ABSTRACT
Nucleolar stress (NS) has been associated with age-related diseases such as cancer or neurodegeneration. To investigate how NS triggers toxicity, we used (PR)n arginine-rich peptides present in some neurodegenerative diseases as inducers of this perturbation. We here reveal that whereas (PR)n expression leads to a decrease in translation, this occurs concomitant with an accumulation of free ribosomal (r) proteins. Conversely, (PR)n-resistant cells have lower rates of r-protein synthesis, and targeting ribosome biogenesis by mTOR inhibition or MYC depletion alleviates (PR)n toxicity in vitro. In mice, systemic expression of (PR)97 drives widespread NS and accelerated aging, which is alleviated by rapamycin. Notably, the generalized accumulation of orphan r-proteins is a common outcome of chemical or genetic perturbations that induce NS. Together, our study presents a general model to explain how NS induces cellular toxicity and provides in vivo evidence supporting a role for NS as a driver of aging in mammals.
Subject(s)
Neoplasms , Ribosomes , Mice , Animals , Ribosomes/metabolism , Aging/genetics , Peptides/metabolism , Sirolimus/pharmacology , Neoplasms/metabolism , Cell Nucleolus/genetics , MammalsABSTRACT
Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family mainly targeting cytosolic nonhistone substrates, such as α-tubulin, cortactin, and heat shock protein 90 to regulate cell proliferation, metastasis, invasion, and mitosis in tumors. We describe the identification and characterization of a series of 2-(difluoromethyl)-1,3,4-oxadiazoles (DFMOs) as selective nonhydroxamic acid HDAC6 inhibitors. By comparing structure-activity relationships and performing quantum mechanical calculations of the HDAC6 catalytic mechanism, we show that potent oxadiazoles are electrophilic substrates of HDAC6 and propose a mechanism for the bioactivation. We also observe that the inherent electrophilicity of the oxadiazoles makes them prone to degradation in water solution and the generation of potentially toxic products cannot be ruled out, limiting the developability for chronic diseases. However, the oxadiazoles demonstrate high oral bioavailability and low in vivo clearance and are excellent tools for studying the role of HDAC6 in vitro and in vivo in rats and mice.
Subject(s)
Neoplasms , Oxadiazoles , Rats , Mice , Animals , Histone Deacetylase 6 , Oxadiazoles/pharmacology , Tubulin/metabolism , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/chemistryABSTRACT
Cathelicidin antimicrobial peptides have an extended and/or unstructured conformation in aqueous solutions but fold into ordered conformations, such as the α-helical structure, when interacting with cellular membranes. These structural transitions can be directly correlated to their antimicrobial activity and its underlying mechanisms. SMAP-18, the N-terminal segment (residues 1-18) of sheep cathelicidin (SMAP-29), is known to kill microorganisms by translocating across membranes and interacting with their nucleic acids. The amino acid sequence of SMAP-18 contains three Gly residues (at positions 2, 7, and 13) that significantly affect the flexibility of its peptide structure. This study investigated the role of Gly residues in the structure, membrane interaction, membrane translocation, and antimicrobial mechanisms of SMAP-18. Five analogs were designed and synthesized through Gly â Ala substitution (i.e., G2A, G7A, G13A, G7,13A, and G2,7,13A); these substitutions altered the helical content of SMAP-18 peptides. We found that G7,13A and G2,7,13A changed their mode of action, with circular dichroism and nuclear magnetic resonance studies revealing that these analogs changed the structure of SMAP-18 from a random coil to an α-helical structure. The results of this experiment suggest that the Gly residues at positions 7 and 13 in SMAP-18 are the structural and functional determinants that control its three-dimensional structure, strain-specific activity, and antimicrobial mechanism of action. These results provide valuable information for the design of novel peptide-based antibiotics.
Subject(s)
Anti-Infective Agents , Cathelicidins , Animals , Sheep , Cathelicidins/chemistry , Antimicrobial Peptides , Anti-Infective Agents/pharmacology , Amino Acid Sequence , Cell Membrane/metabolism , Circular DichroismABSTRACT
The expansion of GGGGCC repeats within the first intron of C9ORF72 constitutes the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Through repeat-associated non-ATG translation, these expansions are translated into dipeptide repeats (DPRs), some of which accumulate at nucleoli and lead to cell death. We here performed a chemical screen to identify compounds reducing the toxicity of ALS-related poly(PR) peptides. Our screening identified sodium phenylbutyrate, currently in clinical trials, and BET Bromodomain inhibitors as modifiers of poly(PR) toxicity in cell lines and developing zebrafish embryos. Mechanistically, we show that BET Bromodomain inhibitors rescue the nucleolar stress induced by poly(PR) or actinomycin D, alleviating the effects of the DPR in nucleolus-related functions such as mRNA splicing or translation. Our work suggests that BET Bromodomain inhibitors might have beneficial effects in diseases linked to nucleolar stress such as ALS/FTD.
