What Negative Charges on NHD Can Impart to LC3
Date Issued
2012
Date
2012
Author(s)
Wang, Hsu-Chi
Abstract
Autophagy is ubiquitous in all eukaryotic cells. It is crucial for cells to clear less necessary organic particles, damaged organelles, or intracellular pathogens for emergent energy needs, nutrient recycling, immune response, etc. In autophagy, cellular components to be degraded are first surrounded by a double membrane structure called phagophore; this membrane gradually expands and is sealed into a close structure called autophagosome, which subsequently fuses with lysosome, becoming autolysosome; the content within the autolysosome is then digested with hydrolytic enzymes, and freed from the structure into cytosol. Autophagy has been linked to numerous human pathological processes, such as neurodegeneration, cancer, and aging.
LC3, a ubiquitin-like autophagic protein, is decorated on the phagophore during membrane expansion. In an E1-E3-like enzyme system, LC3 is conjugated to the phosphatidylethanolamine (PE) on the phagophore. LC3 is suggested to promote membrane tethering and hemifusion between phagophores through its multimerization. Previous studies showed that LC3 consists of two domains: the N-terminal helical domain (NHD) and the C-terminal ubiquitin-like domain (ULD). During lipidation, the NHD may dissociate from the ULD, adopting an “open” form, and exposing a region on the ULD surface to induce LC3 multimerization.
In 2010, two studies showed that, both in vitro and in vivo, protein kinase A (PKA) and protein kinase C (PKC) could phosphorylate LC3’s Ser12 and Thr6, respectively. These imply a novel mode of LC3 regulation, possibly through changing its NHD dynamics.
In this study, the phosphomimetic human LC3s, T6D and T12D, were used for comparison with the LC3 wild type and the ULD, to investigate whether the additional negative charges on the NHD influence their dynamics, or stabilities, and hence the activities of LC3. The technique circular dichroism (CD) was utilized for examining the collective behavior of their secondary structures.
The results showed that compared with the ULD, the wild type, T6D, and T12D all had much stronger and similar signals around the wavelength 222 nm, which largely arises from the presence of α-helices, and decreases drastically in their loss or denaturation. Further examining the urea and the guanidine hydrochloride denaturation curves by CD222 of these LC3 variants found that there are significant differences between the wild type and the phosphomimetic mutants, showing the later more readily denatured. Compared with the denaturation curve of the ULD, the observed early unfolding of the LC3 could be, at least partially, accounted by the NHD, which might begin massive denaturation before the ULD does.
Subjects
autophagy
circular dichroism
denaturation curve
phosphomimetic
SDGs
Type
thesis
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