Detergent-free extraction of a functional low-expressing GPCR from a human cell line.

Dopamine receptors (DRs) are class A G-Protein Coupled Receptors (GPCRs) prevalent in the central nervous system (CNS). These receptors mediate physiological functions ranging from voluntary movement and reward recognition to hormonal regulation and hypertension. Drugs targeting dopaminergic neurotransmission have been employed to treat several neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, Huntington's disease, attention deficit hyperactivity disorder (ADHD), and Tourette's syndrome. In vivo, incorporation of GPCRs into lipid membranes is known to be key to their biological function and, by inference, maintenance of their tertiary structure. A further significant challenge in the structural and biochemical characterization of human DRs is their low levels of expression in mammalian cells. Thus, the purification and enrichment of DRs whilst retaining their structural integrity and function is highly desirable for biophysical studies. A promising new approach is the use of styrene-maleic acid (SMA) copolymer to solubilize GPCRs directly in their native environment, to produce polymer-assembled Lipodisqs (LQs). We have developed a novel methodology to yield detergent-free D1-containing Lipodisqs directly from HEK293f cells expressing wild-type human dopamine receptor 1 (D1). We demonstrate that D1 in the Lipodisq retains activity comparable to that in the native environment and report, for the first time, the affinity constant for the interaction of the peptide neurotransmitter neurotensin (NT) with D1, in the native state.

Engineering synucleinopathy-resistant human dopaminergic neurons by CRISPR-mediated deletion of the SNCA gene.

An emerging treatment for Parkinson's disease (PD) is cell replacement therapy. Authentic midbrain dopaminergic (mDA) neuronal precursors can be differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These laboratory-generated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD. However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting host-to-graft transfer of α-synuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for α-synuclein, in a clinical-grade hESC line to generate SNCA+/- and SNCA-/- cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant α-synuclein preformed fibrils (PFFs) to seed the formation for Lewy-like pathology as measured by phosphorylation of serine-129 of α-synuclein (pS129-αSyn). Wild-type neurons were fully susceptible to the formation of protein aggregates positive for pS129-αSyn, while SNCA+/- and SNCA-/- neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9n-mediated gene editing confers a measure of resistance to Lewy pathology.

A mixture of functionally oligoclonal humanized monoclonal antibodies that neutralize Clostridium difficile TcdA and TcdB with high levels of in vitro potency shows in vivo protection in a hamster infection model.

Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.

Product inhibition in the radical S-adenosylmethionine family.

Members of the radical S-adenosylmethionine (AdoMet) superfamily reductively cleave AdoMet to generate the highly reactive 5'-deoxyadenosyl radical (DOA()) which initiates biological transformations by abstraction of a hydrogen atom. We demonstrate that three members of the family: biotin synthase (BioB), lipoyl synthase (LipA) and tyrosine lyase (ThiH) are inhibited in vitro by a combination of the products 5'-deoxyadenosine (DOA) and methionine. These results suggest the observed inhibition is a common feature of the radical AdoMet proteins that form DOA and methionine as products. Addition of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) to BioB, LipA or ThiH activity assays removed the product inhibition by catalysing the hydrolysis of DOA and gave an increase in activity.

Thiazole synthase from Escherichia coli: an investigation of the substrates and purified proteins required for activity in vitro.

