Antiproliferative factor (APF) binds specifically to sites within the cytoskeleton-associated protein 4 (CKAP4) extracellular domain.
Antiproliferative factor (APF) binds specifically to sites within the cytoskeleton-associated protein 4 (CKAP4) extracellular domain.
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DOI：
10.1186/s12858-017-0088-y
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发表时间：
2017-09-11
期刊：
影响因子：
--
通讯作者：
Planey SL
中科院分区：
文献类型：
--
作者：
Chavda B;Ling J;Majernick T;Planey SL
抗增殖因子（APF）是间质性膀胱炎（一种慢性、疼痛的膀胱疾病）患者尿液中升高的唾液糖肽。APF在体外抑制正常膀胱上皮细胞和癌细胞的增殖，可能是通过与其细胞受体细胞骨架相关蛋白4 （CKAP4）结合；然而，APF与CKAP4的生物物理相互作用尚未被表征。在这项研究中，我们使用表面等离子体共振（SPR）来探索APF和as-APF（一种具有全活性的去乙酰化APF类似物）与CKAP4相互作用的结合动力学。我们将非糖基化APF （TVPAAVVVA）固定在Fc1通道上作为对照，将as-APF固定在Fc2通道上作为配体，以测量CKAP4重组蛋白仅包含胞外结构域（Aa 127-602）或胞外结构域加跨膜结构域（Aa 106-602）的结合情况。检测到与CKAP4126-602和CKAP4106-602的阳性结合，表明as-APF可以特异性结合CKAP4，并且潜在的结合位点位于细胞外结构域。为了确定CKAP4胞外结构域内APF的主要结合位点，根据结构预测设计了缺失突变体，并将纯化的重组蛋白通过胺偶联固定在CM5芯片上，以测量as-APF的结合活性。重要的是，CKAP4127-360和CKAP4361-524都表现出快速的结合速率（k on）和缓慢的解离速率（k off），从而产生高的结合亲和力，这表明这两个区域对整体as-APF结合的贡献相对相等。因此，在CKAP4胞外结构域的Aa 127-524区域内可能存在两个或多个as-APF结合位点。我们确定，与全长胞外结构域相比，CKAP4127-360和CKAP4361-524突变体对as- apf的结合活性有所提高，这使得检测尿液中低浓度的as- apf成为可能，从而为IC的非侵入性诊断检测奠定了基础。这些数据揭示了新的APF结合位点，表明靶向CKAP4的这一区域抑制APF结合可能是治疗ic相关膀胱病理的有效策略。
Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis—a chronic, painful bladder disease. APF inhibits the proliferation of normal bladder epithelial cells and cancer cells in vitro, presumably by binding to its cellular receptor, cytoskeleton associated-protein 4 (CKAP4); however, the biophysical interaction of APF with CKAP4 has not been characterized previously. In this study, we used surface plasmon resonance (SPR) to explore the binding kinetics of the interaction of APF and as-APF (a desialylated APF analogue with full activity) to CKAP4. We immobilized non-glycosylated APF (TVPAAVVVA) to the Fc1 channel as the control and as-APF to Fc2 channel as the ligand in order to measure the binding of CKAP4 recombinant proteins encompassing only the extracellular domain (Aa 127–602) or the extracellular domain plus the transmembrane domain (Aa 106–602). Positive binding was detected to both CKAP4126–602 and CKAP4106–602, suggesting that as-APF can bind specifically to CKAP4 and that the potential binding site(s) are located within the extracellular domain. To identify the primary APF binding site(s) within the CKAP4 extracellular domain, deletion mutants were designed according to structural predictions, and the purified recombinant proteins were immobilized on a CM5 chip through amine-coupling to measure as-APF binding activity. Importantly, both CKAP4127–360 and CKAP4361–524 exhibited a fast association rate (k on) and a slow dissociation rate (k off), thus generating high binding affinity and suggesting that both regions contribute relatively equally to overall as-APF binding. Therefore, two or more as-APF binding sites may exist within the Aa 127–524 region of the CKAP4 extracellular domain. We determined that the CKAP4127–360 and CKAP4361–524 mutants exhibit improved binding activity to as-APF as compared to the full-length extracellular domain, making it possible to detect low concentrations of as-APF in urine, thereby establishing a foundation for a non-invasive diagnostic assay for IC. Further, these data have revealed novel APF binding site(s) suggesting that targeting this region of CKAP4 to inhibit APF binding may be a useful strategy for treating IC-related bladder pathology.
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抗增生因子降低了T24膀胱癌细胞中CKAP4的AKT磷酸化并改变基因表达。
DOI：
10.1186/1756-9966-29-160
发表时间：
2010-12-10
期刊：
Journal of experimental & clinical cancer research : CR
影响因子：
--
作者：
Shahjee HM;Koch KR;Guo L;Zhang CO;Keay SK
通讯作者：
Keay SK
影响因子：
11.4
作者：
Klopfenstein, DRC;Kappeler, F;Hauri, HP
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Hauri, HP
影响因子：
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作者：
Mallajosyula, Sairam S.;Adams, Kristie M.;MacKerell, Alexander D.
通讯作者：
MacKerell, Alexander D.
影响因子：
3.3
作者：
Vedrenne, C;Klopfenstein, DR;Hauri, HP
通讯作者：
Hauri, HP
影响因子：
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作者：
Keay, S;Zhang, CO;Chai, TC
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