Although the stress-mediated activation mechanism is not clear, it has been hypothesized that stress factors induce receptor clustering and internalization, which lead to JNK activation. Mammalian JNK proteins are activated by various extracellular stimuli, including growth factors, cytokines, and cellular stresses such as heat shock, hyperosmolarity, UV radiation, and ischemia/reperfusion. JNKs, like ERK proteins, are activated by phosphorylation on threonine and tyrosine residues, which are separated by a proline within the tripeptide activation loop in the kinase domain. The small N-terminal lobe helps in the orientation and binding of ATP, whereas the large C-terminal lobe aids in substrate recognition. JNKs have a similar core structure as that of ERK proteins but there are differences in the conformation of their activation loop, resulting in differences in the mechanism of regulation. Chad Hancock, in Encyclopedia of Endocrine Diseases, 2004 JNK Structure and Activation In experimental models, specific inhibitors of JNK prevented Bak induction, Bid degradation, caspase-3 activation, and mitochondrial cytochrome c release, eventually attenuating hepatocyte necrosis and apoptosis after ischemia-reperfusion or liver transplantation. Furthermore, ischemia-reperfusion liver injury has been shown to cause JNK1 activation. Recruitment of activated JNK to the outer membrane of mitochondria is an important step in induction of JNK-mediated hepatocyte death, and mitochondrial Bcl-xL and Mcl-1 are substrates for JNK. 22 Sustained JNK activation leads to cell death and occurs via modulation of Bcl-2 family proteins, with subsequent mitochondrial permeabilization. Both of these can be activated by ER stress pathways of apoptosis and may also be the pathway of caspase-independent reactive oxygen species–mediated cell death. Two of the three known JNK proteins are expressed in the liver. Although the signal transduction pathways can produce a variety of physiological outcomes, membrane/organelle-initiated cytotoxic signaling pathways often converge on JNK. JNKs are known to regulate signaling molecules, such as Mcl-1 and Bid by phosphorylation. JNK signaling is associated with cell death, survival, differentiation, proliferation, and tumorigenesis in hepatocytes. It is possible to remove N-terminal pyroglutamic acid enzymatically 23 to obtain the remaining N-terminal sequence however, the yield of the enzymatic removal is variable.Ĭonstance Mobley, Ali Zarrinpar, in Transplantation of the Liver (Third Edition), 2015 JNK Chemically modified N-termini, such as cyclization of Glu (pyroglutamic acid) or carbamylation, often cannot be successfully sequenced. N-terminal sequencing has some limitations. In the case of HCPs, identity can sometimes be assigned based on BLAST searches using the N-terminal sequence. It is especially useful in the identification of unknown bands in SDS-PAGE (after blotting), allowing the important distinction of product-related (such as fragments) versus nonproduct impurities (such as host–cell proteins (HCP)). It provides complementary information to accurate mass analysis of intact proteins and subunits. The amino acid in each hydrolysis cycle is identified and quantitated based on comparison to amino acid standards.įor analysis of biopharmaceuticals, N-terminal sequencing is typically used to confirm the identity of the protein, as well as to assess N-terminal heterogeneity. The peptide bonds are sequentially hydrolyzed from the N-terminus, and the released amino acid is derivatized and analyzed by HPLC. N-terminal sequencing by Edman degradation 21,22 is frequently used to determine the N-terminal amino acid sequence of a protein. John Steckert, in Separation Science and Technology, 2011 b N-Terminal Sequencing
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