GAPDH Human, Active

Introduction to GAPDH Human, Active

Recombinant Human GAPDH protein is a full-length human protein encompassing amino acids 1 to 335, typically expressed in Escherichia coli expression systems with greater than 95% purity . The active form specifically refers to preparations that retain both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase enzymatic activities, making it suitable for functional studies and applications requiring catalytically competent enzyme . GAPDH Human, Active is commonly utilized in research settings for investigating glycolytic pathways, protein-protein interactions, and as a reference standard in various biochemical and cellular assays.

Protein Characteristics

Commercial preparations of GAPDH Human, Active typically possess the following characteristics:

These specifications ensure the protein maintains its native conformation and enzymatic activity, making it valuable for diverse research applications .

Primary Structure

Human GAPDH is encoded by a single gene located on chromosome 12 (position 6,534,512-6,538,374 on the forward strand in the GRCh38 reference genome) . The gene has multiple transcripts (11 splice variants), with the primary protein-coding transcript (GAPDH-201) producing the 335-amino acid polypeptide .

The amino acid sequence of human GAPDH begins with MGKVKVGVNGFGRIGRLVTRAA and contains several functionally important regions, including the NAD+ binding domain and catalytic sites .

Tertiary and Quaternary Structure

Under normal cellular conditions, active GAPDH exists primarily as a homotetramer composed of four identical 37-kDa subunits . This tetrameric structure is essential for the protein's glycolytic function. Each subunit contains a critical catalytic cysteine residue (C152) in the active site that is required for enzymatic activity and is also implicated in the protein's role in oxidative stress responses and apoptosis .

The first high-resolution (1.75 Angstroms) crystal structure of human placental GAPDH revealed that the intersubunit selectivity cleft is closed in the human enzyme, which provides insights into targeting GAPDH for drug design . Water-mediated intersubunit hydrogen bonds assist in the closure of this cleft in the human enzyme .

Domains and Functional Regions

GAPDH contains two major domains:

1. N-terminal NAD+ binding domain (Pfam: PF00044)

2. C-terminal catalytic domain (Pfam: PF02800)

The NAD+ binding domain facilitates cofactor binding essential for the oxidation reaction in glycolysis, while the C-terminal domain contains the catalytic machinery necessary for substrate conversion .

Glycolytic Function

The primary enzymatic function of active GAPDH is to catalyze the sixth step of glycolysis, converting D-glyceraldehyde 3-phosphate (G3P) to 3-phospho-D-glyceroyl phosphate while reducing NAD+ to NADH . This reaction occurs in two coupled steps:

3.1.1 Oxidation

First, the active site cysteine residue attacks the carbonyl group of G3P, creating a hemithioacetal intermediate that is subsequently deprotonated by a histidine residue in the active site. This leads to the reformation of a carbonyl group and ejection of a hydride ion, which is accepted by NAD+ to form NADH, while the hemithioacetal is oxidized to a thioester .

3.1.2 Phosphorylation

In the second step, inorganic phosphate attacks the thioester, forming a tetrahedral intermediate that collapses to release 1,3-bisphosphoglycerate and regenerate the enzyme's active site .

This reaction mechanism highlights the critical nature of the active site cysteine and the dependence on NAD+ as a cofactor for catalytic activity.

Non-Glycolytic Functions

Beyond glycolysis, active GAPDH participates in several non-metabolic cellular processes:

1. Nitrosylase activity: GAPDH mediates cysteine S-nitrosylation of nuclear target proteins such as SIRT1, HDAC2, and PRKDC .

2. Cytoskeletal organization: The protein modulates the organization and assembly of the cytoskeleton and facilitates CHP1-dependent microtubule and membrane associations .

3. Translational regulation: GAPDH is a component of the GAIT (gamma interferon-activated inhibitor of translation) complex, which mediates interferon-gamma-induced transcript-selective translation inhibition during inflammation .

4. Innate immunity: The protein promotes TNF-induced NF-kappa-B activation and type I interferon production through interactions with TRAF2 and TRAF3 .

5. Nuclear functions: GAPDH participates in transcription, RNA transport, DNA replication, and apoptosis .

6. Mitochondrial quality control: GAPDH plays a role in eliminating damaged mitochondria, a process that can be inhibited by phosphorylation by δ protein kinase C (δPKC) under oxidative stress conditions .

Post-Translational Modifications

GAPDH activity is regulated by various post-translational modifications:

1. Phosphorylation: δPKC-mediated phosphorylation has been shown to decrease GAPDH tetramerization and reduce glycolytic activity . This modification also inhibits GAPDH-dependent mitochondrial elimination under oxidative stress .

2. S-nitrosylation: Under cellular stress, GAPDH is S-nitrosylated by nitric oxide, which causes it to bind to the protein SIAH1, a ubiquitin ligase. This complex moves into the nucleus where it contributes to controlled cell shutdown .

3. Oxidation: Oxidative modifications to the active site cysteine can inhibit GAPDH's glycolytic activity, potentially redirecting metabolic flux from glycolysis to the pentose phosphate pathway to generate more NADPH for antioxidant systems .

4. Crotonylation: This modification has been linked to human embryonic stem cell endodermal lineage differentiation and metabolic switching .

Oligomeric State Regulation

The functional switch between GAPDH's metabolic and non-metabolic roles appears to be related to changes in its oligomeric state. The active tetrameric form predominantly functions in glycolysis, while monomeric or dimeric forms may be involved in non-glycolytic processes . Phosphorylation by δPKC decreases GAPDH tetramerization, reducing its glycolytic activity while potentially promoting alternative functions .

