IRE1 Biochemistry

To transition from an alcohol to an alkene, the alcohol must be dehydrated with the help of an acid through a reaction known as an E1 mechanism.1 The first step of an E1 reaction is the formation of a carbocation intermediate. This carbocation is produced by the removal of a halogen or a substituted group.2 In this experiment, the hydroxy (OH ) group of the alcohol is removed and this produces the carbocation. The OH- group is removed due to the presence of phosphoric acid. The phosphoric acid is used in the process of adding an additional H+ to the OH- group on the alcohol and assists the OH- in leaving, making the reaction an acid catalyzed dehydration. Another reagent used along with phosphoric acid is heat, which is often used in acid catalyzed dehydration.1

To understand the AMPK dependence of ULK1, different cultures human osteosarcoma U2OS cells were introduced to only one of the following: the AMPK nonphosphorylatable (4SA)ULK1 cDNA , wild type AMPK and ULK1, or catalytically inactive (KI) ULK1 and AMPK. The endogenous ULK1 and 2 were also reduced with lentiviral short hairpin RNAs. ULK1 MEFs that were knocked down for endogenous ULK2 was also reconstituted with wild type, KI (catalytically inactive), or 4SA ULK1 cDNAs. They were also examined for the extent of autophagy after the cells were starved.

In humans/animals, the main cells that store fat for energy are adipocytes. These fat cells are found under the skin, in the abdominal cavity and surround major organs. The fatty tissue is the body’s main means of storing energy for long periods of time. Lipids, like triglycerides are stored in the adipocytes until ready to be used by the body for energy. Fat is broken down through metabolism in the mitochondria of the cell. The triglycerides are broken down into glycerol and 3 fatty acids. The glycerol can be easily converted to glyceraldehyde 3 phosphate, an intermediate of glycolysis. From there it can go through the Krebs Cycle and electron transport chain to make ATP. The 3 fatty acids can be broken

Chaperones are proteins that ensure the correct folding of the CFTR within the endoplasmic reticulum. Hsp70 is an important cytosolic chaperone that complexes with CFTR and reduces aggregation [5]. The CFTR passes through the endoplasmic reticulum-associated degradation (ERAD) after folding in the ER. This quality control system involves the ubiquitin proteasome system (UPS) for which CFTR is a substrate [16]. If a protein is molded and targeted for degradation, then ubiquitin will covalently attach to lysine residues on the CFTR. Three enzymes are required for the process of ubiquitylation: E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin protein ligases. E1 enzymes are activated through hydrolysis of ATP, which creates an activated ubiquitin that is transferred to an E2 active site. The activated ubiquitin is then covalently bound to a lysine on the protein by an E3 ligase. A polyubiquitin chain is then formed as ubiquitin molecules link together, and if there are four or more then the misfolded CFTR chain is removed form the ER membrane and targeted for degradation by the 26S proteasome

The body of an adult human contains almost 2-3 g of zinc. However, only 0,1% of it is found in the serum. As for other transition metals, circulating zinc is not free, but is complexed to proteins that maintain it in a non-reactive form in order to prevent their toxicity: almost 80% is bound to albumin and the remaining 20% is bound to α2-macroglobulin (Kambe et al., 2015). As zinc is required for many functions in living organisms, its transport across biological membranes is mediated by several proteins that can be classified in two major families of transporters: the SLC39 (Zrt-Irt-related Proteins, ZIPs) family, with 13 members identified, and the SLC30 (Zn-transporters, ZnTs) family, with 10 members (Fukada et al., 2011; Kambe et al.,

Cell growth, migration, proliferation and mRNA levels of YAP regulated genes were tested using LPA treated cells that had YAP/TAZ knockdown. Cell growth, migration, proliferation and mRNA levels of YAP regulated genes were all significantly repressed compared to the controls suggesting that LPA acts through YAP/TAZ. Similar to YAP, TAZ was shown to localize in the nuclear fractions of cells in immunofluorescence experiments. MST 1/2 and Lats 1/2 levels were tested in response to LPA treatment. LPA was shown to inhibit Lats 1/2 but not MST 1/2 in immunoblot experiments. Then to test how LPA and S1P were modulating YAP/TAZ immunoblotting experiments were utilized. LPA and S1P were found to modulate YAP/TAZ through G-protein-coupled receptor signaling and Rho GTPases. More specifically, G12/13 receptors appeared to be most involved while Gq/11 receptors were only slightly involved in dephosphorylating YAP/TAZ. On the other hand, Gs receptors had the opposite effect, phosphorylating and inhibiting

The results presented here are logical extension of previous studies focusing on Nrf2 as a control point for the maintenance of GSH homeostasis in response to E-induced redox shifts in cultured neurons and in the developing brain. The Nrf2-related studies illustrated that the transcription factor does play a vital role in neuroprotection against E damage [7]. Yet, the neuroprotective setting remained incomplete in that GSH homeostasis was not preserved in a subpopulation of neurons in the E-exposed fetal brain [7], even in the presence of E-dependent up-regulation of Nrf2 expression. To mitigate this gap in our understanding, these new studies address a role of Cys homeostasis and their control points which could be