Subject(s)
Apoptosis/drug effects , C9orf72 Protein/chemistry , Peptides/toxicity , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , C9orf72 Protein/genetics , C9orf72 Protein/metabolism , Cell Line, Tumor , Cell Nucleolus/drug effects , Cell Nucleolus/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Repeat Expansion , Dactinomycin/toxicity , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/physiology , Frontotemporal Dementia/metabolism , Frontotemporal Dementia/pathology , Histone Deacetylase Inhibitors/pharmacology , Humans , Peptides/chemical synthesis , Proteins/antagonists & inhibitors , Proteins/metabolism , Zebrafish/growth & developmentABSTRACT
Trafficking of the G protein-coupled receptor (GPCR) Smoothened (Smo) to the primary cilium (PC) is a potential target to inhibit oncogenic Hh pathway activation in a large number of tumors. One drawback is the appearance of Smo mutations that resist drug treatment, which is a common reason for cancer treatment failure. Here, we undertook a high content screen with compounds in preclinical or clinical development and identified ten small molecules that prevent constitutive active mutant SmoM2 transport into PC for subsequent Hh pathway activation. Eight of the ten small molecules act through direct interference with the G protein-coupled receptor associated sorting protein 2 (Gprasp2)-SmoM2 ciliary targeting complex, whereas one antagonist of ionotropic receptors prevents intracellular trafficking of Smo to the PC. Together, these findings identify several compounds with the potential to treat drug-resistant SmoM2-driven cancer forms, but also reveal off-target effects of established drugs in the clinics.
Subject(s)
Cilia/metabolism , Hedgehog Proteins/metabolism , Oncogenes , Smoothened Receptor/metabolism , Animals , Mice , Pancreatic Neoplasms/metabolism , Protein Transport , Signal TransductionABSTRACT
The seven-transmembrane receptor Smoothened (Smo) activates all Hedgehog (Hh) signaling by translocation into the primary cilia (PC), but how this is regulated is not well understood. Here we show that Pitchfork (Pifo) and the G protein-coupled receptor associated sorting protein 2 (Gprasp2) are essential components of an Hh induced ciliary targeting complex able to regulate Smo translocation to the PC. Depletion of Pifo or Gprasp2 leads to failure of Smo translocation to the PC and lack of Hh target gene activation. Together, our results identify a novel protein complex that is regulated by Hh signaling and required for Smo ciliary trafficking and Hh pathway activation.
Subject(s)
Hedgehog Proteins/metabolism , Homeodomain Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/physiology , Animals , Cell Line , Cilia/genetics , Cilia/metabolism , Hedgehog Proteins/genetics , Homeodomain Proteins/genetics , Intracellular Signaling Peptides and Proteins , Mice , Protein Transport/physiology , Receptors, G-Protein-Coupled/genetics , Smoothened ReceptorABSTRACT
To find out whether the expressions of these adipocyte markers are influenced by oriental medicine, obesity rats induced by high fat diet (HFD) for 8 weeks were injected with 50 mg/100 g body weight adlay seed crude extract (ACE), daily for 4 weeks. The results are summarized as follows: HFD + ACE group significantly reduced food intakes and body weights. Weights of epididymal and peritoneal fat were dramatically increased in HFD groups compared with those of normal diet (ND) group but significantly decreased more in HFD + ACE group than those of HFD + saline group (sham). Those of brown adipocytes were increased in HFD + ACE group compared to ND and sham groups but there was no significant difference. The sizes in white adipose tissue (WAT) by microscope were markedly larger in HFD groups than ND group but considerably reduced in HFD + ACE group compared with sham group. The levels of triglyceride, total-cholesterol and leptin in blood serum were significantly decreased in HFD + ACE group compared to those of sham group. Leptin and TNF-alpha mRNA expressions in WAT of rats were remarkably increased more in sham group than in those of ND group. Those of HFD + ACE group were significantly decreased compared with those of sham group, especially. TNF-alpha mRNA expression in HFD + ACE group was declined more than that of ND group. In conclusion, treatments of ACE modulated expressions of leptin and TNF-alpha and reduced body weights, food intake, fat size, adipose tissue mass and serum hyperlipidemia in obesity rat fed HFD. Accordingly, the oriental medicine extract, adlay seed crude extract, can be considered for obesity therapies controlling.
