G6PC mRNA Therapy Positively Regulates Fasting Blood Glucose and Decreases Liver Abnormalities in a Mouse Model of Glycogen Storage Disease 1a.

Glycogen storage disease type Ia (GSD1a) is an inherited metabolic disorder caused by the deficiency of glucose-6-phosphatase (G6Pase). GSD1a is associated with life-threatening hypoglycemia and long-term liver and renal complications. We examined the efficacy of mRNA-encoding human G6Pase in a liver-specific G6Pase-/- mouse model (L-G6PC-/-) that exhibits the same hepatic biomarkers associated with GSD1a patients, such as fasting hypoglycemia, and elevated levels of hepatic glucose-6-phosphate (G6P), glycogen, and triglycerides. We show that a single systemic injection of wild-type or native human G6PC mRNA results in significant improvements in fasting blood glucose levels for up to 7 days post-dose. These changes were associated with significant reductions in liver mass, hepatic G6P, glycogen, and triglycerides. In addition, an engineered protein variant of human G6Pase, designed for increased duration of expression, showed superior efficacy to the wild-type sequence by maintaining improved fasting blood glucose levels and reductions in liver mass for up to 12 days post-dose. Our results demonstrate for the first time the effectiveness of mRNA therapy as a potential treatment in reversing the hepatic abnormalities associated with GSD1a.

Optimization of mRNA untranslated regions for improved expression of therapeutic mRNA.

mRNA based therapies hold great promise for the treatment of genetic diseases. However, this therapeutic approach suffers from multiple challenges including the short half-life of exogenously administered mRNA and subsequent protein production. Modulation of untranslated regions (UTR) represents one approach to enhance both mRNA stability and translation efficiency. The current studies describe and validate screening methods using a diverse set of 5'UTR and 3'UTR combinations for improved expression of the Arginase 1 (ARG1) protein, a potential therapeutic mRNA target. Data revealed a number of critical aspects which need to be considered when developing a screening approach for engineering mRNA improvements. First, plasmid-based screening methods do not correlate with protein expression driven by exogenously expressed mRNA. Second, improved ARG1 protein production was driven by increased translation and not improved mRNA stability. Finally, the 5' UTR appears to be the key driver in protein expression for exogenously delivered mRNA. From the testing of the combinatorial library, the 5'UTR for complement factor 3 (C3) and cytochrome p4502E1 (CYP2E1) showed the largest and most consistent increase in protein expression relative to a reference UTR. Collectively, these data provide important information for the development and optimization of therapeutic mRNAs.

Panoramic view of a superfamily of phosphatases through substrate profiling.

Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified iso-functional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.

Structure of the trehalose-6-phosphate phosphatase from Brugia malayi reveals key design principles for anthelmintic drugs.

Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible.

Covalent docking predicts substrates for haloalkanoate dehalogenase superfamily phosphatases.

Enzyme function prediction remains an important open problem. Though structure-based modeling, such as metabolite docking, can identify substrates of some enzymes, it is ill-suited to reactions that progress through a covalent intermediate. Here we investigated the ability of covalent docking to identify substrates that pass through such a covalent intermediate, focusing particularly on the haloalkanoate dehalogenase superfamily. In retrospective assessments, covalent docking recapitulated substrate binding modes of known cocrystal structures and identified experimental substrates from a set of putative phosphorylated metabolites. In comparison, noncovalent docking of high-energy intermediates yielded nonproductive poses. In prospective predictions against seven enzymes, a substrate was identified for five. For one of those cases, a covalent docking prediction, confirmed by empirical screening, and combined with genomic context analysis, suggested the identity of the enzyme that catalyzes the orphan phosphatase reaction in the riboflavin biosynthetic pathway of Bacteroides.

Enzyme promiscuity: engine of evolutionary innovation.

Catalytic promiscuity and substrate ambiguity are keys to evolvability, which in turn is pivotal to the successful acquisition of novel biological functions. Action on multiple substrates (substrate ambiguity) can be harnessed for performance of functions in the cell that supersede catalysis of a single metabolite. These functions include proofreading, scavenging of nutrients, removal of antimetabolites, balancing of metabolite pools, and establishing system redundancy. In this review, we present examples of enzymes that perform these cellular roles by leveraging substrate ambiguity and then present the structural features that support both specificity and ambiguity. We focus on the phosphatases of the haloalkanoate dehalogenase superfamily and the thioesterases of the hotdog fold superfamily.

