Heterobifunctional molecules that recruit E3 ubiquitin ligases, such as cereblon, for targeted protein degradation represent an emerging pharmacological strategy. A major unanswered question is how generally applicable this strategy is to all protein targets. In this study, we designed a multi-kinase degrader by conjugating a highly promiscuous kinase inhibitor with a cereblon-binding ligand, and used quantitative proteomics to discover 28 kinases, including BTK, PTK2, PTK2B, FLT3, AURKA, AURKB, TEC, ULK1, ITK, and nine members of the CDK family, as degradable. This set of kinases is only a fraction of the intracellular targets bound by the degrader, demonstrating that successful degradation requires more than target engagement. The results guided us to develop selective degraders for FLT3 and BTK, with potentials to improve disease treatment. Together, this study demonstrates an efficient approach to triage a gene family of interest to identify readily degradable targets for further studies and pre-clinical developments.
Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase which has multiple functions, including inhibition of the mitogen-activated protein (MAP) kinase pathway. Simian virus 40 small t antigen specifically inhibits PP2A function by binding to the PP2A regulatory subunit, interfering with the ability of PP2A to associate with its cellular substrates. We have reported that the expression of small t antigen inhibits PP2A association with Shc, leading to augmentation of insulin and epidermal growth factor-induced Shc phosphorylation with enhanced activation of the Ras/MAP kinase pathway. However, the potential involvement of PP2A in insulin's metabolic signaling pathway is presently unknown. To assess this, we overexpressed small t antigen in 3T3-L1 adipocytes by adenovirus-mediated gene transfer and found that the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C (PKC ) activity was also augmented in small-t-antigenexpressing 3T3-L1 adipocytes. Consistent with this result, both basal and insulin-stimulated glucose uptake were enhanced in these cells. In support of this result, when inhibitory anti-PP2A antibody was microinjected into 3T3-L1 adipocytes, we found a twofold increase in GLUT4 translocation in the absence of insulin. The small-t-antigen-induced increase in Akt and PKC activities was not inhibited by wortmannin, while the ability of small t antigen to enhance glucose transport was inhibited by dominant negative Akt (DN-Akt) expression and Akt small interfering RNA (siRNA) but not by DN-PKC expression or PKC siRNA. We conclude that PP2A is a negative regulator of insulin's metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.Protein phosphorylation plays a key role in many cellular processes, including insulin signal transduction (24), and the phosphorylation state of a target protein is regulated by opposing kinase and phosphatase activities (24). Thus, the balance of enzyme activity between kinases and phosphatases is critical for the mediation of insulin's effects and, in turn, for the pathogenesis of insulin-resistant states.Tyrosine phosphorylation is essential for insulin action, and several lines of evidence have demonstrated that protein tyrosine phosphatases can play a role in insulin-resistant states (3, 4). For example, protein tyrosine phosphatase 1B (PTP1B) directly interacts with the activated insulin receptor and exhibits high specific activity for IRS-1 (22, 49). It has been reported previously that hyperglycemia can impair insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1, at least in part because of the increased expression and activity of PTP1B (37,41), and that overexpression of PTP1B inhibits insulin-stimulated glucose metabolism in 3T3-L1 adipocytes and L6 myocytes (12,18,51).Serine/thre...
