GSTM5 Human

What is GSTM5 and what is its functional role in human metabolism?

GSTM5 belongs to the Mu class of Glutathione S-transferases (GSTs), a family of Phase II detoxification enzymes that catalyze the conjugation of glutathione to various electrophilic compounds. These enzymes play crucial roles in detoxifying carcinogens, therapeutic drugs, environmental toxins, and products of oxidative stress .

The GSTM gene family in humans consists of five members (GSTM1-5) located on chromosome 1. These genes encode enzymes that function primarily in xenobiotic metabolism and cellular protection against oxidative damage . In the broader context of human physiology, GSTs operate within the endocrine system where hormones regulate body growth, development, metabolism, homeostasis, and reproduction .

To study GSTM5's basic function, researchers typically employ enzyme activity assays using specific substrates like styrene 7,8-oxide and trans-stilbene oxide . When investigating GSTM5 at the cellular level, it's important to consider that its activity may vary significantly between individuals due to genetic polymorphisms, potentially influencing detoxification capacity and disease susceptibility.

How does the structure of GSTM5 differ from other members of the GSTM family?

While all GSTM family members share basic structural similarities as cytosolic GST enzymes, GSTM5 possesses unique features that distinguish it from its paralogs:

A particularly distinctive characteristic of GSTM5 is the presence of a long inverted repeat (LIR) in intron 5. This structural element can form a stable stem-loop configuration with a 31-bp stem and a 9-nt loop . This feature is not present in other GSTM genes and appears to be primate-specific, suggesting specialized regulatory functions that evolved relatively recently .

Sequence analysis reveals that the intronic LIR in GSTM5 contains inverted exons derived from exon 5 of GSTM4 and exon 5 of GSTM1, indicating a complex evolutionary history involving exon acquisition and rearrangement . This structural peculiarity suggests that GSTM5 may be subject to unique regulatory mechanisms not shared by other GSTM family members.

For researchers investigating GSTM family proteins, it's important to note that in some non-human primates (like macaques), GSTM5 serves as the predominant GSTM isoform instead of GSTM1 (which is pseudogenized in these species), indicating potential functional compensation between family members across different primate lineages .

What is the genomic organization of GSTM5 and its evolutionary significance?

The human GSTM5 gene, along with other GSTM family members, is located on chromosome 1p13.3 . The gene contains multiple exons and introns in a structure typical of GST genes. What makes GSTM5 particularly interesting from an evolutionary perspective is the presence of unique structural elements:

The long inverted repeat (LIR) in intron 5 of GSTM5 represents a distinctive genomic feature that is present in primates but absent in non-primates . Phylogenetic analysis indicates that this LIR contains inverted exons derived from GSTM4 and GSTM1, suggesting a complex evolutionary process involving exon duplication and rearrangement .

The human and chimpanzee GSTM5 genes show evidence of compensating mutations that have made the stem-loop structure more stable, suggesting that this feature has been maintained by natural selection and likely serves a functional purpose .

To study the evolutionary significance of GSTM5, researchers should employ comparative genomics approaches to analyze orthologous sequences across multiple species. Methods such as phylogenetic analysis, selective pressure analysis (dN/dS ratios), and functional assays comparing GSTM5 activity across species can provide insights into how this gene has evolved and potentially acquired new functions in primates.

What genetic variants of GSTM5 have been identified and characterized?

While comprehensive data on human GSTM5 variants is somewhat limited in the provided search results, studies in non-human primates provide valuable insights into the types of variants that may affect GSTM5 function:

In cynomolgus and rhesus macaques, re-sequencing of GSTM5 identified 6 non-synonymous variants (amino acid-changing mutations) and 1 variant (IVS5+1) causing exon skipping . Of these variants, 3 were found exclusively in Indochinese cynomolgus macaques and 1 only in Indonesian cynomolgus macaques, indicating population-specific variation patterns .

Functional characterization using recombinant proteins revealed that 4 of the 6 non-synonymous variants (E29Q, L96R, M166V, and S201N) exhibited substantially reduced metabolic activities . Moreover, animals with specific genotypes (homozygotes for E29Q and heterozygotes for S201N or IVS5+1) showed significantly lower glutathione conjugation activities in liver cytosolic fractions compared to wild-type animals .

