Quantitation of AAV in a dual-vector system using SV-AUC.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has become the "gold standard" for characterization of the empty, partial, and full capsids of gene therapy products (e.g., AAV and Adenovirus vectors).

BASIS: BioAnalysis SEDFIT integrated software for cGMP analysis of SV-AUC data.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has long been an important method for characterization of antibody therapeutics. Recently, SV-AUC has experienced a wave of new interest and usage from the gene and cell therapy industry, where SV-AUC has proven itself to be the "gold standard" analytical approach for determining capsid loading ratios for adeno-associated virus (AAV) and other viral vectors. While other more common approaches have existed in the realm of cGMP-compliant techniques for years, SV-AUC has long been used strictly for characterization, but not for release testing.

AAV analysis by sedimentation velocity analytical ultracentrifugation: beyond empty and full capsids.

The recent surge of therapeutic interest in recombinant adeno-associated viral (AAV) vectors for targeted DNA delivery has brought analytical ultracentrifugation (AUC) into the spotlight. A major concern during formulation of AAV therapeutics is purity of the active species (DNA-containing capsid, or "filled capsids"). Insertion of DNA into AAV is not a highly efficient process; thus, a significant amount of empty and partial/intermediate AAV molecules may exist.

Relationships Between Plasminogen-Binding M-Protein and Surface Enolase for Human Plasminogen Acquisition and Activation in .

The proteolytic activity of human plasmin (hPm) is utilized by various cells to provide a surface protease that increases the potential of cells to migrate and disseminate. Skin-trophic Pattern D strains of (GAS), e.g.

Visualizing the enzyme mechanism of mevalonate diphosphate decarboxylase.

Mevalonate diphosphate decarboxylases (MDDs) catalyze the ATP-dependent-Mg-decarboxylation of mevalonate-5-diphosphate (MVAPP) to produce isopentenyl diphosphate (IPP), which is essential in both eukaryotes and prokaryotes for polyisoprenoid synthesis. The substrates, MVAPP and ATP, have been shown to bind sequentially to MDD. Here we report crystals in which the enzyme remains active, allowing the visualization of conformational changes in Enterococcus faecalis MDD that describe sequential steps in an induced fit enzymatic reaction.

Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen .

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from (MDD).

Wolbachia Endosymbionts Modify Drosophila Ovary Protein Levels in a Context-Dependent Manner.

Unlabelled: Endosymbiosis is a unique form of interaction between organisms, with one organism dwelling inside the other. One of the most widespread endosymbionts is Wolbachia pipientis, a maternally transmitted bacterium carried by insects, crustaceans, mites, and filarial nematodes. Although candidate proteins that contribute to maternal transmission have been identified, the molecular basis for maternal Wolbachia transmission remains largely unknown.

Antibacterial activity and mechanism of action of auranofin against multi-drug resistant bacterial pathogens.

Traditional methods employed to discover new antibiotics are both a time-consuming and financially-taxing venture. This has led researchers to mine existing libraries of clinical molecules in order to repurpose old drugs for new applications (as antimicrobials). Such an effort led to the discovery of auranofin, a drug initially approved as an anti-rheumatic agent, which also possesses potent antibacterial activity in a clinically achievable range.

Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination.

Manipulation of the host's ubiquitin network is emerging as an important strategy for counteracting and repurposing the posttranslational modification machineries of the host by pathogens. Ubiquitin E3 ligases encoded by infectious agents are well known, as are a variety of viral deubiquitinases (DUBs). Bacterial DUBs have been discovered, but little is known about the structure and mechanism underlying their ubiquitin recognition.

Exploring simvastatin, an antihyperlipidemic drug, as a potential topical antibacterial agent.

The rapid rise of bacterial resistance to traditional antibiotics combined with the decline in discovery of novel antibacterial agents has created a global public health crisis. Repurposing existing drugs presents an alternative strategy to potentially expedite the discovery of new antimicrobial drugs. The present study demonstrates that simvastatin, an antihyperlipidemic drug exhibited broad-spectrum antibacterial activity against important Gram-positive (including methicillin-resistant Staphylococcus aureus (MRSA)) and Gram-negative pathogens (once the barrier imposed by the outer membrane was permeabilized).

Dynamics of an Active-Site Flap Contributes to Catalysis in a JAMM Family Metallo Deubiquitinase.

The endosome-associated deubiquitinase (DUB) AMSH is a member of the JAMM family of zinc-dependent metallo isopeptidases with high selectivity for Lys63-linked polyubiquitin chains, which play a key role in endosomal-lysosomal sorting of activated cell surface receptors. The catalytic domain of the enzyme features a flexible flap near the active site that opens and closes during its catalytic cycle. Structural analysis of its homologues, AMSH-LP (AMSH-like protein) and the fission yeast counterpart, Sst2, suggests that a conserved Phe residue in the flap may be critical for substrate binding and/or catalysis.

Digestion, Purification, and Enrichment of Protein Samples for Mass Spectrometry.

Proteomic studies rely heavily on the use of liquid chromatography (LC)-mass spectrometry (MS and MS/MS) analyses to provide information about protein composition and function. Profiling the proteome can be the first step to understanding biological pathways, but the challenges scientists face with the complex nature of proteins and proteolysis products can be daunting. Techniques involving fractionation, immunoprecipitation, and phosphopeptide enrichment can simplify complex protein mixtures and enhance the amount of target proteins that are important to the investigator.

Hsp31 Is a Stress Response Chaperone That Intervenes in the Protein Misfolding Process.

The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog.

AMP-Activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma.

