Post-Translational Modifications

  • Histone mutations play important roles in DNA organisation

    Histone proteins help compact and organise the DNA of the nucleus, they also play key roles in orchestrating gene expression. The four core histones, H2AH2BH3 and H4 form a complex around which DNA is wound forming the nucleosome. Between the nucleosome is the internucleosomal DNA which is stabilised by the linker histone H1. Modifications of histones can alter the nature of the chromatin and affect its level of compaction, as can histone variants. An example of such a variant is γH2Ax which is associated with DNA double strand breaks, such as during meiosis; for which CRB has raised an antibody.

    Mutations of histone proteins can also have a huge role in how the chromatin is organised and can impact disease. As part of our histone antibody range we have produced two novel mutation specific antibodies able to specifically detect two somatic missense mutant histone H3 (H3.3) proteins. The mutations are at a critical position within the histone tail, glycine 34, and involve amino acid changes to arginine or valine (G34R and G34V). The conversion of glycine to a large side chain containing residue sterically hinders interactions of H3K36-specific methyltransferases such as STED2 with the downstream H3K36 position. This results in the blocking of H3 lysine 36 (H3K36) dimethylation and trimethylation. H3K36me3 is essential for DNA repair, including DNA mismatch repair (MMR) by interacting with mismatch repair (MMR) protein MutSα, and recruiting the MMR machinery to chromatin. MMR corrects errors created during DNA replication and the resulting MMR deficiency leads to genome instability and tumorigenesis.

    Histone H3.3 G34V/R mutations are a hallmark of paediatric diffuse intrinsic pontine gliomas (DIPG), non-brain stem paediatric high grade gliomas (pHGG) and some adult glioblastoma multiforme (GBM) tumours. The anti-H3.3 G34R antibody offered in our DISCOVERY® antibody catalogue has shown high specificity and selectivity to the G34R mutation in paediatric brain tumour sections by immunohistochemistry. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumour development in vivo.

    Anti-Histone H3.3 G34R mutant-specific antibody can be bought here and anti-Histone H3.3 G34V mutant-specific antibody here as cited in:

    Haque et al., (2017). Acta Neuropathol Commun. 5(1):45. PMID: 28587626

    To read our full case study on the anti-histone H3.3 G34R/V antibodies click here


    Fang et al., (2018). Cancer-driving H3G34V/R/D mutations block H3K36 methylation and H3K36me3–MutSα interaction. Proc Natl Acad Sci U S A. 115(38): 9598. PMID: 30181289

  • Succinocysteine: a PTM and Biomarker of Complex Cellular Dysfunction

    Image above shows the succination reaction which creates the succinocysteine modification.

    Protein succination is a post-translational modification formed by a reaction between the tricarboxylic acid cycle intermediate fumarate with protein cysteines to form S-(2-succino)cysteine (2SC). Cysteine predominantly exists in the thiolate form and can acts as a reactive nucleophile. Cysteine succination can occur non-enzymatically and is formed by a Michael addition reaction between fumarate and the free thiol groups of protein cysteines at physiological pH.  The thioether bond of 2SC is considered to be stable to acid hydrolysis and irreversible. Fumarate is a weak electrophile and its modification of thiols is highly pH dependent.  As a consequence, succination can be selective towards functional, low pKa cysteine residues in proteins, such as catalytic cysteine residues in enzymes.

    Succination at critical cysteine residues can result in the inactivation of enzymatic activity or protein function in many biological processes. For example, the succination of key components of the iron-sulfur cluster biogenesis family of proteins, Iscu and Nfu1, lead to defects in iron-sulfur biosynthesis required for respiratory chain complexes. Succination of glutathione has been shown to increase oxidative stress and cellular senescence. The loss of fumarate hydratase (FH), the enzyme that catalyzes the reversible hydration/dehydration of fumarate to L-malate, contributes to the accumulation of fumarate and succination. FH deficiency leads to the inactivation of the E3 ubiquitin ligase Keap1 by succination, which promotes the stabilization of NRF2 and activation of the antioxidant pathway. Keap1 also plays a key role in controlling tumorigenesis.

    2SC is considered as a biomarker for mitochondrial stress in obesity, insulin resistance and diabetes. The succination of adiponectin is increased in adipocytes and adipose tissue of type 2 diabetic mice. Adiponectin succination blocks the formation of oligomeric species and secreted forms of adiponectin, which contributes to reduced levels of plasma adiponectin in diabetes. Succination causes irreversible inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) resulting in the loss of activity in muscle of diabetic rats. The elucidation of the succinated proteome will provide an insight into the role of succination in regulatory biology and determine its effect on cellular dysfunction.

    Anti-2SC Antibody (crb2005012)

  • Phospho-Specific Antibodies: Focus on CDK1

    Cyclin D1 is a key regulator of cell proliferation as it links the extracellular signaling environment to cell cycle progression.  Cyclin D1 accumulation is the rate-limiting step for cell cycle entry and the transition from G1 to S phase. The levels of cyclin D1 are elevated in G1 phase, where it interacts with the serine-threonine protein kinases, cyclin-dependent kinases (CDK) CDK4 and/or CDK6 to activate its catalytic activity. Active Cyclin D1/CDK complexes then phosphorylate the retinoblastoma protein (Rb). Rb inhibits cell cycle progression through its ability to repress E2F transcription factors activity, which is involved in the regulation of genes required for DNA replication and G2/M progression. Phosphorylation of RB (pRb) promotes the release of E2F and subsequently promotes cell cycle progression. Furthermore, cyclin D1 plays a role in maintaining the integrity of the G1/S checkpoint. Cyclin D1 associates with proliferating cell nuclear antigen (PCNA), a component of the DNA replication and repair machinery. During S phase, cyclin D1 down-regulation is necessary for PCNA translocation, DNA repair and initiation of DNA replication.

    Cyclin D1 has been shown to associate with a number of transcription factors, HATs and HDACs in a CDK independent manner to modulate transcription and epigenetic changes. Cyclin D1 contains an LxxLL motif (251-255) that facilitates coactivator recruitment to mediate transcriptional activation. In addition, cyclin D1 contains a repressor domain (142-253) within its central region, which facilitates the interactions with corepressors to negatively regulate transcription. The levels of cyclin D1 are determined by the rate of expression, protein stability, localization, associations and degradation. The phosphorylation of cyclin D1 at Thr286 has been shown to target it for nuclear export and ubiquitin-proteasome degradation. The diverse roles of cyclin D1 are dependent on its protein level. The various biological processes that cyclin D1 has been implicated in include cell migration, mitochondrial metabolism, cell cycle arrest and apoptosis.

    Given the pivotal role of cyclin D1 in promoting cell proliferation, aberrant cyclin D1 expression and activity frequently occurs in human cancers. The overexpression of cyclin D1 is predominantly associated with tumorigenesis and metastases. Understanding the multifaceted role of cyclin D1 and its dysregulation may provide a better understanding of its involvement in the development and progression of cancer.

    Anti-Cyclin D1 antibody

    Cyclin D1 peptide


3 Item(s)