2,482 research outputs found
O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease.
Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation
Structure-based stabilization of insulin as a therapeutic protein assembly via enhanced aromatic-aromatic interactions
Key contributions to protein structure and stability are provided by weakly polar interactions, which arise from asymmetric electronic distributions within amino acids and peptide bonds. Of particular interest are aromatic side chains whose directional π-systems commonly stabilize protein interiors and interfaces. Here, we consider aromatic-aromatic interactions within a model protein assembly: the dimer interface of insulin. Semi-classical simulations of aromatic-aromatic interactions at this interface suggested that substitution of residue TyrB26 by Trp would preserve native structure while enhancing dimerization (and hence hexamer stability). The crystal structure of a [TrpB26]insulin analog (determined as a T3Rf3 zinc hexamer at a resolution of 2.25 Å) was observed to be essentially identical to that of WT insulin. Remarkably and yet in general accordance with theoretical expectations, spectroscopic studies demonstrated a 150-fold increase in the in vitro lifetime of the variant hexamer, a critical pharmacokinetic parameter influencing design of long-acting formulations. Functional studies in diabetic rats indeed revealed prolonged action following subcutaneous injection. The potency of the TrpB26-modified analog was equal to or greater than an unmodified control. Thus, exploiting a general quantum-chemical feature of protein structure and stability, our results exemplify a mechanism-based approach to the optimization of a therapeutic protein assembly
Diversity through semisynthesis: the chemistry and biological activity of semisynthetic epothilone derivatives
Epothilones are myxobacterial natural products that inhibit human cancer cell growth through the stabilization of cellular microtubules (i.e., a "taxol-like” mechanism of action). They have proven to be highly productive lead structures for anticancer drug discovery, with at least seven epothilone-type agents having entered clinical trials in humans over the last several years. SAR studies on epothilones have included a large number of fully synthetic analogs and semisynthetic derivatives. Previous reviews on the chemistry and biology of epothilones have mostly focused on analogs that were obtained by de novo chemical synthesis. In contrast, the current review provides a comprehensive overview on the chemical transformations that have been investigated for the major epothilones A and B as starting materials, and it discusses the biological activity of the resulting products. Many semisynthetic epothilone derivatives have been found to exhibit potent effects on human cancer cell growth and several of these have been advanced to the stage of clinical development. This includes the epothilone B lactam ixabepilone (Ixempra®, which has been approved by the FDA for the treatment of advanced and metastatic breast cance
Semisynthesis of Taxol (R): an improved procedure far the isolation of 10-deacetylbaccatin III
From the needles of domestic yew, (Taxus baccata), 10-deacetylbaccatin III (10-DAB) call be isolated in quantities of up to 297 mg per kg of fresh needles. Additional quantities of 10-DAB call be obtained from the extract by NaBH4 mediated reductive hydrolysis of baccatin esters. A four-step procedure converts 10-DAB into taxol in 58% overall yield
Bioengineering and Semisynthesis of an Optimized Cyclophilin Inhibitor for Treatment of Chronic Viral Infection.