Subject(s)
Coix , Drugs, Chinese Herbal/therapeutic use , Hypolipidemic Agents/therapeutic use , Leptin/genetics , Obesity/drug therapy , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/genetics , Adipose Tissue/drug effects , Adipose Tissue/metabolism , Adipose Tissue/pathology , Administration, Oral , Animals , Coix/chemistry , Dietary Fats/administration & dosage , Disease Models, Animal , Leptin/metabolism , Lipids/blood , Male , Obesity/metabolism , Plant Extracts/administration & dosage , Plant Extracts/therapeutic use , Rats , Rats, Sprague-Dawley , Seeds/chemistry , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Methionine sulfoxide reductase (MsrA) catalyzes the reduction of methionine sulfoxide to methionine, which is able to scavenge oxidatively damaged proteins. Oxidative stress has been linked to the pathophysiology of Alzheimer's disease, and a decrease in MsrA activity has also been implicated in Alzheimer's disease. The transactivator of transcription (TAT) protein from human immunodeficiency virus 1 has been used to deliver full-length proteins into mammalian cells. We produced genetic in-frame TAT-MsrA fusion protein and successfully transduced it into PC12 cells, where it showed enzymatic activity. We showed that transduction of TAT-MsrA increased cell viability and reduced DNA fragmentation in PC12 cells treated with amyloid-beta (A beta). We suggest that MsrA transduction could reduce the oxidative damage caused to cellular proteins by A beta and could play a role in the treatment of Alzheimer's disease.
Subject(s)
Amyloid beta-Peptides/pharmacology , Gene Products, tat/genetics , Oxidoreductases/genetics , Peptide Fragments/pharmacology , Transduction, Genetic/methods , Amyloid beta-Peptides/physiology , Animals , Cell Survival/genetics , DNA Fragmentation/genetics , Humans , Methionine Sulfoxide Reductases , PC12 Cells , Peptide Fragments/physiology , RatsABSTRACT
Transforming growth factor (TGF)-beta1 is a key regulator of brain response to injury and inflammation. It exerts anti-inflammatory roles by inhibiting microglial proliferation and free radical induction. TGF-beta1 is known to induce apoptotic cell death of microglia in a Bcl-2-independent pathway. The purpose of this study was to examine detailed mechanisms of TGF-beta1-induced microglial apoptosis. Assays for cell viability and DNA fragmentation demonstrated that TGF-beta1 induced apoptotic cell death in primary rat microglial cultures. Reverse transcription (RT)-PCR analysis showed that primary microglial cells expressed mRNAs for rat inhibitor-of-apoptosis protein (RIAP)-1 and RIAP-3 under normal culture conditions and that treatment with TGF-beta1 resulted in a significant reduction in the amounts of RIAP-1 and RIAP-3 mRNAs. Because IAPs are potent suppressor of apoptotic cell death, decrease in IAP expression might provide an important regulatory function in TGF-beta1-mediated microglial death and in attenuation of excessive microglial activation in pathological conditions.
Subject(s)
Apoptosis , Microglia/metabolism , Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Transforming Growth Factor beta/metabolism , Animals , Baculoviral IAP Repeat-Containing 3 Protein , Cell Culture Techniques , DNA Fragmentation , Down-Regulation , Inhibitor of Apoptosis Proteins , Rats , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta1ABSTRACT
Sustained stress induces neuronal atrophy and death, especially in the hippocampus, which impairs hippocampal function. However, underlying mechanisms of stress-induced neuronal damage have not been precisely defined. We analyzed the molecular events related to apoptosis in the hippocampus of rats exposed to immobilization stress. Terminal dUTP nick end-labeling exhibited positive nuclei in the hippocampus of stressed rats, indicating DNA fragmentation. RT-PCR and Western blot analyses showed that immobilization stress increased and decreased the expression of pro-apoptotic gene bax and anti-apoptotic bcl-2 genes, respectively. Western blot analysis demonstrated that the characteristic 85 kDa apoptotic fragment of poly(ADP-ribose) polymerase (PARP) was not observed in the hippocampus subjected to immobilization stress. The amount of PARP protein was significantly reduced following stress. This study may provide a novel insight into molecular mechanisms implicated in hippocampal damage associated with stress.