Thiamine is biosynthesized by combining two heterocyclic precursors. In Escherichia coli and other anaerobes, one of the heterocycles, 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate, is biosynthesized from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine. Genetic evidence has identified thiH, thiG, thiS, and thiF as essential for thiazole biosynthesis in E. coli. In this paper, we describe the measurement of the thiazole phosphate-forming reaction using purified protein components. The activity is shown to require four proteins isolated as heterodimers: ThiGH and ThiFS. Reconstitution of the [4Fe-4S] cluster in ThiH was essential for activity, as was the use of ThiS in the thiocarboxylate form. Spectroscopic studies with ThiGH strongly suggested that S-adenosylmethionine (AdoMet) bound to the [4Fe-4S] cluster, which became more susceptible to reduction to the +1 state. Assays of thiazole phosphate formation showed that, in addition to the proteins, Dxp, tyrosine, AdoMet, and a reductant were required. The analysis showed that no more than 1 mol eq of thiazole phosphate was formed per ThiGH. Furthermore, for each mole of thiazole-P formed, 1 eq of AdoMet and 1 eq of tyrosine were utilized, and 1 eq of 5'-deoxyadenosine was produced. These results demonstrate that ThiH is a member of the "radical-AdoMet" family and support a mechanistic hypothesis in which AdoMet is reductively cleaved to yield a highly reactive 5'-deoxyadenosyl radical. This radical is proposed to abstract the phenolic hydrogen atom from tyrosine, and the resultant substrate radical cleaves to yield dehydroglycine, which is required by ThiG for the thiazole cyclization reaction.

The activity of a thermostable lipoyl synthase from Sulfolobus solfataricus with a synthetic octanoyl substrate.

The protein lipoyl synthase (LipA) is essential for lipoic acid biosynthesis via sulfur insertions into a protein-bound octanoyl group. We have developed an in vitro assay for LipA using a synthetic tetrapeptide substrate, containing an N(epsilon)-octanoyl lysine residue, corresponding in sequence to the lipoyl binding domain of the E2 subunit of pyruvate dehydrogenase. A putative LipA from the hypothermophilic archaea Sulfolobus solfataricus was expressed in Escherichia coli and purified, and the activity was measured using this novel assay. The optimal temperature for the S. solfataricus LipA-dependent formation of the lipoyl group was found to be 60 degrees C.

Optimized conjugation of a fluorescent label to proteins via intein-mediated activation and ligation.

Intein-mediated ligation provides a site-specific method for the attachment of molecular probes to proteins. The method is inherently flexible with regard to either the protein sequence or the attached probe, but practical difficulties have limited the widespread use of this valuable labeling system for the attachment of small- to medium-sized molecules. We report herein studies to improve the efficiency and practical application of these reactions, including the assembly of plasmids for the expression of target-intein fusion proteins and the analysis of their reaction with a fluorescent cysteine derivative under a range of conditions. Optimal ligation of the fluorophore to the target protein is critically dependent on the degree of oxidation of the fluorescent cysteine derivative. Efficient ligation has been achieved with freshly prepared fluorescent cysteine derivative under rigorously anaerobic conditions. Similar ligation yields have also been achieved using more practically convenient conditions including anaerobic reaction with addition of thiophenol, or aerobic reaction with the further addition of tricarboxyethylphosphine.

Effect of iron-sulfur cluster assembly proteins on the expression of Escherichia coli lipoic acid synthase.

Lipoic Acid Synthase (LipA) can accommodate a [4Fe-4S] cluster that is thought to be essential for the insertion of sulfur into an octanoyl substrate during the biosynthesis of lipoic acid. With the objective of improving soluble holo-LipA expression, a series of multi-cistronic plasmids were constructed carrying lipA in combination with one of the three systems: groE/SL, trxA, or the isc operon. Co-expression of lipA with the isc operon approximately trebled the isolated yield of soluble LipA and resulted in efficient assembly of the Fe-S cluster. This strategy may be helpful in the soluble expression of a wide range of Fe-S cluster-dependent proteins.

Thiamine biosynthesis in Escherichia coli: isolation and initial characterisation of the ThiGH complex.

In Escherichia coli, two of the proteins required for the biosynthesis of the thiazole moiety of thiamine (vitamin B(1)) are ThiG and ThiH, encoded as part of the thiCEFSGH operon. In this study, a C-terminally hexahistidine-tagged ThiH (ThiH-His) was expressed in E. coli as a soluble protein from thiGH-His-tag and thiFSGH-His-tag-bearing plasmids. When isolated under anaerobic conditions, ThiG and ThiH-His co-purify as a large multimeric non-covalent complex. Electron paramagnetic resonance and UV-visible spectroscopy together with iron and sulfide analyses revealed the presence of an iron-sulfur cluster within this complex.