Forced intercalation-induced light-up peptides as fluorogenic indicators for the HIV-1 TAR RNA-ligand assay.

Fluorescence indicators capable of binding to human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA are powerful tools for the exploratory studies of the identification of anti-HIV drug candidates. This work presents a new design strategy for fluorogenic indicators with a transactivator of transcription (Tat)-derived peptide based on the forced intercalation of thiazole orange (TO) dyes (FIT). The developed 9-mer FIT peptide (RKKRR-TO-RRR: named FiLuP) features the TO unit integrated onto a Dap (2,3-diaminopropionic acid) residue in the middle of the Tat peptide sequence; the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. This facilitates a significant light-up response (450-fold at λem = 541 nm, Φfree = 0.0057, and Φbound = 0.61) upon binding to TAR RNA. The response of FiLuP is highly selective to TAR RNA over other non-cognate RNAs, and FiLuP maintains strong binding affinity (Kd = 1.0 ± 0.6 nM). Significantly, in contrast to previously developed Tat peptide-based FRET probes, FiLuP is able to discriminate between "competitive" and "noncompetitive" inhibitors when used in the fluorescence indicator displacement (FID) assay. The FID assay under stringent screening conditions is also possible, enabling super-strong competitive binders toward TAR RNA to be sieved out.

Targeting neddylation sensitizes colorectal cancer to topoisomerase I inhibitors by inactivating the DCAF13-CRL4 ubiquitin ligase complex.

Colorectal cancers (CRCs) are prevalent worldwide, yet current treatments remain inadequate. Using chemical genetic screens, we identify that co-inhibition of topoisomerase I (TOP1) and NEDD8 is synergistically cytotoxic in human CRC cells. Combination of the TOP1 inhibitor irinotecan or its bioactive metabolite SN38 with the NEDD8-activating enzyme inhibitor pevonedistat exhibits synergy in CRC patient-derived organoids and xenografts. Mechanistically, we show that pevonedistat blocks the ubiquitin/proteasome-dependent repair of TOP1 DNA-protein crosslinks (TOP1-DPCs) induced by TOP1 inhibitors and that the CUL4-RBX1 complex (CRL4) is a prominent ubiquitin ligase acting on TOP1-DPCs for proteasomal degradation upon auto-NEDD8 modification during replication. We identify DCAF13, a DDB1 and Cullin Associated Factor, as the receptor of TOP1-DPCs for CRL4. Our study not only uncovers a replication-coupled ubiquitin-proteasome pathway for the repair of TOP1-DPCs but also provides molecular and translational rationale for combining TOP1 inhibitors and pevonedistat for CRC and other types of cancers.

Small molecule-PNA oligomer conjugates for rRNA A-site at neutral pH for FID assays.

A triplex-forming PNA oligomer conjugated with a naphthyridine derivative (ATMND-C2-NH2) showed high selectivity and strong binding for the bacterial rRNA A-site at pH 7.0 (Kd = 190 ± 72 nM), which was accompanied by fluorogenic signaling that allowed the potential use of this conjugate probe in fluorescent indicator displacement assays.

Aryl Sulfonamides Degrade RBM39 and RBM23 by Recruitment to CRL4-DCAF15.

Indisulam and related sulfonamides recruit the splicing factor RBM39 to the CRL4-DCAF15 E3 ubiquitin ligase, resulting in RBM39 ubiquitination and degradation. Here, we used a combination of domain mapping and random mutagenesis to identify domains or residues that are necessary for indisulam-dependent RBM39 ubiquitination. DCAF15 mutations at Q232 or D475 prevent RBM39 recruitment by indisulam. RBM39 is recruited to DCAF15 by its RRM2 (RNA recognition motif 2) and is ubiquitinated on its N terminus. RBM23, which is an RBM39 paralog, is also recruited to the CRL4-DCAF15 ligase through its RRM2 domain and undergoes sulfonamide-dependent degradation. Indisulam alters the expression of more than 3,000 genes and causes widespread intron retention and exon skipping. All of these changes can be attributed to RBM39, and none are the consequence of RBM23 degradation. Our findings demonstrate that indisulam selectively degrades RBM23 and RBM39, the latter of which is critically important for splicing and gene expression.

Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established1,2. Here we report lifespan and healthspan extension in a mouse model with increased basal autophagy. To determine the effects of constitutively increased autophagy on mammalian health, we generated targeted mutant mice with a Phe121Ala mutation in beclin 1 (Becn1F121A/F121A) that decreases its interaction with the negative regulator BCL2. We demonstrate that the interaction between beclin 1 and BCL2 is disrupted in several tissues in Becn1 F121A/F121A knock-in mice in association with higher levels of basal autophagic flux. Compared to wild-type littermates, the lifespan of both male and female knock-in mice is significantly increased. The healthspan of the knock-in mice also improves, as phenotypes such as age-related renal and cardiac pathological changes and spontaneous tumorigenesis are diminished. Moreover, mice deficient in the anti-ageing protein klotho 3 have increased beclin 1 and BCL2 interaction and decreased autophagy. These phenotypes, along with premature lethality and infertility, are rescued by the beclin 1(F121A) mutation. Together, our data demonstrate that disruption of the beclin 1-BCL2 complex is an effective mechanism to increase autophagy, prevent premature ageing, improve healthspan and promote longevity in mammals.

Author Correction: Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

In this Letter, the graphs in Fig. 2a and c were inadvertently the same owing to a copy and paste error from the original graphs in Prism. The Source Data files containing the raw data were correct. Fig. 2c has been corrected online.

A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease.

Impairment of the autophagy pathway has been observed during the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disorder characterized by abnormal deposition of extracellular and intracellular amyloid ß (Aß) peptides. Yet the role of autophagy in Aß production and AD progression is complex. To study whether increased basal autophagy plays a beneficial role in Aß clearance and cognitive improvement, we developed a novel genetic model to hyperactivate autophagy in vivo. We found that knock-in of a point mutation F121A in the essential autophagy gene Beclin 1/Becn1 in mice significantly reduces the interaction of BECN1 with its inhibitor BCL2, and thus leads to constitutively active autophagy even under non-autophagy-inducing conditions in multiple tissues, including brain. Becn1F121A-mediated autophagy hyperactivation significantly decreases amyloid accumulation, prevents cognitive decline, and restores survival in AD mouse models. Using an immunoisolation method, we found biochemically that Aß oligomers are autophagic substrates and sequestered inside autophagosomes in the brain of autophagy-hyperactive AD mice. In addition to genetic activation of autophagy by Becn1 gain-of-function, we also found that ML246, a small-molecule autophagy inducer, as well as voluntary exercise, a physiological autophagy inducer, exert similar Becn1-dependent protective effects on Aß removal and memory in AD mice. Taken together, these results demonstrate that genetically disrupting BECN1-BCL2 binding hyperactivates autophagy in vivo, which sequestrates amyloid oligomers and prevents AD progression. The study establishes new approaches to activate autophagy in the brain, and reveals the important function of Becn1-mediated autophagy hyperactivation in the prevention of AD.

Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15.

Indisulam is an aryl sulfonamide drug with selective anticancer activity. Its mechanism of action and the basis for its selectivity have so far been unknown. Here we show that indisulam promotes the recruitment of RBM39 (RNA binding motif protein 39) to the CUL4-DCAF15 E3 ubiquitin ligase, leading to RBM39 polyubiquitination and proteasomal degradation. Mutations in RBM39 that prevent its recruitment to CUL4-DCAF15 increase RBM39 stability and confer resistance to indisulam's cytotoxicity. RBM39 associates with precursor messenger RNA (pre-mRNA) splicing factors, and inactivation of RBM39 by indisulam causes aberrant pre-mRNA splicing. Many cancer cell lines derived from hematopoietic and lymphoid lineages are sensitive to indisulam, and their sensitivity correlates with DCAF15 expression levels. Two other clinically tested sulfonamides, tasisulam and chloroquinoxaline sulfonamide, share the same mechanism of action as indisulam. We propose that DCAF15 expression may be a useful biomarker to guide clinical trials of this class of drugs, which we refer to as SPLAMs (splicing inhibitor sulfonamides).

PERK-eIF2α-ATF4-CHOP signaling contributes to TNFα-induced vascular calcification.