Substrate deconstruction and the nonadditivity of enzyme recognition.

Predicting substrates for enzymes of unknown function is a major postgenomic challenge. Substrate discovery, like inhibitor discovery, is constrained by our ability to explore chemotypes; it would be expanded by orders of magnitude if reactive sites could be probed with fragments rather than fully elaborated substrates, as is done for inhibitor discovery. To explore the feasibility of this approach, substrates of six enzymes from three different superfamilies were deconstructed into 41 overlapping fragments that were tested for activity or binding. Surprisingly, even those fragments containing the key reactive group had little activity, and most fragments did not bind measurably, until they captured most of the substrate features. Removing a single atom from a recognized substrate could often reduce catalytic recognition by 6 log-orders. To explore recognition at atomic resolution, the structures of three fragment complexes of the ß-lactamase substrate cephalothin were determined by X-ray crystallography. Substrate discovery may be difficult to reduce to the fragment level, with implications for function discovery and for the tolerance of enzymes to metabolite promiscuity. Pragmatically, this study supports the development of libraries of fully elaborated metabolites as probes for enzyme function, which currently do not exist.

Targeted transcriptional downregulation of MYC using epigenomic controllers demonstrates antitumor activity in hepatocellular carcinoma models.

Dysregulation of master regulator c-MYC (MYC) plays a central role in hepatocellular carcinoma (HCC) and other cancers but remains an elusive target for therapeutic intervention. MYC expression is epigenetically modulated within naturally occurring DNA loop structures, Insulated Genomic Domains (IGDs). We present a therapeutic approach using an epigenomic controller (EC), a programmable epigenomic mRNA medicine, to precisely modify MYC IGD sub-elements, leading to methylation of MYC regulatory elements and durable downregulation of MYC mRNA transcription. Significant antitumor activity is observed in preclinical models of HCC treated with the MYC-targeted EC, as monotherapy or in combination with tyrosine kinase or immune checkpoint inhibitors. These findings pave the way for clinical development of MYC-targeting epigenomic controllers in HCC patients and provide a framework for programmable epigenomic mRNA therapeutics for cancer and other diseases.

Divergence of structure and function in the haloacid dehalogenase enzyme superfamily: Bacteroides thetaiotaomicron BT2127 is an inorganic pyrophosphatase.

The explosion of protein sequence information requires that current strategies for function assignment evolve to complement experimental approaches with computationally based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown haloalkanoate dehalogenase superfamily member BT2127 (Uniprot accession code Q8A5 V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics-structure-mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with a k(cat)/K(m) value for pyrophosphate of ~1 × 10(5) M(-1) s(-1)), together with the gene context, support the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure-guided site-directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domain-domain association. On the basis of this structure-function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172, and Glu47 were used as markers to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biological range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in the divergence of BT2127 and its closest homologue, ß-phosphoglucomutase, control the leaving group size (phosphate vs glucose phosphate) and the position of the Asp acid/base in the open versus closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases.

Structure and function of interacting IcmR-IcmQ domains from a type IVb secretion system in Legionella pneumophila.

During infection, Legionella pneumophila creates a replication vacuole within eukaryotic cells and this requires a Type IVb secretion system (T4bSS). IcmQ plays a critical role in the translocase and associates with IcmR. In this paper, we show that the N-terminal domain of IcmQ (Qn) mediates self-dimerization, whereas the C-terminal domain with a basic linker promotes membrane association. In addition, the binding of IcmR to IcmQ prevents self-dimerization and also blocks membrane permeabilization. However, IcmR does not completely block membrane binding by IcmQ. We then determined crystal structures of Qn with the interacting region of IcmR. In this complex, each protein forms an alpha-helical hairpin within a parallel four-helix bundle. The amphipathic nature of helices in Qn suggests two possible models for membrane permeabilization by IcmQ. The Rm-Qn structure also suggests how IcmR-like proteins in other L. pneumophila species may interact with their IcmQ partners.

Dissociation between serotonin neurotoxicity and brain-derived neurotrophic factor induction following neonatal MDMA exposure in rats.

