Vitamin C induced lysine vitcylation, protein post-translational modification, in immune response against tumor cells.

 

 

A recent study published in the journal Cell unveils the discovery of vitamin C’s ability to directly modify proteins through a process known as vitcylation. This breakthrough marks a significant advancement in our understanding of protein post-translational modifications and their influence on cellular function. It also paves the way for new insights into vitamin C’s biological effects and its potential therapeutic applications, particularly in cancer treatment and immunotherapy.

The Multifaceted Functions of Vitamin C

Vitamin C is essential for humans, who lack the ability to synthesize it due to a mutation in the L-gulonolactone oxidase gene. While it prevents scurvy, the effects of high-dose supplementation, particularly in cancer therapy, remain under investigation. The loss of endogenous production raises safety questions but does not imply harm from supplementation. Vitamin C primarily exists as ascorbate, with dehydroascorbic acid (DHA) as its oxidized form. It acts as an antioxidant at physiological levels but induces reactive oxygen species (ROS) at pharmacological doses, selectively targeting cancer cells. Beyond redox functions, vitamin C is a cofactor for enzymes like ten-eleven translocation (TET) enzymes, which regulate DNA demethylation, and prolyl hydroxylases, which control hypoxia-inducible factor 1-alpha (HIF1α). Its potential as an anti-cancer agent is linked to ROS-mediated cytotoxicity, HIF1α suppression, and immune modulation, though its molecular mechanisms require further study.

Vitamin C in Health and Disease: From Essential Nutrient to Therapeutic Agent

Vitamin C (ascorbic acid) is essential for numerous physiological functions and plays a therapeutic role in several diseases. It is the primary treatment for scurvy, a severe deficiency condition that leads to fatigue, swollen gums, joint pain, and poor wound healing. Vitamin C also enhances iron absorption, aiding in the management of iron deficiency anemia, and supports wound healing by promoting collagen synthesis, making it beneficial for ulcers, burns, and post-surgical recovery. In infectious diseases, vitamin C may help reduce the severity and duration of the common cold and respiratory infections, and it is being studied for potential benefits in COVID-19 and pneumonia due to its anti-inflammatory properties. High-dose intravenous vitamin C is under investigation as an adjuvant cancer therapy, showing potential in reducing tumor growth and oxidative stress. Additionally, it may contribute to cardiovascular health by lowering blood pressure and arterial stiffness, while also offering neuroprotective effects in conditions such as Alzheimer’s and Parkinson’s disease. In critical care, vitamin C has been examined for its role in sepsis and critical illness, where it may reduce organ failure and mortality. It also has potential benefits for diabetes and metabolic syndrome by reducing oxidative stress and improving insulin sensitivity. In inflammatory conditions like gout and arthritis, vitamin C helps lower uric acid levels, potentially preventing flare-ups. Its antioxidant properties may also be protective in asthma, allergic reactions, and eye diseases like cataracts and age-related macular degeneration (AMD). While vitamin C has numerous health benefits, high doses should be used with caution, particularly in individuals prone to kidney stones or those with pre-existing kidney disease. Furthermore, additional research is needed to analyze whether vitamin C induces lysine VitCylation, a potential post-translational modification of proteins, and to determine its clinical significance in various physiological and pathological processes.

The Discovery of Vitamin C-Induced Lysine Vitcylation in Proteins

The discovery that vitamin C directly modifies lysine residues in proteins advances our understanding of post-translational modifications (PTMs). He et al. found that the ascorbate anion, with a reactive lactone structure, resembles compounds like succinic anhydride and homocysteine thiolactone, suggesting its ability to modify lysine’s ε-amine group.
Vitcylation refers to lysine modification by ascorbate, forming “vitcyl-lysine.” This was confirmed via MALDI-TOF/TOF MS, revealing a 175 Da mass shift when lysine-containing peptides were incubated with vitamin C at physiological pH. Isotope-labeled vitamin C further validated this modification. Unlike DHA-induced ascorbylation, vitcylation is specific to lysine and occurs at neutral to mildly alkaline pH.

Vitcylation is dose-dependent (EC50 ~2 mM) and pH-sensitive, peaking at pH 7.0–10.0 before declining. Not all lysine-containing peptides undergo vitcylation, suggesting sequence specificity. In cellular studies, vitamin C induced lysine vitcylation in human and murine cancer cells, affecting proteins across subcellular compartments, with higher modification in mitochondrial proteins. A key finding was STAT1 vitcylation at lysine-298 (K298), enhancing its phosphorylation and activation, leading to stronger IFN-mediated immune responses. This links vitamin C to immune regulation, particularly in cancer.

STAT Signaling: Mechanism and Functions in Immune Response and Disease

STAT proteins are cytoplasmic transcription factors mediating cytokine and growth factor signaling. STAT1 is central to interferon (IFN) signaling, crucial for antiviral defense and immune regulation. STAT1 activation starts with IFN binding to receptors, triggering Janus kinase (JAK) activation. JAKs phosphorylate STAT1 at Y701, enabling dimerization and nuclear translocation. STAT1 dimers bind gamma IFN activation sites (GAS) to drive IFN-responsive gene transcription. Regulation occurs through dephosphorylation by T cell protein-tyrosine phosphatase (TCPTP), ensuring controlled signaling. STAT1 promotes antigen presentation via MHC class I and enhances immune surveillance by recruiting immune cells. Dysregulation contributes to disease: gain-of-function mutations cause chronic mucocutaneous candidiasis (CMC), while deficiency leads to immunodeficiency. In cancer, STAT1 can act as a tumor suppressor or promote immune evasion by inducing IDO1 and PD-L1, depending on context.