BACKGROUND: Vascular calcification is a common feature in patients with chronic kidney disease (CKD). CKD increases serum levels of tumor necrosis factor-α (TNFα), a critical mediator of vascular calcification. However, the molecular mechanism by which TNFα promotes CKD-dependent vascular calcification remains obscure. The purpose of the present study was to investigate whether TNFα-induced vascular calcification in CKD is caused by the endoplasmic reticulum response involving protein kinase RNA-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). METHODS AND RESULTS: We examined the effects of TNFα on the endoplasmic reticulum (ER) stress response of vascular smooth muscle cells (VSMCs). TNFα treatment drastically induced the PERK-eIF2α-ATF4-CHOP axis of the ER stress response in VSMCs. PERK, ATF4, and CHOP shRNA-mediated knockdowns drastically inhibited mineralization and osteogenesis of VSMCs induced by TNFα. CKD induced by 5/6 nephrectomies activated the PERK-eIF2α-ATF4-CHOP axis of the ER stress response in the aortas of ApoE-/- mice with increased aortic TNFα expression and vascular calcification. Treatment of 5/6 nephrectomized ApoE-/- mice with the TNFα neutralizing antibody or chemical Chaperones reduced aortic PERK-eIF2α-ATF4-CHOP signaling of the ER stress increased by CKD. This resulted in the inhibition of CKD-dependent vascular calcification. CONCLUSIONS: These results suggest that TNFα induces the PERK-eIF2α-ATF4-CHOP axis of the ER stress response, leading to CKD-dependent vascular calcification.

Activating transcription factor 4 regulates stearate-induced vascular calcification.

Previously, we reported that stearate, a saturated fatty acid, promotes osteoblastic differentiation and mineralization of vascular smooth muscle cells (VSMC). In this study, we examined the molecular mechanisms by which stearate promotes vascular calcification. ATF4 is a pivotal transcription factor in osteoblastogenesis and endoplasmic reticulum (ER) stress. Increased stearate by either supplementation of exogenous stearic acid or inhibition of stearoyl-CoA desaturase (SCD) by CAY10566 induced ATF4 mRNA, phosphorylated ATF4 protein, and total ATF4 protein. Induction occurred through activation of the PERK-eIF2α pathway, along with increased osteoblastic differentiation and mineralization of VSMCs. Either stearate or the SCD inhibitor but not oleate or other fatty acid treatments also increased ER stress as determined by the expression of p-eIF2α, CHOP, and the spliced form of XBP-1, which were directly correlated with ER stearate levels. ATF4 knockdown by lentiviral ATF4 shRNA blocked osteoblastic differentiation and mineralization induced by stearate and SCD inhibition. Conversely, treatment of VSMCs with an adenovirus containing ATF4 induced vascular calcification. Our results demonstrated that activation of ATF4 mediates vascular calcification induced by stearate.

Role of cellular cholesterol metabolism in vascular cell calcification.

Vascular calcification impairs vessel compliance and increases the risk of cardiovascular events. We found previously that liver X receptor agonists, which regulate intracellular cholesterol homeostasis, augment PKA agonist- or high phosphate-induced osteogenic differentiation of vascular smooth muscle cells. Because cholesterol is an integral component of the matrix vesicles that nucleate calcium mineral, we examined the role of cellular cholesterol metabolism in vascular cell mineralization. The results showed that vascular smooth muscle cells isolated from LDL receptor null (Ldlr(-/-)) mice, which have impaired cholesterol uptake, had lower levels of intracellular cholesterol and less osteogenic differentiation, as indicated by alkaline phosphatase activity and matrix mineralization, compared with WT cells. PKA activation with forskolin acutely induced genes that promote cholesterol uptake (LDL receptor) and biosynthesis (HMG-CoA reductase). In WT cells, inhibition of cholesterol uptake by lipoprotein-deficient serum attenuated forskolin-induced matrix mineralization, which was partially reversed by the addition of cell-permeable cholesterol. Prolonged activation of both uptake and biosynthesis pathways by cotreatment with a liver X receptor agonist further augmented forskolin-induced matrix mineralization. Inhibition of either cholesterol uptake, using Ldlr(-/-) cells, or of cholesterol biosynthesis, using mevastatin-treated WT cells, failed to inhibit matrix mineralization due to up-regulation of the respective compensatory pathway. Inhibition of both pathways simultaneously using mevastatin-treated Ldlr(-/-) cells did inhibit forskolin-induced matrix mineralization. Altogether, the results suggest that up-regulation of cholesterol metabolism is essential for matrix mineralization by vascular cells.