For researchers studying GSTM5 variants, it's recommended to:

• Employ next-generation sequencing to identify variants across the entire gene, including regulatory regions

• Use site-directed mutagenesis to recreate variants in expression systems

• Conduct enzyme activity assays with physiologically relevant substrates

• Assess protein stability and expression levels for different variants

• Correlate genotypes with clinical or physiological parameters in population studies

How does DNA methylation affect GSTM5 expression in normal and disease states?

DNA methylation represents an important epigenetic mechanism regulating GSTM5 expression with significant implications for disease processes:

GSTM5 DNA hypermethylation has been identified as a potential tumor biomarker in urothelial carcinoma, suggesting that epigenetic silencing of this gene may contribute to carcinogenesis . This finding is consistent with the known roles of GST enzymes in detoxifying carcinogens and protecting cells from oxidative damage.

To investigate GSTM5 methylation status, researchers typically employ methods such as:

• Bisulfite sequencing - the gold standard for comprehensive methylation analysis

• Methylation-specific PCR - for targeted assessment of specific CpG sites

• Pyrosequencing - for quantitative methylation analysis

• Genome-wide methylation arrays - to study GSTM5 methylation in broader context

When studying the relationship between GSTM5 methylation and disease, it's important to correlate methylation patterns with gene expression levels (using qRT-PCR or RNA-seq) and to validate findings across multiple patient samples and diverse tissue types. Functional studies using demethylating agents (like 5-aza-2'-deoxycytidine) can help establish causality between methylation status and expression changes.

How can GSTM5 expression be accurately quantified in different human tissues and experimental models?

Accurate quantification of GSTM5 expression requires tailored methodological approaches depending on the level of analysis (DNA, RNA, protein, or activity) and the experimental context:

For mRNA expression analysis, quantitative real-time PCR (qRT-PCR) using primers specific to unique regions of GSTM5 is essential to avoid cross-reactivity with other GSTM family members . RNA sequencing provides a more comprehensive view, capable of detecting splice variants and novel transcripts. As demonstrated in diabetic foot ulcer (DFU) studies, microarray data from repositories like the Gene Expression Omnibus (GEO) can be valuable for analyzing GSTM5 expression patterns across different conditions .

Protein expression can be assessed through Western blotting using antibodies specific to GSTM5. Immunohistochemistry (IHC) has been successfully employed to visualize and validate GSTM5 protein expression in tissue sections, as shown in the DFU research . For functional assessment, enzyme activity assays using specific substrates provide insight into GSTM5's catalytic capacity. In macaque studies, styrene 7,8-oxide and trans-stilbene oxide conjugation activities were correlated with GSTM protein levels quantified immunochemically in liver samples .

When designing experiments to quantify GSTM5 expression, researchers should:

• Include appropriate housekeeping genes or reference proteins for normalization

• Validate antibody specificity to avoid cross-reactivity with other GSTM proteins

• Consider tissue heterogeneity when interpreting results

• Account for potential post-transcriptional regulation by comparing mRNA and protein levels

• Assess both expression levels and enzymatic activity when possible

What is GSTM5's role in immune regulation and inflammatory processes?

Emerging evidence suggests that GSTM5 may play important roles in immune regulation and inflammatory responses beyond its classical detoxification function:

In diabetic foot ulcer (DFU) research, GSTM5 has been identified as a potential immune-related key target using machine learning and bioinformatics approaches . The gene was found to be downregulated in DFU compared to normal samples, and subsequent validation through external datasets and immunohistochemistry experiments confirmed GSTM5 as a critical biomarker for this condition .

Functional analysis indicates that GSTM5 may be associated with specific immune cell populations, particularly regulatory T cells (Tregs) and T follicular helper cells . Additionally, GSTM5 appears to influence several signaling pathways crucial for immune function, including NF-κB, GnRH, and MAPK pathways .

To investigate GSTM5's immunoregulatory functions, researchers should consider:

• Immune cell profiling in tissues with differential GSTM5 expression

• In vitro models manipulating GSTM5 expression in immune cells

• Cytokine production assessment following GSTM5 modulation

• Pathway analysis focusing on NF-κB and MAPK signaling

• Animal models with tissue-specific GSTM5 knockout in immune compartments

The identification of GSTM5 as an immune-related target in DFU suggests potential for therapeutic development aimed at modulating inflammatory responses in this and potentially other inflammatory conditions.