Early developmental treatment of rats with 3,4-methylenedioxymethamphetamine (MDMA) was previously found to cause an abnormal pattern of forebrain serotonergic axon density in adulthood consisting of a cortical hypoinnervation and a striatal hyperinnervation. The present study tested the hypothesis that this reorganization was due to regional differences in brain-derived neurotrophic factor (BDNF) expression. Rats received MDMA (10 mg/kg, s.c., b.i.d.) on postnatal days (PD) 1-4, after which brain tissues were collected on PD 11, 30, and 67 for analysis. BDNF protein levels were found to be elevated in the occipital cortex but not in the hippocampus or striatum following MDMA administration. Serotonin transporter binding (an index of serotonergic fiber integrity) was significantly reduced in the hippocampus at PD 11 but returned to normal by PD 30, whereas the cortex exhibited a delayed reduction that was not manifested until PD 30. These results do not support the view that a region-specific enhancement in BDNF expression mediates the abnormal serotonergic reinnervation observed following neonatal MDMA exposure.

Leveraging Rational Protein Engineering to Improve mRNA Therapeutics.

Messenger RNA (mRNA) is a promising new class of therapeutics that has potential for treatment of diseases in fields such as immunology, oncology, vaccines, and inborn errors of metabolism. mRNA therapy has several advantages over DNA-based gene therapy, including the lack of the need for nuclear import and transcription, as well as limited possibility of genomic integration. One drawback of mRNA therapy, especially in cases such as metabolic disorders where repeated dosing will be necessary, is the relatively short in vivo half-life of mRNA (∼6-12 h). We hypothesize that protein engineering designed to improve translation, yielding longer-lasting protein, or modifications that would increase enzymatic activity would be helpful in alleviating this issue. In this study, we present two examples where sequence engineering improved the expression and duration, as well as enzymatic activity of target proteins in vitro. We then confirmed these findings in wild-type mice. This work shows that rational engineering of proteins can lead to improved therapies in vivo.

Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.

A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.

IcmQ in the Type 4b secretion system contains an NAD+ binding domain.

A Type 4b secretion system (T4bSS) is required for Legionella growth in alveolar macrophages. IcmQ associates with IcmR, binds to membranes, and has a critical role in the T4bSS. We have now solved a crystal structure of IcmR-IcmQ to further our understanding of this complex. This structure revealed an amphipathic four-helix bundle, formed by IcmR and the N-terminal domain of IcmQ, which is linked to a novel C-terminal domain of IcmQ (Qc) by a linker helix. The Qc domain has structural homology with ADP ribosyltransferase domains in certain bacterial toxins and binds NAD(+) with a dissociation constant in the physiological range. Structural homology and molecular dynamics were used to identify an extended NAD(+) binding site on Qc, and the resulting model was tested by mutagenesis and binding assays. Based on the data, we suggest that IcmR-IcmQ binds to membranes, where it may interact with, or perhaps modify, a protein in the T4bSS when NAD(+) is bound.

On the unyielding hydrophobic core of villin headpiece.

Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system for understanding the fundamental tenets governing protein folding. In this communication, we explore the role that Leu61 plays in the structure and stability of the construct. Deletion of Leu61 results in a completely unfolded protein that cannot be expressed in Escherichia coli. Omission of only the aliphatic leucine side chain (HP67 L61G) perturbed neither the backbone conformation nor the orientation of local hydrophobic side chains. As a result, a large, solvent-exposed hydrophobic pocket, a negative replica of the leucine side-chain, was created on the surface. The loss of the hydrophobic interface between leucine 61 and the hydrophobic pocket destabilized the construct by ~3.3 kcal/mol. Insertion of a single glycine residue immediately before Leu61 (HP67 L61[GL]) was also highly destabilizing and had the effect of altering the backbone conformation (α-helix to π-helix) in order to precisely preserve the wild-type position and conformation of all hydrophobic residues, including Leu61. In addition to demonstrating that the hydrophobic side-chain of Leu61 is critically important for the stability of villin headpiece, our results are consistent with the notion that the precise interactions present within the hydrophobic core, rather than the hydrogen bonds that define the secondary structure, specify a protein's fold.

On unsatisfied hydrogen bonds in the N-terminal subdomain of villin headpiece.

Villin headpiece is a small autonomously folding protein that has emerged as a model system for understanding the fundamental tenets governing protein folding. In this communication, we employ NMR and X-ray crystallography to characterize a point mutant, H41F, which retains actin-binding activity, is more thermostable but, interestingly, does not exhibit the partially folded intermediate observed of either wild-type or other similar point mutants.