1 deficiency leads to upregulation of IGF1R, which promotes cancer cell survival. Targeting IGF1R with inhibitors like PPP or RNA interference reversed the antiapoptotic effects of GSTZ1 depletion

• NRF2: As a key mediator in the pathway between GSTZ1 deficiency and IGF1R upregulation, NRF2 represents another potential target. The research explicitly states that "targeting NRF2 or IGF1R may be a promising treatment approach for this subset HCC"

• Downstream signaling molecules: GSTZ1 deficiency affects key survival pathways through altered phosphorylation of AKT, ERK, BAD, and CREB . These signaling nodes could potentially serve as targets for therapeutic intervention.

The most compelling evidence supports targeting the NRF2/IGF1R axis in HCC cases with GSTZ1 deficiency, with the experimental work demonstrating efficacy of IGF1R inhibition in reversing the tumor-promoting effects of GSTZ1 loss .