• 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)

  • CTLA-4 and its Role in Immune Homeostasis

    Cytotoxic T lymphocyte associated gene-4 (CTLA-4), also known as CD152, is a type I glycoprotein that belongs to the immunoglobulin superfamily. CTLA-4 is required for immune homeostasis as it functions as a checkpoint for T-cell activation and a critical inhibitor of autoimmunity. CTLA-4 is constitutively expressed in Foxp3+ regulatory T cells, where it mediates cell extrinsic control of effector responses to regulate immune suppression. In contrast, CTLA-4 expression is only induced in T cells following activation, where CLTA-4 primarily acts as a co-inhibitory molecule to transmit a negative feedback signal. CLTA-4 counteracts with the B7:CD28 pathway by binding to the B7 ligand on antigen-presenting cells with a higher affinity to antagonise CD28 positive co-stimulatory signalling. CTLA4 any also function by downregulating ligand expression and transmitting inhibitory signals.

    The membrane bound CTLA-4, mCTLA-4, contains an extracellular V domain, a transmembrane domain and a cytoplasmic tail. An alternatively spliced isoform has also been identified to be a secreted soluble form, sCTLA-4, which lacks the transmembrane domain and possess a different cytoplasmic tail. sCTLA-4 can still bind B7 and modulate the strength and durability of the B7:CD28-mediated costimulation. However, sCTLA-4 may also interfere with B7:mCTLA-4 interactions, causing a reduction in the negative signal. sCTLA-4 also exhibits a dual effect on cytokine production to modulate T cell proliferation.  sCTLA4 can inhibit the secretion of activator cytokines IFN-γ , IL-2, IL-7, and IL-13 and activate the secretion of TGF-β and IL-10.

    The levels of sCTLA-4 play a role in determining the fate of immune responses. Low levels of sCTLA-4 have been detected in normal human serum. Increased levels of sCTLA-4 have been observed in several autoimmune diseases such as Graves' disease, type 1 diabetes, celiac disease, Crohn’s disease, systemic lupus erythematosus, and systemic sclerosis. Understanding the role of sCTLA-4 in modulating the immune response may provide an insight to its relevance in autoimmune disease pathogenesis.

    Monoclonal Antibodies:

    Anti-sCTLA-4 antibody CRB4B8 (crb5005104)

    Anti-sCTLA-4 antibody CRB10D1  (crb5005105)

    Polyclonal Antibodies:

    Anti-sCTLA-4 antibody 4017 (crb2005177)

    Anti-sCTLA-4 antibody 4018 (crb2005178)

  • 10% Discount On Your First DISCOVERY® Purchase

    We are offering a 10% discount off your first purchase from DISCOVERY® Peptides & Antibodiesusing the code : ENDEAVOUR10

    The code is valid across all products on the DISCOVERY® Antibodies site and also valid on our sister site DISCOVERY® Peptides.

  • Antibody of the Month: ERK1/2 Antibody

    Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) super family that regulate cell proliferation and apoptosis. The 44 kDa ERK1 and 42 kDa ERK2 share 83% amino acid identity with higher homology in the core regions and are expressed in almost all tissues. ERK1/2 are protein-Ser/Thr kinases that participate in the Ras-Raf-MEK-ERK signal transduction cascade. Extracellular stimuli such as growth factors, mitogens, cytokines, hormones, and oxidative or heat stress can trigger signal transduction pathways that lead to ERK1/2 activation. Human MEK1/2 phosphorylates the effector MAPKs ERK1/2 on Tyr204/187 and Thr202/185 in the TEY sequence to activate its enzymatic function.

    Activated ERK1/2 preferentially phosphorylate substrates containing the Ser/Thr-Pro motif, with the optimal consensus sequence identified as Pro-Xxx-Ser/Thr-Pro. The ERK1/2 proline-directed kinases phosphorylate a multitude of cytoplasmic and nuclear protein substrates including signaling effectors, protein kinases, receptors and cytoskeletal proteins. ERK1/2 can also translocate into the nucleus and phosphorylate many transcription factors.  ERK1/2 phosphorylates an array of proteins involved in various processes including cell adhesion, cell cycle progression, proliferation, migration, differentiation, cell survival and metabolism. The co-ordinated variations of ERK1/2 signalling from its duration, magnitude, subcellular localization and protein interactions can determine the final outcome of cellular fate.

     

    Anti-ERK 1/2 Antibody (crb2005025f)

    Alexa Fluor® 488 Anti-ERK1/2 antibody (crb1200306e)

    ERK 1 peptide (crb1200306e)

  • Phospho-Specific Antibodies: Focus on CDK2

    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

     

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