The ATPase cycle of the chaperone Hsc70 is regulated by co-chaperones; Hsp40/DnaJ-related proteins stimulate ATP hydrolysis by Hsc70 and can bind unfolded polypeptides themselves. Conversely, various nucleotide exchange factors (NEFs) stimulate ADP-ATP exchange by Hsc70. We analyzed the purified Hsp40-related co-chaperones DJA1 (Hdj2) and DJA2 (Hdj3) and found that they had a distinct pattern of binding to a range of polypeptides. DJA2 alone could stimulate Hsc70-mediated refolding of luciferase in the absence of NEF, whereas DJA1 was much less active. The addition of the Bag1 NEF increased refolding by Hsc70 and DJA2, as did the newly characterized NEF Hsp110, but each NEF had a different optimal concentration ratio to Hsc70. Notably, the NEF HspBP1 could not increase refolding by Hsc70 and DJA2 at any concentration, and none of the NEFs improved the refolding activity with DJA1. Instead, DJA1 was inhibitory of refolding with DJA2 and Hsc70. All combinations of DJA1 or DJA2 with the three NEFs stimulated the Hsc70 ATPase rate, although Hsp110 became less effective with increasing concentrations. A chimeric DJA2 having its Hsc70-stimulatory J domain replaced with that of DJA1 was functional for polypeptide binding and ATPase stimulation of Hsc70. However, it could not support efficient Hsc70-mediated refolding and also inhibited refolding with DJA2 and Hsc70. These results suggest a more complex model of Hsc70 mechanism than has been previously thought, with notable functional divergence between Hsc70 co-chaperones.The Hsp70 family of proteins are ATP-dependent molecular chaperones that assist the folding of polypeptides. Hsp70 chaperones have a typical structure divided into ATPase and substrate-binding domains that work in an ATP-driven substrate binding cycle. The mechanism of Hsp70 proteins has been well established in studies of the Escherichia coli homolog DnaK. In the ATP-bound state, an Hsp70 chaperone has low affinity for unfolded polypeptide. After hydrolysis of ATP, Hsp70 in the ADP-bound state binds substrate with high affinity. Exchange of ADP for ATP then reverts Hsp70 to its low polypeptide affinity state. Conversion of an Hsp70 between these two nucleotide states is controlled by different co-chaperone proteins. The Hsp40/DnaJ-related co-chaperones, including E. coli DnaJ, contain J domains that stimulate ATP hydrolysis by Hsp70, and consequently substrate binding. Nucleotide exchange factors (NEFs), 2 such as GrpE in E. coli, trigger the dissociation of bound ADP from Hsp70 to allow the binding of ATP, resetting the cycle. The principles of this mechanism appear to be conserved in Hsp70 chaperones, including the major cytosolic form in humans, Hsc70 (HSPA8) (1, 2).The DnaJ-related co-chaperones are also conserved between species. Type 1 J domain co-chaperones are homologous to DnaJ throughout their sequence and have the same domain architecture. Following their N-terminal J domains, they contain a linker sequence, zinc finger and central regions, and a C-terminal homodimerization region. Unfolde...
Aberrant activation of the Wnt/b-catenin signaling pathway is a common event in human tumor progression. Wnt signaling has also been implicated in maintaining a variety of adult and embryonic stem cells by imposing a restraint to differentiation. To understand the function and mechanism of Wnt/b-catenin signaling on the pathogenesis of teratocarcinoma, we used the mouse teratocarcinoma P19 cell line as a model in vitro. Gsk3b specific inhibitor (SB216763) was used to activate Wnt/b-catenin signaling. All trans-retinoic acid (RA) was used to induce P19 cell differentiation. At different culture times, gene expression was examined by immunofluorescence staining, quantitative real-time PCR, and Westernblotting; BrdU incorporation assays were performed to measure P19 cell proliferation. Small interference RNA technology was used to downregulate c-myc expression. The results showed that SB216763 induced the nuclear translocation of b-catenin, upregulated the expression of c-myc and pluripotency related genes, oct4, sox2 and nanog, and blocked cell differentiation induced by all trans-RA. The proliferation of P19 cells was significantly enhanced by SB216763, as well as c-myc overexpression. C-myc downregulation inhibited P19 cell proliferation caused by activation of Wnt/b-catenin signaling and induced P19 cell differentiation. In conclusion, activation of the Wnt/b-catenin pathway could promote the proliferation and inhibit the differentiation of mouse teratocarcinoma cells by upregulation of c-myc expression. Anat Rec, 295:2104Rec, 295: -2113