Inhibition of host-encoded targets, such as the cyclophilins, provides an opportunity to generate potent high barrier to resistance antivirals for the treatment of a broad range of viral diseases. However, many host-targeted agents are natural products, which can be difficult to optimize using synthetic chemistry alone. We describe the orthogonal combination of bioengineering and semisynthetic chemistry to optimize the drug-like properties of sanglifehrin A, a known cyclophilin inhibitor of mixed nonribosomal peptide/polyketide origin, to generate the drug candidate NVP018 (formerly BC556). NVP018 is a potent inhibitor of hepatitis B virus, hepatitis C virus (HCV), and HIV-1 replication, shows minimal inhibition of major drug transporters, and has a high barrier to generation of both HCV and HIV-1 resistance
Mechanistic Insights into the Stimulation of Dot1L-Mediated Methylation of Histone H3 by Semisynthetically Ubiquitylated Histone H2b
Post-translational modification of histones plays an integral role in regulation of chromatin-templated processes through modulation of chromatin structure and function. One such modification, ubiquitylation of histone H2B on lysine 120 (uH2B), has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 by K79-specific methyltransferase, disruptor of telomeric silencing-like (Dot1L/KMT4). However, the specific function of uH2B in this crosstalk pathway was not understood, in part due to the challenges associated with isolating or generating homogeneously ubiquitylated H2B for use in biochemical studies. As both modifications are integral to transcriptional regulation and DNA damage repair, full elucidation of their functions is critical to understanding their roles in development and disease. In this thesis, a chemical strategy is presented for the preparation of native uH2B. Two traceless orthogonal expressed protein ligation (EPL) reactions were used for this purpose, one employing a photolytically removable ligation auxiliary, and the other, a cysteine-mediated ligation followed by a desulfurization to restore the native sequence. Reconstitution of semisynthetic uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed a direct role for uH2B in the stimulation of Dot1L-mediated methylation of H3K79. Although recruitment of Dot1L to the nucleosomal surface by uH2B could be excluded, comprehensive mechanistic analysis was precluded by systematic limitations in the ability to generate native uH2B in large-scale. To overcome this shortcoming, a highly optimized synthesis of ubiquitylated H2B bearing a Gly76Ala point mutation (uH2BG76A) was developed, yielding tens of milligrams of ubiquitylated protein. This mutant was indistinguishable from native uH2B by Dot1L, allowing for detailed studies of the resultant trans-histone crosstalk pathway. Kinetic and structure activity relationship analyses using uH2BG76A suggest a non-canonical role for ubiquitin in the enhancement of the chemical step of H3K79 methylation. This enhancement likely results from an allosteric change in the nucleosome and/or Dot1L following H2B ubiquitylation. Current work is aimed at further elucidation of the molecular mechanism of uH2B-mediated stimulation of Dot1L and the role of uH2B in other chromatin templated-processes
Characterization of the Central Cavity of a Potassium Channel: Helix Dipoles, Conformational Plasticity and Inhibition
Potassium channels are important for regulating the flow of potassium ions across semi-permeable cell membranes in an efficient and selective manner. Potassium channels form a conduction pore comprised of a selectivity filter responsible for the strong preference for potassium, and a water-filled central cavity that contributes to rapid conduction by lowering the energy barrier for potassium ions to cross the low dielectric membrane environment. In the high resolution structure of the potassium channel KcsA, a hydrated potassium ion was observed in the central cavity. It was proposed that some electrostatic stabilization for this potassium ion may come from the backbone of nearby α- helix C-termini, through the helix dipole effect. We studied the role of helix dipoles in KcsA using protein semisynthesis in order to modify the backbone of KcsA and reduce the dipoles of the implicated helix termini. The modified protein was studied by both X-ray crystallography and electrophysiology, demonstrating that the pore helix dipoles may play an important role in potassium conductance. In the course of these experiments, a new conformation for the KcsA cavity was discovered: a phenylalanine (Phe) from each subunit flipped into the center of the cavity, the cavity ion was no longer observed, and a new non-peptidic density extended into the cavity through lateral openings exposed by the conformational change of the Phe. Subsequent structural studies identified conditions that induce or prevent this conformational change. In particular, mutations were incorporated into KcsA that make the protein less likely to enter the alternative conformation, while not greatly affecting potassium conductance. Crystal structures of KcsA in complex with cavity blocking small molecules revealed that certain inhibitors bind to the cavity in its alternative conformation, and electrophysiology confirmed inhibition by one such molecule in the membrane environment. A sequence alignment between KcsA and several human potassium channels identified a subset of channels where this mode of cavity block may be conserved, including BK and HERG channels. Thus, this new conformation of block could have important implications for the pharmacology of human potassium channels. This work furthers our understanding of electrostatic interactions, structural plasticity, and a new mode of action for a family of inhibitors, within the cavity of KcsA. The study of helix dipoles has relevance for the function of a wide range of proteins, and characterization of conformational dependent cavity block has particular relevance to some pharmacologically relevant human potassium channels
Diversity through semisynthesis: The chemistry and biological activity of semisynthetic epothilone derivatives
ISSN:1381-1991ISSN:1573-501
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