A brain-targeting lipidated peptide for neutralizing RNA-mediated toxicity in Polyglutamine Diseases

Abstract Polyglutamine (PolyQ) diseases are progressive neurodegenerative disorders caused by both protein- and RNA-mediated toxicities. We previously showed that a peptidyl inhibitor, P3, which binds directly to expanded CAG RNA can inhibit RNA-induced nucleolar stress and suppress RNA-induced neurotoxicity. Here we report a N-acetylated and C-amidated derivative of P3, P3V8, that showed a more than 20-fold increase in its affinity for expanded CAG RNA. The P3V8 peptide also more potently alleviated expanded RNA-induced cytotoxicity in vitro, and suppressed polyQ neurodegeneration in Drosophila with no observed toxic effects. Further N-palmitoylation of P3V8 (L1P3V8) not only significantly improved its cellular uptake and stability, but also facilitated its systemic exposure and brain uptake in rats via intranasal administration. Our findings demonstrate that concomitant N-acetylation, C-amidation and palmitoylation of P3 significantly improve both its bioactivity and pharmacological profile. L1P3V8 possesses drug/lead-like properties that can be further developed into a lead inhibitor for the treatment of polyQ diseases

SR protein kinase 1 : conformation, substrate recognition and catalysis

Serine/arginines-rich (SR) proteins play important roles in constitutive and alternative pre-mRNA splicing, mRNA export and translation. The C-terminal RS domains of these proteins contain repeats of consecutive arginine-serine (RS) dipeptides, which are extensively phosphorylated and mediate protein-protein and protein-RNA interactions. One of the kinase families that phosphorylate SR proteins is the SR protein kinase family. SRPKs are unusual members of the kinase superfamily in that their kinase domains are bifurcated by spacer regions of 250 to 300 residues. Furthermore, they are constitutively active and do not require any post-translational modification or interaction with regulatory factors. In this study, I employed a combination of X-ray crystallography and biochemical techniques to investigate the molecular basis of constitutive activity, catalytic mechanism and substrate recognition of human SRPK1. An appropriate fragment of SRPK1 was designed for crystallization. The determination of the SRPK1 X-ray structure at 1.73 Å revealed that SRPK1 contains unique secondary structural elements that mimic in-trans activation mechanisms observed in different kinases. Moreover, the global network of interactions allows the activation loop to adopt a catalytically competent conformation without relying upon any direct stabilization mechanism. SRPK1 binds its SR protein substrate, ASF/SF2, stably and processively phosphorylates only half of the serines of the RS domain. The crystal structure of SRPK1 bound to a peptide and ADP has led to the identification of a docking groove in SRPK1 and a docking motif in ASF/SF2. Detailed biochemical studies of the docking interactions suggested mechanisms for the restriction in the number of phosphorylated sites and the mode of regulation of phosphorylation. Finally, the current model of the X-ray crystal structure of SRPK1 bound to AMP-PNP and a natural substrate, ASF/SF2, has revealed the structural basis for substrate recognition. Unexpectedly, ASF/SF2 engages SRPK1 at both small and large lobes upon binding. Detailed analysis of the complex structure is underway and will provide insights into the substrate specificity and the mechanism of processive phosphorylatio

Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila

Polyglutamine (polyQ) diseases represent a group of progressive neurodegenerative disorders that are caused by abnormal expansion of CAG triplet nucleotides in disease genes. Recent evidence indicates that not only mutant polyQ proteins, but also their corresponding mutant RNAs, contribute to the pathogenesis of polyQ diseases. Here, we describe the identification of a 13-amino-acid peptide, P3, which binds directly and preferentially to long-CAG RNA within the pathogenic range. When administered to cell and Drosophila disease models, as well as to patient-derived fibroblasts, P3 inhibited expanded-CAG-RNA-induced nucleolar stress and suppressed neurotoxicity. We further examined the combined therapeutic effect of P3 and polyQ-binding peptide 1 (QBP1), a well-characterized polyQ protein toxicity inhibitor, on neurodegeneration. When P3 and QBP1 were co-administered to disease models, both RNA and protein toxicities were effectively mitigated, resulting in a notable improvement of neurotoxicity suppression compared with the P3 and QBP1 single-treatment controls. Our findings indicate that targeting toxic RNAs and/or simultaneous targeting of toxic RNAs and their corresponding proteins could open up a new therapeutic strategy for treating polyQ degeneration

SRPK2 Mediates HBV Core Protein Phosphorylation and Capsid Assembly via Docking Interaction.

