Verma Amit Kumar, Sharma Priya, Islam Zeyaul, Biswal Anup Kumar, Tak Yogesh, Sahi Chandan
Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, Madhya Pradesh 462066, India.
Department of Internal Medicine, UT Southwestern Medical Center, Dallas, Texas 75390-9096, United States.
ACS Omega. 2024 Aug 28;9(36):37650-37661. doi: 10.1021/acsomega.4c01776. eCollection 2024 Sep 10.
J-domain proteins (JDPs) are obligate cochaperones of Hsp70s with a wide range of functions in protein homeostasis. Although the J-domain is required for the stimulation of Hsp70s ATPase activity, the functional specificity of JDPs is governed by domains or regions other than the J-domain. Jjj3/Dph4, a class III JDP, is required for diphthamide (DPH) biosynthesis in eukaryotes, including yeast and mammals. Dph4 has a conserved N-terminal J-domain and an uncharacterized C-terminal domain containing a signature CSL zinc finger motif. Previously, we showed that the Dph4 ortholog in (atDjC13/Jjj3/Dph4) could restore DPH biosynthesis in yeast Δ mutant in a J-domain-dependent manner. Here, we characterize the C-terminal CSL motif of Dph4 using yeast genetic and biochemical approaches. The CSL motif of Dph4 is essential for DPH biosynthesis, and like human Dph4, Dph4 showed distinct iron-binding activity, which is not present in its yeast counterpart. Dph4 and Dph4 proteins exhibit distinct iron-binding capabilities, as evidenced by UV-vis spectrophotometry, SEM-EDS (energy-dispersive spectroscopy function on the scanning electron microscope) and electron paramagnetic resonance (EPR) spectra analyses. Collectively, our data suggests that beyond their role as an Hsp70 cochaperone, Dph4 homologues in complex eukaryotes may have iron-binding abilities, indicating a potential role in iron-sulfur cluster assembly and iron homeostasis.
J结构域蛋白（JDPs）是热休克蛋白70（Hsp70s）的必需共伴侣蛋白，在蛋白质稳态中具有广泛功能。尽管J结构域是刺激Hsp70s ATP酶活性所必需的，但JDPs的功能特异性由J结构域以外的结构域或区域决定。Jjj3/Dph4是III类JDP，在包括酵母和哺乳动物在内的真核生物中，它是白喉酰胺（DPH）生物合成所必需的。Dph4具有保守的N端J结构域和一个含有特征性CSL锌指基序的未表征的C端结构域。此前，我们发现（atDjC13/Jjj3/Dph4）中的Dph4直系同源物能够以J结构域依赖的方式恢复酵母Δ突变体中的DPH生物合成。在此，我们使用酵母遗传学和生物化学方法对Dph4的C端CSL基序进行了表征。Dph4的CSL基序对DPH生物合成至关重要，并且与人类Dph4一样，Dph4表现出独特的铁结合活性，而其酵母对应物中不存在这种活性。紫外可见分光光度法、扫描电子显微镜能量色散光谱（SEM-EDS）和电子顺磁共振（EPR）光谱分析表明，Dph4和Dph4蛋白表现出不同的铁结合能力。总的来说，我们的数据表明，除了作为Hsp70共伴侣蛋白的作用外，复杂真核生物中的Dph4同源物可能具有铁结合能力，这表明它们在铁硫簇组装和铁稳态中可能发挥作用。
