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  • RAMP's: G-Protein coupled receptor accessory proteins with vital biological roles

    Receptor activity-modifying proteins (RAMPs) are a family of single-transmembrane receptor accessory proteins which include RAMP-1, RAMP-2 and RAMP-3 that are involved in several cellular pathways of high importance. Here at Discovery Antibodies® we offer high-quality specific antibodies to each of the three RAMP proteins which are able to detect their target proteins in Western blot and immuno-histochemical applications. RAMPs act by binding to G-protein coupled receptors (GPCRs), which are vital in intercellular signalling, and alter their pharmacology such as changing ligand specificity and mediating receptor trafficking. RAMPs play essential roles in several body systems and mutations in RAMP-GPCR pairings have been identified and linked to human disease. Therefore this interesting protein family is of high value for studying and understanding many human disease pathways.

    RAMPs interact with class B GPCRs and several GPCRs from other families, including: calcitonin receptor (CTR) and calcitonin-like receptor (CLR), the vasoactive intestinal polypeptide/pituitary AC-activating peptide 1 (VPAC1) receptor, glucagon receptor (GCGR), corticotropin-releasing factor receptors (CRF) parathyroid hormone receptors, secretin receptor, and pituitary adenylate cyclase activating peptide (PACAP) receptors.

    Perhaps the most well studied role for RAMPs is their binding to and regulation of CTR and CLR. RAMPs are able to modulate several aspects of receptor function including receptor trafficking to the cell surface, recycling, ligand binding preference, signal transduction, post-translational modifications, and G-protein coupling. The peptide ligands that bind to CTR and CLR: include calcitonin (CT), amylin, calcitonin gene-related peptides α and β (αCGRP and βCGRP), adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD), many of which are available in our Discovery Peptides catalogue.

    References:

    Hay and Pioszak (2015). RAMPs (Receptor-Activity Modifying Proteins): New Insights and Roles. Annu. Rev. Pharmacol. Toxicol. 56: 469 PMID: 26514202

    McLatchie et al., (1998). RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature. 393(6683): 333. PMID: 9620797

    Suekane et al., (2019). CGRP-CRLR/RAMP1 signal is important for stress-induced hematopoiesis. Sci Rep. 9(1). PMID: 30674976

  • 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

    References:

    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

  • The diverse role of cell cycle regulator, p21

    The multi-functional cyclin-dependent kinase (CDK) inhibitor, p21 plays a diverse role in regulating the DNA damage response, senescence, DNA repair, transcription and apoptosis. p21, also known as p21WAF1/Cip1 or CDKN1A, belongs to the CIP/Kip family of CDK inhibitors.

    In normal cells, p21 functions as a cell cycle inhibitor and anti-proliferative effector. Upon DNA damage and genotoxic stress, the tumour suppressor p53 is activated and induces the expression of p21. p21 can bind to cyclin A/CDK2, E/CDK2, D1/CDK4 and D2/CDK4 complexes and suppress their catalytic activity. This prevents the phosphorylation of Retinoblastoma protein (Rb) and leads to the arrest in G1/S cell cycle progression and G2/M transitions. In addition, p21 also inactivates Rb by mediating its degradation. Studies also show that p21 can mediate cellular senescence via p53-dependent and p53-independent pathways.

    p21 can also acts as either an enhancer or a suppressor of various DNA repair pathways. In DNA damaged cells, p21 is recruited to damaged sites and co-localises with double strand break (DSB) repair proteins to facilitate various repair pathways. p21 has been implicated in nucleotide excision repair (NER), base excision repair (BER) and DNA translesion synthesis (TLS) by disrupting the proliferating cell nuclear antigen (PCNA) interaction with other DNA repair factors and promoting PCNA degradation. PCNA-dependent DNA replication is also inhibited by p21. Another important function of p21 is regulating the transcription of genes involved in various biological process such as cell cycle progression, DNA repair and regulation of apoptosis, such as E2F family, NF-κB, c-myc, STAT and p300/CPB.

    In response to DNA damage and stress signals, p21 has been shown to exert anti-apoptotic activity. The cytoplasmic interaction of p21 with pro-caspase 3 and caspase 2 prevents their activation of apoptosis. Fas-induced apoptosis is also inhibited via p21 interaction with caspase 3 and inhibition of its activity. p21 also forms a complex with stress induced kinases, such as apoptosis signal regulating kinase 1 (ASK1) to inhibit apoptosis. In addition, the phosphorylation of p21 by Akt1/PKB enhances its stability and promotes cell survival.

    Anti-p21 Antibody (crb2005030)

    Read more: Phospho-specific Antibodies: Focus on CDK1

  • 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 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

     

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