Members of the serine-arginine protein kinase (SRPK) family, SRPK1 and SRPK2, phosphorylate the hepatitis B core protein (Cp) and are crucial for pregenomic RNA encapsidation during viral nucleocapsid assembly. Among them, SRPK2 exhibits higher kinase activity toward Cp. In this study, we identified Cp sites that are phosphorylated by SRPK2 and demonstrated that the kinase utilizes an SRPK-specific docking groove to interact with and regulate the phosphorylation of the C-terminal arginine rich domain of Cp. We determined that direct interaction between the docking groove of SRPK2 and unphosphorylated Cp inhibited premature viral capsid assembly in vitro, whereas the phosphorylation of the viral protein reactivated the process. Pull-down assays together with the new cryo-electron microscopy structure of the HBV capsid in complex with SRPK2 revealed that the kinases decorate the surface of the viral capsid by interacting with the C-terminal domain of Cp, underscoring the importance of the docking interaction in regulating capsid assembly and pregenome packaging. Moreover, SRPK2-knockout in HepG2 cells suppressed Cp phosphorylation, indicating that SRPK2 is an important cellular kinase for HBV life cycle

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.status: publishe

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rin r), modules consisting of tandem RNA recognition motifs (RRMs; RRM1– The splicing of precursor mRNA (pre-mRNA) is family of splicing cofactors are the SR proteins doi:10.1016/j.jmb.2008.07.055 Available online at wwwessential for proteome diversity and many cellular regulatory processes, but is also associated with numerous diseases when mistakes are propagated into themature splicedmRNA.1–4 Splicing occurs in a macromolecular complex known as the spliceosome, (splicing factors containing arginine-serine repeats), so named because they contain a C-terminal domain composed largely of arginine-serine dipeptide repeats (RS domain, a domain rich in arginine-serine repeats). In addition to these repetitive sequences, SR proteins contain one or two N-terminal RNA recognition motifs (RRMs) that are essential for recognition of exonic enhancer sequences in pre-mRNA.6–8 The SR proteins play roles in both constitutive and alternative splicing.9,10 While SR proteins are critical for the earl

Phosphorylation of FE65 Ser(610) by serum- and glucocorticoid-induced kinase 1 modulates Alzheimer's disease amyloid precursor protein processing

Alzheimer's disease (AD) is a fatal neurodegenerative disease affecting 36 million people worldwide. Genetic and biochemical research indicate that the excessive generation of amyloid-β peptide (Aβ) from amyloid precursor protein (APP), is a major part of AD pathogenesis. FE65 is a brain-enriched adaptor protein that binds to APP. However, the role of FE65 in APP processing and the mechanisms that regulate binding of FE65 to APP are not fully understood. In the present study, we show that serum- and glucocorticoid-induced kinase 1 (SGK1) phosphorylates FE65 on Ser(610) and that this phosphorylation attenuates FE65 binding to APP. We also show that FE65 promotes amyloidogenic processing of APP and that FE65 Ser(610) phosphorylation inhibits this effect. Furthermore, we found that the effect of FE65 Ser(610) phosphorylation on APP processing is linked to a role of FE65 in metabolic turnover of APP via the proteasome. Thus FE65 influences APP degradation via the proteasome and phosphorylation of FE65 Ser(610) by SGK1 regulates binding of FE65 to APP, APP turnover and processing

Brain-Targeting Delivery of Two Peptidylic Inhibitors for Their Combination Therapy in Transgenic Polyglutamine Disease Mice via Intranasal Administration

Polyglutamine diseases are a set of progressive neurodegenerative disorders caused by misfolding and aggregation of mutant CAG RNA and polyglutamin protein. To date, there is a lack of effective therapeutics that can counteract the polyglutamine neurotoxicity. Two peptidylic inhibitors, QBP1 and P3, targeting the protein and RNA toxicities, respectively, have been previously demonstrated by us with combinational therapeutic effects on the Drosophila polyglutamine disease model. However, their therapeutic efficacy has never been investigated in vivo in mammals. The current study aims to (a) develop a brain-targeting delivery system for both QBP1 and L1P3V8 (a lipidated variant of P3 with improved stability) and (b) evaluate their therapeutic effects on the R6/2 transgenic mouse model of polyglutamine disease. Compared with intravenous administration, intranasal administration of QBP1 significantly increased its brain-to-plasma ratio. In addition, employment of a chitosan-containing in situ gel for the intranasal administration of QBP1 notably improved its brain concentration for up to 10-fold. Further study on intranasal cotreatment with the optimized formulation of QBP1 and L1P3V8 in mice found no interference on the brain uptake of each other. Subsequent efficacy evaluation of 4-week daily QBP1 (16 μmol/kg) and L1P3V8 (6 μmol/kg) intranasal cotreatment in the R6/2 mice demonstrated a significant improvement on the motor coordination and explorative behavior of the disease mice, together with a full suppression on the RNA- and protein-toxicity markers in their brains. In summary, the current study developed an efficient intranasal cotreatment of the two peptidylic inhibitors, QBP1 and L1P3V8, for their brain-targeting, and such a novel therapeutic strategy was found to be effective on a transgenic polyglutamine disease mouse model