Cellular Localization

While primarily cytosolic, active GAPDH exhibits dynamic subcellular distribution:

1. Cytosolic: The majority of GAPDH is found in the cytosol, where it participates in glycolysis .

2. Nuclear: Under specific conditions, GAPDH can translocate to the nucleus, where it participates in transcription, DNA replication, and apoptotic processes .

3. Membrane-associated: In red blood cells, GAPDH and other glycolytic enzymes assemble in complexes on the inside of the cell membrane, potentially enhancing glycolytic efficiency .

4. Cell surface: GAPDH can be expressed on the cell exterior in an iron-dependent manner, where it plays a role in maintaining cellular iron homeostasis .

5. Mitochondria-associated: During cellular stress, GAPDH can associate with damaged mitochondria as part of quality control mechanisms .

Cancer

Active GAPDH is overexpressed in multiple human cancers, including cutaneous melanoma, where its expression positively correlates with tumor progression . Its glycolytic and antiapoptotic functions contribute to tumor cell proliferation and protection. Notably:

1. GAPDH can bind to active Akt, limiting its dephosphorylation and leading to Bcl-xL overexpression, which protects a subset of mitochondria from permeabilization .

2. This GAPDH-dependent Bcl-xL overexpression can protect cells from caspase-independent cell death (CICD), potentially facilitating tumor survival and chemotherapeutic resistance .

3. Depletion of GAPDH has been shown to induce senescence in tumor cells, presenting a potential therapeutic strategy for controlling tumor growth .

Neurodegenerative Disorders

GAPDH has been implicated in several neurodegenerative diseases:

1. Parkinson's disease: Nuclear translocation of GAPDH has been reported in Parkinson's disease, and anti-apoptotic drugs like rasagiline function by preventing this translocation .

2. Alzheimer's disease: The SNP rs3741916 in the 5' UTR of the GAPDH gene may be associated with late-onset Alzheimer's disease . GAPDH can interact with beta-amyloid precursor protein (betaAPP), potentially interfering with cytoskeletal and membrane transport functions .

3. Huntington's disease: GAPDH interactions with huntingtin could interfere with processes related to apoptosis, nuclear tRNA transport, and DNA replication/repair .

Immune System Regulation

Active GAPDH plays roles in immune function and inflammation:

1. It is a component of the GAIT complex, which mediates interferon-gamma-induced transcript-selective translation inhibition during inflammation .

2. GAPDH promotes TNF-induced NF-kappa-B activation and type I interferon production through interactions with TRAF2 and TRAF3 .

3. GAPDH inhibition has been proposed as a strategy to modulate immune responses, with inhibitors like Koningic acid (KA) demonstrating decreased cytokine production by Th1 effector cells in autoimmune models .

As a Molecular Tool

Active human GAPDH finds application in various research contexts:

1. Reference standard: As a housekeeping gene product for normalization in expression studies .

2. Activity assays: For investigating glycolytic flux and metabolic pathways .

3. Protein interaction studies: To identify and characterize binding partners and regulatory mechanisms .

4. Drug screening: As a target for developing inhibitors with potential therapeutic applications .

Monitoring GAPDH Activity

Recent developments include tools for monitoring GAPDH activity in complex biological systems:

1. Cysteine-reactive probes: Electrophilic peptide-based probes like SEC1 can covalently modify the active-site cysteine (C152) of GAPDH to directly report on its activity within a proteome .

2. Activity assays: Commercial kits like the KDalert GAPDH assay kit enable quantitative measurements of GAPDH enzymatic activity .

These tools allow researchers to assess changes in GAPDH activity in response to oncogenic transformation, reactive oxygen species (ROS), and small-molecule GAPDH inhibitors .

Chemical Inhibitors

Several compounds have been identified that can inhibit human GAPDH activity:

1. Koningic acid (KA): A highly effective irreversible inhibitor of GAPDH that acts through a NAD+-uncompetitive and G3P-competitive mechanism. Proteome-wide evaluations have demonstrated its high selectivity for the active-site cysteine of GAPDH .

2. CGP-3466: This compound inhibits apoptosis by preventing nuclear accumulation of GAPDH, as suggested by computational ligand-docking studies .

3. ψGAPDH peptide: An inhibitor of the interaction between δPKC and GAPDH that prevents phosphorylation of GAPDH by δPKC. This peptide also inhibits GAPDH oligomerization and glycolytic activity .

Therapeutic Potential

Modulation of GAPDH activity shows promise in several therapeutic contexts:

1. Anti-cancer strategies: Targeting GAPDH's metabolic and anti-apoptotic functions could sensitize cancer cells to chemotherapy .

2. Immunomodulation: GAPDH inhibitors like Koningic acid have demonstrated decreased cytokine production by Th1 effector cells in autoimmune models .

3. Neuroprotection: Compounds that prevent nuclear translocation of GAPDH, such as deprenyl, have been used clinically to treat Parkinson's disease .

Future Research Directions

Several promising avenues for future research on GAPDH Human, Active include:

1. Further elucidation of the structural transitions between GAPDH's various functional states and how these relate to its diverse cellular roles.

2. Development of selective modulators that can target specific functions of GAPDH while preserving others.

3. Investigation of GAPDH's role in emerging areas such as metabolic reprogramming, cellular stress responses, and age-related diseases.

4. Exploitation of GAPDH's non-canonical functions for therapeutic applications beyond metabolism.

5. Understanding the regulatory networks that control GAPDH's multifunctional nature and how these networks are perturbed in disease states.