Insulin also binds to IGF-1 receptors with low affinity (Smith, 1988) and stimulates the formation of an active Ras-GTP complex and ERK-MAPK pathway (Porras, 1996) and the PI3K pathway. Both these pathways appear to be required for adipogenesis (Sale, 1995; Sakaue, 1998; Tang, 2005). The Ras-GTP complex activates RAF kinase which is phosphorylated to induce MAPK. The phosphorylated MAPK interacts with Elk1. Phohosphorylation of Ekl1 facilitates binding to the mediator complex via the Med23 subunit (Stevens, 2002; Wang, 2005). The mediator complex is a multi-protein complex which bridges gene specific transcription factors and transcriptional machinery. The Mediator Med23 (Sur2) subunit was originally identified as a genetic suppressor of a hyperactive Ras phenotype in C.elegans (Singh, 1995). Elk1 is a member of the ternary complex factor family and an important regulator of adipogenesis. The Elk1/Med23 interaction controls the expression of Krox20 in a MAPK dependent manner by promoting a productive preinitiation complex which leads to transcription (Wang, 2009). Krox20 is expressed in adipose tissue (Soukas, 2001) and is the first transcriptional factor expressed during adipogenesis after insulin stimulation (Chen, 2005; Gonzalez, 2005). Krox20 expression has been reported to regulate

A study released in the beginning of this month has discovered a certain gene that turns hair gray. The IRF4 gene has previously been the cause for having light hair as well as gray hair. Now that this trait has been linked to a gene, researchers are looking into the future of genetically modified hair.

Obesity is known to produce a number of stress and inflammation responses in the body that lead to the activation of the inflammatory signalling molecules, Jun NH2-terminal kinase (JNK) and inhibitory kB kinase (IKK). Activation of these pathways plays a key part in the development of insulin resistance followed by progression to diabetes as they greatly affect inflammatory responses, insulin signalling, and lipid and glucose homeostatis (Nakamura et al. 2014). For example, activating the JNK pathway leads to serine phosphorylation of insulin receptor substrate (IRS) proteins which results in inhibited insulin signalling and thus insulin resistance. Double-stranded RNA-dependent protein kinase (PKR) has recently been shown to control the JNK and other major inflammatory pathways, to directly inhibit insulin signalling, to be activated by fatty acids as well as endoplasmic reticulum (ER) stress, and to be necessary for the activity of inflammasomes (Nakamura et al. 2010; Komiya et al. 2010; Lu et al. 2012). Moreover, PKR has been found to be highly activated in obese people as well as mice with genetically and diet-induced obesity, especially in adipose and hepatic tissues (Boden et al. 2008; Nakamura et al. 2010; Carvalho-Filho et al. 2012). Finally, recent studies have found that PKR knockout obese mice were protected against both IR and obesity-triggered inflammation and that administration of PKR inhibitors reduced JNK activation, reduced inflammation in adipose

Endoplasmic reticulum (ER) stress response may be mediated by HRV16 nonstructural proteins 2B, which share similar characteristics to CVB3 2B. To understand whether HRV16 2B induces an ER stress response, a HRV16 2B fusion protein with GFP were generated and its effects on ATF6a (activating transcription factor 6a), IRE1 (Inositol-requiring enzyme 1), and PERK (PKR-like ER protein kinas) expression in BHK cells were determined. The results showed that HRV16 2B induced significantly high expression of glucose-regulated protein 78, and activated ATF4 and ATF6 in a time-dependent manner. HRV16 2B also significantly activated the PERK-eIF2α pathway with marked increased in expression of p-PERK, p-eIF2α, and CHOP. Additionally, HRV16 2B decreased the expression of p-IRE1 and ATF6 p50. These results demonstrated for the first time that the PERK-eIF2α signaling pathway, but not IRE1 and ATF6, mediates ER stress in BHK cells.

Discuss in detail the effects that an activating mutation in the IKBKB gene would be expected to have on the downstream signalling pathway components and target gene expression, and how these effects are measured in the article.

In an immunoprecipitation assay, 293T cells testing wild type and mutated TQ/SQ sites in TRAF6 was utilized to determine the effect of genotoxic agents on the phosphorylation of TRAF6 at TQ/SQ motifs. The authors immunoprecipitated for flag asTRAF6 was tagged with the flag antibody. CDDP and ATM/ATR were used as our tested variables. The assay elucidated that the double mutation of S13 and T330 on TRAF6 significantly reduced phosphorylation of TRAF6 in the presence of CDDP and absence of ATM/ATR. Consequently, this data provides information for belief that mutated TRAF6 is not sufficient for phosphorylation to occur in the presence of CDDP. Finally, a cellular fractionation assay for 293T wild type and mutant TRAF6 cells with or without CDDP treatment tested whether such mutation in the presence of genotoxic stress inhibits or promotes TRAF6 expression. As in the previous fractionation studies, tubulin and laminB were loading controls. These results concluded that the mutant displayed resistance to the CDDP mediated reduction of TRAF6 in the cytosol. Overall, these studies suggest that CDDP induces phosphorylation of TRAF6 in the cytosol, which may contribute to the reduction of K63-linked ubiquitination in the cytosol. The information provided thus far suggests that K63-linked ubiquitination of p53 is accomplished by TRAF6, which is hypothesized to suppress spontaneous apoptosis. Moreover, genotoxic stress is shown to promote TRAF6

The endoplasmic reticulum (ER) is an essential organelle that is a major place for the biogenesis of cellular components including proteins, lipids, and carbohydrates and internal calcium storage. ER is primarily responsible for protein translocation, protein folding and protein post modification. Proper folding of protein in the ER is accomplished with the aid of ER resident proteins or enzymes such as chaperones. Binding of chaperones to

Molecular chaperones stabilize unfolded or misfolded proteins until native conformations have been obtained to promote cell survival during and after stress conditions. They do not change or add to the folding principles encoded by a protein because polypeptide chains inherently carry within them all the information that is necessary for achieving the native state of a protein. Instead, they optimize the folding process by stabilizing folding intermediates and are involved in every aspect of proteome maintenance including de novo folding, refolding of stress-induced misfolded proteins, and targeting proteins for degradation (Hartl 2009, Hartl 2011). Chaperones, many of which are induced or upregulated only during stress conditions, work in cooperative networks when protein-aggregate concentration