What advanced bioinformatic approaches are most effective for studying GSTM5 in disease contexts?

Modern bioinformatic and computational biology approaches offer powerful tools for investigating GSTM5's role in various disease contexts:

Machine learning models have demonstrated particular utility in identifying GSTM5 as a significant biomarker. In DFU research, LASSO and Support Vector Machine-Recursive Feature Elimination (SVM-RFE) models successfully identified GSTM5 as a key gene associated with this condition . These approaches can efficiently analyze complex gene expression datasets to identify genes with the strongest disease associations.

Pathway enrichment techniques, including Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA), provide valuable insights into the biological processes and signaling pathways associated with GSTM5 expression . In the DFU study, these methods helped establish connections between GSTM5 and immune function.

For immunological assessment, computational algorithms like CIBERSORT can be employed to estimate the relative abundance of different immune cell types and correlate these with GSTM5 expression . This approach revealed potential associations between GSTM5 and specific T cell populations in DFU.

When implementing bioinformatic approaches for GSTM5 research, consider:

• Integration of multi-omics data (transcriptomics, proteomics, epigenomics)

• Appropriate data preprocessing to address batch effects and technical variations

• External validation using independent datasets

• Experimental validation of computational predictions

• Network analysis to understand GSTM5's position in broader biological systems

The table below summarizes key bioinformatic approaches for GSTM5 research:

How do GSTM5 variants affect enzyme kinetics and substrate specificity?

Understanding how GSTM5 genetic variants impact enzyme function requires detailed biochemical characterization:

Studies in non-human primates have provided valuable insights into how GSTM5 variants affect enzymatic function. Four non-synonymous variants (E29Q, L96R, M166V, and S201N) exhibited substantially reduced metabolic activities compared to the wild-type enzyme when tested with model substrates . Animals with specific genotypes showed significantly lower conjugation activities in liver cytosolic fractions, confirming the functional impact of these variants in a more physiological context .

To characterize GSTM5 variants biochemically, researchers should:

• Express recombinant wild-type and variant GSTM5 proteins in suitable systems

• Purify proteins and confirm structural integrity

• Conduct enzyme kinetics assays (determining Km and Vmax) with multiple substrates

• Assess protein stability under various conditions

• Investigate potential changes in protein-protein interactions

• Consider using molecular dynamics simulations to predict structural impacts

Variations in GSTM5 may affect:

• Substrate binding affinity

• Catalytic rate

• Allosteric regulation

• Protein stability and half-life

• Interactions with other cellular components

These biochemical differences can translate to altered detoxification capacity at the cellular and organismal levels, potentially influencing susceptibility to environmental toxins, drug metabolism, and disease risk.

What experimental models are most appropriate for studying GSTM5 functional genomics?

Selecting appropriate experimental models is critical for meaningful GSTM5 research. Various systems offer different advantages depending on the specific research questions:

For in vitro studies, recombinant protein expression systems allow detailed biochemical characterization of GSTM5 and its variants. This approach was successfully employed in macaque studies to assess the functional impact of genetic variants on enzymatic activity . Human cell lines with endogenous GSTM5 expression or engineered for controlled expression provide cellular contexts for studying regulation and function.

Tissue samples and subcellular fractions offer more physiologically relevant systems. Liver cytosolic fractions have been effectively used to measure GSTM5-mediated conjugation activities in correlation with protein levels quantified immunochemically . For disease-specific studies, relevant tissue samples (like diabetic foot ulcer specimens) provide valuable insights into altered GSTM5 expression in pathological states .

Computational approaches complement experimental models. Structural modeling can predict the impacts of mutations, while systems biology approaches place GSTM5 in broader metabolic and signaling networks. Machine learning models, as demonstrated in DFU research, can identify complex patterns in gene expression data .

When selecting experimental models for GSTM5 research, consider:

• The specific aspect of GSTM5 biology being investigated

• The relevance of primate-specific features to your research question

• The need for high-throughput vs. physiological relevance

• Technical feasibility and available resources

• Ethical considerations, particularly for animal models

• Complementary approaches to strengthen findings

How does the intronic long inverted repeat in GSTM5 influence gene regulation?