Increased lipogenesis and stearate accelerate vascular calcification in calcifying vascular cells.

Vascular calcification is recognized as an independent predictor of cardiovascular mortality, particularly in subjects with chronic kidney disease. However, the pathways by which dysregulation of lipid and mineral metabolism simultaneously occur in this particular population remain unclear. We have shown that activation of the farnesoid X receptor (FXR) blocks mineralization of bovine calcifying vascular cells (CVCs) and in ApoE knock-out mice with 5/6 nephrectomy. In contrast to FXR, this study showed that liver X receptor (LXR) activation by LXR agonists and adenovirus-mediated LXR overexpression by VP16-LXRα and VP16-LXRß accelerated mineralization of CVCs. Conversely, LXR inhibition by dominant negative (DN) forms of LXRα and LXRß reduced calcium content in CVCs. The regulation of mineralization by FXR and LXR agonists was highly correlated with changes in lipid accumulation, fatty acid synthesis, and the expression of sterol regulatory element binding protein-1 (SREBP-1). The rate of lipogenesis in CVCs through the SREBP-1c dependent pathway was reduced by FXR activation, but increased by LXR activation. SREBP-1c overexpression augmented mineralization in CVCs, whereas SREBP-1c DN inhibited alkaline phosphatase activity and mineralization induced by LXR agonists. LXR and SREBP-1c activations increased, whereas FXR activation decreased, saturated and monounsaturated fatty acids derived from lipogenesis. In addition, we found that stearate markedly promoted mineralization of CVCs as compared with other fatty acids. Furthermore, inhibition of either acetyl-CoA carboxylase or acyl-CoA synthetase reduced mineralization of CVCs, whereas inhibition of stearoyl-CoA desaturase induced mineralization. Therefore, a stearate metabolite derived from lipogenesis might be a risk factor for the development of vascular calcification.

Farnesoid X receptor activation prevents the development of vascular calcification in ApoE-/- mice with chronic kidney disease.

RATIONALE: Vascular calcification is highly associated with cardiovascular morbidity and mortality, especially in patients with chronic kidney disease. The nuclear receptor farnesoid X receptor (FXR) has been implicated in the control of lipid, carbohydrate and bile acid metabolism in several cell types. Although recent studies have shown that FXR is also expressed in vascular smooth muscle cells, its physiological role in vasculature tissue remains obscure. OBJECTIVE: Here, we have examined the role of FXR in vascular calcification. METHODS AND RESULTS: The FXR gene, a bile acid nuclear receptor, was highly induced during osteogenic differentiation of bovine calcifying vascular cells (CVCs) and in the aorta of apolipoprotein (Apo)E(-/-) mice with chronic kidney disease which are common tissue culture and mouse model, respectively, for aortic calcification. FXR activation by a synthetic FXR agonist, 6alpha-ethyl chenodeoxycholic acid (INT-747) inhibited phosphate induced-mineralization and triglyceride accumulation in CVCs. FXR dominant negative expression augmented mineralization of CVCs and blocked the anticalcific effect of INT-747 whereas VP16FXR that is a constitutively active form reduced mineralization of CVCs. INT-747 treatment also increased phosphorylated c-Jun N-terminal kinase (JNK). SP600125 (specific JNK inhibitor) significantly induced mineralization of CVCs and alkaline phosphatase expression, suggesting that the anticalcific effect of INT-747 is attributable to JNK activation. We also found that INT-747 ameliorates chronic kidney disease induced-vascular calcification in 5/6 nephrectomized ApoE(-/-) mice without affecting the development of atherosclerosis. CONCLUSIONS: These observations provide direct evidence that FXR is a key signaling component in regulation of vascular osteogenic differentiation and, thus representing a promising target for the treatment of vascular calcification.