The Hedgehog (Hh) pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP-activated protein kinase (AMPK) is an important sensor of energy stores and controls protein synthesis and other energy-intensive processes.

Ligand-induced Dimerization of Middle East Respiratory Syndrome (MERS) Coronavirus nsp5 Protease (3CLpro): IMPLICATIONS FOR nsp5 REGULATION AND THE DEVELOPMENT OF ANTIVIRALS.

All coronaviruses, including the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) from the β-CoV subgroup, require the proteolytic activity of the nsp5 protease (also known as 3C-like protease, 3CL(pro)) during virus replication, making it a high value target for the development of anti-coronavirus therapeutics. Kinetic studies indicate that in contrast to 3CL(pro) from other β-CoV 2c members, including HKU4 and HKU5, MERS-CoV 3CL(pro) is less efficient at processing a peptide substrate due to MERS-CoV 3CL(pro) being a weakly associated dimer. Conversely, HKU4, HKU5, and SARS-CoV 3CL(pro) enzymes are tightly associated dimers.

Characterization of the proteome of cytoplasmic lipid droplets in mouse enterocytes after a dietary fat challenge.

Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown.

A conserved acidic residue in phenylalanine hydroxylase contributes to cofactor affinity and catalysis.

The catalytic domains of aromatic amino acid hydroxylases (AAAHs) contain a non-heme iron coordinated to a 2-His-1-carboxylate facial triad and two water molecules. Asp139 from Chromobacterium violaceum PAH (cPAH) resides within the second coordination sphere and contributes key hydrogen bonds with three active site waters that mediate its interaction with an oxidized form of the cofactor, 7,8-dihydro-l-biopterin, in crystal structures. To determine the catalytic role of this residue, various point mutants were prepared and characterized.

The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers.

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.

Perturbation of the monomer-monomer interfaces of the benzoylformate decarboxylase tetramer.

The X-ray structure of benzoylformate decarboxylase (BFDC) from Pseudomonas putida ATCC 12633 shows it to be a tetramer. This was believed to be typical of all thiamin diphosphate-dependent decarboxylases until recently when the structure of KdcA, a branched-chain 2-keto acid decarboxylase from Lactococcus lactis, showed it to be a homodimer. This lent credence to earlier unfolding experiments on pyruvate decarboxylase from Saccharomyces cerevisiae that indicated that it might be active as a dimer.

Insights into the mechanism of deubiquitination by JAMM deubiquitinases from cocrystal structures of the enzyme with the substrate and product.

AMSH, a conserved zinc metallo deubiquitinase, controls downregulation and degradation of cell-surface receptors mediated by the endosomal sorting complexes required for transport (ESCRT) machinery. It displays high specificity toward the Lys63-linked polyubiquitin chain, which is used as a signal for ESCRT-mediated endosomal-lysosomal sorting of receptors. Herein, we report the crystal structures of the catalytic domain of AMSH orthologue Sst2 from fission yeast, its ubiquitin (product)-bound form, and its Lys63-linked diubiquitin (substrate)-bound form at 1.

Effect of cross-linking of interfacial sodium caseinate by natural processing on the oxidative stability of oil-in-water (o/w) emulsions.

This study investigated how enzymatic cross-linking of interfacial sodium caseinate and emulsification, via high-pressure homogenization, influenced the intrinsic oxidative stability of 4% (w/v) menhaden oil-in-water emulsions stabilized by 1% (w/v) caseinate at pH 7. Oil oxidation was monitored by the ferric thiocyanate perioxide value assay. Higher homogenization pressure resulted in improved intrinsic emulsion oxidative stability, which is attributed to increased interfacial cross-linking as indicated by higher weighted average sedimentation coefficients of interfacial protein species (from 11.

An asymmetric heterodomain interface stabilizes a response regulator-DNA complex.

Two-component signal transduction systems consist of pairs of histidine kinases and response regulators, which mediate adaptive responses to environmental cues. Most activated response regulators regulate transcription by binding tightly to promoter DNA via a phosphorylation-triggered inactive-to-active transition. The molecular basis for formation of stable response regulator-DNA complexes that precede the assembly of RNA polymerases is unclear.

Mechanism of recruitment and activation of the endosome-associated deubiquitinase AMSH.

AMSH, a deubiquitinating enzyme (DUB) with exquisite specificity for Lys63-linked polyubiquitin chains, is an endosome-associated DUB that regulates sorting of activated cell-surface signaling receptors to the lysosome, a process mediated by the members of the endosomal sorting complexes required for transport (ESCRT) machinery. Whole-exome sequencing of DNA samples from children with microcephaly capillary malformation (MIC-CAP) syndrome identified recessive mutations encoded in the AMSH gene causatively linked to the disease. Herein, we report a number of important observations that significantly advance our understanding of AMSH within the context of the ESCRT machinery.

An additional substrate binding site in a bacterial phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes oxidation of phenylalanine to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH has a regulatory domain in which binding of the substrate leads to allosteric activation of the enzyme. However, the existence of PAH regulation in evolutionarily distant organisms, for example some bacteria in which it occurs, has so far been underappreciated.

Stabilization of an unusual salt bridge in ubiquitin by the extra C-terminal domain of the proteasome-associated deubiquitinase UCH37 as a mechanism of its exo specificity.

Ubiquitination is countered by a group of enzymes collectively called deubiquitinases (DUBs); ∼100 of them can be found in the human genome. One of the most interesting aspects of these enzymes is the ability of some members to selectively recognize specific linkage types between ubiquitin in polyubiquitin chains and their endo and exo specificity. The structural basis of exo-specific deubiquitination catalyzed by a DUB is poorly understood.