The primate-specific long inverted repeat (LIR) in intron 5 of GSTM5 represents a fascinating structural feature with potential regulatory implications:

This unique element can form a stable stem-loop structure with a 31-bp stem and a 9-nt loop . The human and chimpanzee LIRs have undergone compensating mutations that enhance the stability of this structure, suggesting functional significance maintained by natural selection .

Sequence analysis reveals that the LIR consists of inverted exons acquired by the intron, specifically derived from exon 5 of GSTM4 and exon 5 of GSTM1 . This indicates a complex evolutionary history involving exon duplication and rearrangement events.

The presence of this intronic LIR may potentially introduce complexity in the expression of the GSTM gene family . Inverted repeats can influence various aspects of gene regulation, including:

• mRNA processing and stability

• Alternative splicing patterns

• Formation of regulatory RNAs

• Recruitment of RNA-binding proteins

• Chromatin structure and accessibility

To investigate the regulatory impact of this LIR, researchers should consider:

• Mini-gene splicing assays to assess effects on exon inclusion/exclusion

• RNA structure probing techniques to confirm stem-loop formation in vivo

• RNA-protein interaction studies to identify LIR-binding factors

• CRISPR-based editing to modify or remove the LIR and evaluate functional consequences

• Comparative studies across primate species with varying LIR structures

Understanding the regulatory role of this primate-specific feature may provide insights into the evolutionary adaptations of detoxification mechanisms in humans and other primates.

What is the relationship between GSTM5 and cancer development or progression?

The relationship between GSTM5 and cancer involves both its classical detoxification function and potential epigenetic regulation:

GSTM5 DNA hypermethylation has been suggested as a tumor biomarker in urothelial carcinoma, indicating that epigenetic silencing of this gene may contribute to cancer development . This finding aligns with the established roles of GST enzymes in detoxifying carcinogens and protecting cells from oxidative damage that could lead to DNA mutations and genomic instability.

As a member of the GST family, GSTM5 likely contributes to the cellular defense against carcinogens through:

• Direct detoxification of carcinogenic compounds

• Reduction of oxidative stress that can damage DNA

• Modulation of signaling pathways involved in cell proliferation and apoptosis

The link between GSTs and drug resistance is also relevant to cancer therapy. GSTs have been associated with resistance to chemotherapy agents, potentially through direct drug metabolism or effects on cellular redox status . GSTM5 may contribute to this process, affecting treatment efficacy.

For researchers investigating GSTM5 in cancer contexts, consider:

• Methylation analysis across different cancer types and stages

• Correlation of GSTM5 expression with clinical outcomes

• Functional studies manipulating GSTM5 levels in cancer cell lines

• Assessment of GSTM5's role in detoxifying specific carcinogens relevant to the cancer type

• Investigation of GSTM5 variants in cancer susceptibility studies

• Potential for targeting GSTM5 or its pathways for therapeutic development

How do environmental factors influence GSTM5 expression and function?

As a detoxification enzyme, GSTM5's expression and function can be influenced by various environmental factors:

GST enzymes, including those in the GSTM family, can be induced by a wide range of compounds associated with chemical stress and carcinogenesis, including phenobarbital, planar aromatic compounds, ethoxyquin, BHA, and trans-stilbene oxide . This induction represents an adaptive response mechanism to enhance detoxification capacity in the presence of potentially harmful substances.

Interestingly, many compounds that induce GSTs are themselves substrates for these enzymes . This relationship suggests a feedback mechanism where exposure to substrates increases the expression of enzymes that metabolize them, potentially enhancing protective capacity.

To study environmental influences on GSTM5, researchers should consider:

• Cell culture models exposed to various xenobiotics

• Dose-response and time-course experiments to characterize induction patterns

• Reporter gene assays to identify regulatory elements responsive to environmental stimuli

• Human or animal studies with controlled exposures

• Epigenetic analysis to assess environmentally-induced alterations in DNA methylation or histone modifications

• Population studies correlating environmental exposures with GSTM5 variants and expression levels

Understanding how environmental factors influence GSTM5 may help explain individual variations in susceptibility to toxins, drugs, and certain diseases. This knowledge could contribute to personalized approaches in environmental health, pharmacology, and disease prevention.