Role of Vitamin C-Induced Lysine Vitcylation in STAT1 Signaling

The discovery of STAT1 K298 vitcylation reveals a novel regulatory mechanism. Mass spectrometry confirmed this modification in human and mouse cells, highlighting its significance as K298 is a conserved, surface-exposed residue. Vitcylation enhances STAT1 phosphorylation by preventing dephosphorylation. Structural analysis shows that K298 vitcylation destabilizes the antiparallel STAT1 dimer, blocking TCPTP binding. This prevents dephosphorylation by disrupting salt bonds with E281/E284.
Experiments confirmed that vitamin C treatment disrupts STAT1-TCPTP interaction while preserving JAK1 binding. STAT1 phosphorylation persisted even after staurosporine treatment, which normally induces rapid dephosphorylation.

Vitcylation’s effect mirrors the STAT1-K298N mutation linked to autoimmune disease, both leading to sustained phosphorylation and stronger interferon signaling. Functionally, vitcylation increases STAT1 nuclear translocation and upregulates interferon-responsive genes, confirmed by gene set enrichment analysis. These effects require both K298 vitcylation and Y701 phosphorylation, as mutating either site abolished vitamin C-induced transcriptional responses.

STAT1 Vitcylation Enhances Tumor Immunogenicity

STAT1 vitcylation plays a crucial role in cancer immunotherapy by enhancing tumor antigen presentation and immune activation. Vitamin C treatment upregulates antigen processing and MHC/HLA class I genes, leading to increased MHC I surface expression and improved tumor antigen presentation to CD8+ T cells. In co-culture experiments, vitamin C-treated cancer cells stimulated dendritic cell (DC) and T cell activation, with DCs exhibiting elevated MHC II, CD86, and CD80 expression, while CD8+ T cells demonstrated increased proliferation and cytokine production, including IFN-γ and TNF-α. In vivo, high-dose vitamin C suppressed tumor growth and extended survival in tumor-bearing mice, but only in immunocompetent models, confirming an immune-dependent mechanism. This effect was linked to increased STAT1 phosphorylation, MHC I expression, and interferon responses within tumors. Furthermore, vitamin C preferentially accumulated in tumors, enhancing immune activation while minimizing systemic effects, which led to greater DC and T cell activation in the tumor microenvironment. Notably, the combination of vitamin C with anti-PD1 therapy significantly improved tumor suppression, suggesting that STAT1 vitcylation enhances immune checkpoint blockade by increasing tumor immunogenicity and promoting T cell-mediated responses.

Clinical Applications of Vitcylation in Cancer Immunotherapy

Lysine vitcylation presents new clinical opportunities, particularly in cancer treatment and biomarker development. High-dose vitamin C, known for its safety and tolerability, enhances conventional therapies, reduces chemotherapy side effects, and improves cancer patients’ quality of life. Vitcylation provides a molecular basis for vitamin C’s effects, with potential therapeutic applications. Modulating STAT1 vitcylation could enhance anti-tumor immunity without triggering chronic immune activation seen in genetic STAT1 mutations. Additionally, vitcylation patterns may serve as diagnostic biomarkers, aiding in disease detection, treatment monitoring, and personalized vitamin C dosing for cancer therapy.

Beyond disease treatment, the dose-dependent nature of vitcylation suggests vitamin C influences biological processes beyond basic nutrition, supporting customized supplementation strategies tailored to individual health needs. However, further research is needed to fully understand its clinical potential. Key areas include identifying vitcylated proteins, exploring possible enzymatic regulation, and assessing its impact on tumor and immune cells. Single-cell sequencing could provide deeper insights into vitamin C’s role in the tumor microenvironment.

Conclusions

The discovery of lysine vitcylation redefines vitamin C’s role, extending beyond its antioxidant and cofactor functions to a direct protein modifier. This insight clarifies its influence on cellular signaling and immune regulation. STAT1 vitcylation enhances interferon signaling by blocking TCPTP-mediated dephosphorylation, sustaining immune activation. In cancer, vitamin C boosts tumor immunogenicity, strengthening anti-tumor responses.

Reference

He, X. et al. Lysine vitcylation is a vitamin C-derived protein modification that enhances STAT1-mediated immune response. Cell; Published online February 28, 2025; DOI: 10.1016/j.cell.2025.01.043. https://www.cell.com/cell/abstract/S0092-8674(25)00145-X

 

About Marin Biologic Laboratories

Our Recent Publication/Meeting Presentation on Gene Therapy

American Society of Hematology (ASH) Annual Meeting 2024.

Abstract link: Using Gene Therapy to Solve Challenges with CAR-T Cell Immunotherapy: Lead Selection and Preclinical Development of an Adeno-Associated Virus with Reduced Immunogenicity Exhibiting Efficient and Long-Term Expression of an Anti-CD19 T-Cell Engager.

Comprehensive Assay Solutions for In Vitro and Cell Based Potency Assays and Pharmacokinetics Studies- Our Expertise

With 30 years of expertise in cell culture, cell-based assays, and preclinical/clinical PK/PD analysis, we specialize in offering assay services essential for a wide variety of therapeutic drug development programs, preclinical studies, IND/BLA applications, and commercialization. Our comprehensive services include both preclinical non-GLP and GLP assays, as well as non-GMP and GMP assays, providing critical support throughout the entire development pipeline.

Watch the following video and explore our latest presentation on the development and validation of potency and pharmacokinetic (PK) assays for AAV vectors, highlighting innovative methodologies and industry-leading expertise.

 

 

Download the full presentation: Development of Custom Cell Based and In vitro Potency and Pharmacokinetics (PK) Assays for AAV vectors- Marin biologic Laboratories

 

Development of Cell-Based Potency Assays: Case Studies and Blogs from Marin Biologic Laboratories (MarinBio)

 

Drug Discovery & Development Assays Offered by Marin Biologic Laboratories (MarinBio)