Like hyperglycemia, postprandial (diet-induced) hypertriglyceridemia is thought to play crucial roles in the pathogenesis of insulin resistant/metabolic syndrome. Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor to induce postprandial hypertriglyceridemia. We found that insulin-resistant rats fed a diet high in fructose showed an increased proteintyrosine phosphatase 1B (PTP1B) content with strong expression of SREBP-1 mRNA in the liver. To clarify the association of PTP1B with SREBP-1 gene expression, we overexpressed PTP1B in rat hepatocytes, which led to increased mRNA content and promoter activity of SREBP-1a and -1c, resulting in the increased mRNA expression of fatty-acid synthase, one of the SREBP-1-responsive lipogenic genes. Because PTP1B overexpression increased phosphatase 2A (PP2A) activity, we inhibited PP2A activity by expression of its selective inhibitor, SV40 small t antigen and found that this normalized the PTP1B-enhanced SREBP-1a and -1c mRNA expressions through activation of the Sp1 site. These results indicate that PTP1B may regulate gene expression of SREBP-1 via enhancement of PP2A activity, thus mediating hepatic lipogenesis and postprandial hypertriglyceridemia. We demonstrate here a unique serial activation of the PTP1B-PP2A axis as a novel mechanism for the regulation of gene expression in the biosynthesis of triglyceride.The insulin resistance/metabolic syndrome is characterized by the variable coexistence of hyperinsulinemia, hyperlipidemia, obesity, and hypertension. Many individuals with obesity and insulin resistance suffer from fatty liver (steatohepatosis). Like hyperglycemia, postprandial (diet-induced) hypertriglyceridemia is thought to play a crucial role in the pathogenesis of the insulin resistance/metabolic syndrome.Several lines of evidence indicate that fatty liver in insulinresistant states is caused by activation of the sterol regulatory element-binding protein (SREBP)-1c, 1 which is elevated in response to high insulin levels (1, 2). Although insulin resistance is present in peripheral tissues, compensatory hyperinsulinemia is postulated to persistently activate SREBP-1c transcription and cleavage, which increases lipogenic gene expression, enhances fatty acid synthesis, and accelerates triglyceride accumulation in the liver in obese insulin-resistant mice (3-5). However, the reason that expression of hepatic SREBP-1c mRNA remains high in insulin-resistant states is little understood.We have reported that high fructose feeding causes insulin resistance with increased hepatic SREBP-1 mRNA content (6), and we recently found that, as reported (7), protein-tyrosine phosphatase 1B (PTP1B) abundance is also increased in the liver. PTP1B is a major regulator of insulin sensitivity and body fat content, and deletion of its gene leads to augmented insulin sensitivity and resistance to obesity induced by high fat feeding (8 -11). Moreover, at least one genetic polymorphism of PTP1B is associated with several features of the insulin resistance...
According to the theories of traditional Chinese medicine, spleen deficiency often leads to diarrhea, and deep-fried Atractylodis Rhizoma (DAR) is commonly used for the treatment. However, the association between spleen deficiency and diarrhea remains unclear. The present study aimed to investigate the therapeutic effect of DAR for the treatment of diarrhea caused by spleen deficiency and analyze the related mechanisms. It was found that a high dose group of an ethanolic extract of deep-fried Atractylodis Rhizoma (EEDAR-H) significantly inhibited weight loss, diarrhea, and pathological changes in colon tissue induced by rhubarb. EEDAR-H was found to significantly reduce the level of intestinal inflammatory cytokines and increase the expression of gastrointestinal motility hormones. In addition, EEDAR-H significantly increased the expression of aquaporin 3 (AQP3) and aquaporin 8 (AQP8) and restored abnormal water metabolism; Shen-Ling-Bai-Zhu-San (SLBZS) induced the same effect as EEDAR-H. Additional tests on the mechanism found that EEDAR-H and SLBZS promoted the integrity of the intestinal barrier. Both significantly increased the expression of the tight junction protein ZO-1 and Occludin, inhibited the phosphorylation of p38MAPK and MLC, and significantly reduced the expression levels of PAR-2. Analysis of the gut microbiota indicated that overall changes in its structure were reversed after treatment with EEDAR-H or SLBZS, in addition to significant modulation of the abundance of different phyla. At the genus level, EEDAR-H or SLBZS significantly reduced the levels of potential pathogens and increased those of beneficial bacteria.
Aminoglycosides are a class of antibiotics with a broad spectrum of antimicrobial activity. Unfortunately, resistance in clinical isolates is pervasive, rendering many aminoglycosides ineffective. The most widely disseminated means of resistance to this class of antibiotics is inactivation of the drug by aminoglycoside-modifying enzymes (AMEs). There are two principal strategies to overcoming the effects of AMEs. The first approach involves the design of novel aminoglycosides that can evade modification. Although this strategy has yielded a number of superior aminoglycoside variants, their efficacy cannot be sustained in the long term. The second approach entails the development of molecules that interfere with the mechanism of AMEs such that the activity of aminoglycosides is preserved. Although such a molecule has yet to enter clinical development, the search for AME inhibitors has been greatly facilitated by the wealth of structural information amassed in recent years. In particular, aminoglycoside phosphotransferases or kinases (APHs) have been studied extensively and crystal structures of a number of APHs with diverse regiospecificity and substrate specificity have been elucidated. In this review, we present a comprehensive overview of the available APH structures and recent progress in APH inhibitor development, with a focus on the structure-guided strategies.
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