One of the most common mutations in the serine protease inhibitor Kazal type 1 (SPINK1) gene is the N34S variant which is strongly associated with chronic pancreatitis. Although it is assumed that N34S mutation constitutes a high-risk factor, the underlying pathologic mechanism is still unknown. In the present study, we investigated the impact of physiological stress factors on SPINK1 protein structure and trypsin inhibitor function using biophysical methods. Our circular dichroism spectroscopy data revealed differences in the secondary structure of SPINK1 and N34S mutant suggesting protein structural changes induced by the mutation as an impairment that could be disease-relevant. We further confirmed that both SPINK1 (KD of 0.15 ± 0.06 nM) and its N34S variant (KD of 0.08 ± 0.02 nM) have similar binding affinity and inhibitory effect towards trypsin as shown by surface plasmon resonance and trypsin inhibition assay studies, respectively. We found that stress conditions such as altered ion concentrations (i.e. potassium, calcium), temperature shifts, as well as environmental pH lead to insignificant differences in trypsin inhibition between SPINK1 and N34S mutant. However, we have shown that the environmental pH induces structural changes in both SPINK1 constructs in a different manner. Our findings suggest protein structural changes in the N34S variant as an impairment of SPINK1 and environmental pH shift as a trigger that could play a role in disease progression of pancreatitis.

中文翻译：

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生理应激条件对丝氨酸蛋白酶抑制剂Kazal 1型（SPINK1）及其N34S变体的蛋白质结构和胰蛋白酶抑制的影响。

丝氨酸蛋白酶抑制剂Kazal 1型（SPINK1）基因中最常见的突变之一是N34S变异体，它与慢性胰腺炎密切相关。尽管假定N34S突变构成高危因素，但其潜在的病理机制仍是未知的。在本研究中，我们使用生物物理方法研究了生理应激因素对SPINK1蛋白结构和胰蛋白酶抑制剂功能的影响。我们的圆二色光谱数据显示SPINK1和N34S突变体的二级结构存在差异，表明该突变诱导的蛋白质结构变化为可能与疾病相关的损伤。我们进一步证实了SPINK1（KD为0.15±0.06 nM）和其N34S变异体（KD为0.08±0。如表面等离振子共振和胰蛋白酶抑制试验研究分别所示，O 2（n 2 O 2）对胰蛋白酶具有相似的结合亲和力和抑制作用。我们发现应激条件，例如改变的离子浓度（即钾，钙），温度变化以及环境pH值，导致SPINK1和N34S突变体在胰蛋白酶抑制方面的差异不明显。但是，我们已经表明，环境pH值会以不同的方式诱导两个SPINK1构造的结构变化。我们的发现表明，N34S变异体中的蛋白质结构变化是对SPINK1的损害，而环境pH的变化则可能在胰腺炎的疾病进展中起作用。我们发现应激条件，例如改变的离子浓度（即钾，钙），温度变化以及环境pH值，导致SPINK1和N34S突变体在胰蛋白酶抑制方面的差异不明显。但是，我们已经表明，环境pH值会以不同的方式诱导两个SPINK1构造的结构变化。我们的发现表明，N34S变异体中的蛋白质结构变化是对SPINK1的损害，而环境pH的变化则可能在胰腺炎的疾病进展中起作用。我们发现应激条件，例如改变的离子浓度（即钾，钙），温度变化以及环境pH值，导致SPINK1和N34S突变体在胰蛋白酶抑制方面的差异不明显。但是，我们已经表明，环境pH值会以不同的方式诱导两个SPINK1构造的结构变化。我们的发现表明，N34S变异体中的蛋白质结构变化是对SPINK1的损害，而环境pH的变化则可能在胰腺炎的疾病进展中起作用。