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. This manuscript describes the challenges faced in bringing SV-AUC to a cGMP environment and describes a new program, "BASIS", which allows for 21 CFR Part 11-compliant data handling and data analysis using the well-known and frequently cited SEDFIT analysis software.

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. Recent guidance from the FDA includes limiting the presence of empty AAV capsids and other impurities to reduce immunotoxicity. While chromatographic techniques (SEC, SEC-MALS, AEX) are often used for empty and full capsid quantitation due to the ease of accessibility and familiarity among most biochemists, the resolution and sensitivity attained by sedimentation velocity (SV-AUC) in the formulation buffer and purification buffers is unmatched. Approaches for using SV-AUC to determine the empty-to-full capsid ratio have already been discussed by others; however, in this report, we focus on the importance of characterizing other impurities, such as free DNA, partially filled capsids, and aggregates that are recognized as species of concern for immunotoxicity. We also demonstrate the usefulness of applying multiple analyses (e.g., c(s), g(s*), WDA) in confirming the presence of and determining the hydrodynamic parameters of these various species.

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

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 Streptococcus pyogenes (GAS), e.g., GAS isolate AP53, contain a surface M-protein (PAM) that directly and strongly interacts (Kd ~ 1 nM) with human host plasminogen (hPg), after which it is activated to hPm by a specific coinherited bacterial activator, streptokinase (SK2b), or by host activators. Another ubiquitous class of hPg binding proteins on GAS cells includes "moonlighting" proteins, such as the glycolytic enzyme, enolase (Sen). However, the importance of Sen in hPg acquisition, especially when PAM is present, has not been fully developed. Sen forms a complex with hPg on different surfaces, but not in solution. Isogenic AP53 cells with a targeted deletion of PAM do not bind hPg, but the surface expression of Sen is also greatly diminished upon deletion of the PAM gene, thus confounding this approach for defining the role of Sen. However, cells with point deletions in PAM that negate hPg binding, but fully express PAM and Sen, show that hPg binds weakly to Sen on GAS cells. Despite this, Sen does not stimulate hPg activation by SK2b, but does stimulate tissue-type plasminogen activator-catalyzed activation of hPg. These data demonstrate that PAM plays the dominant role as a functional hPg receptor in GAS cells that also contain surface enolase.

Visualizing the enzyme mechanism of mevalonate diphosphate decarboxylase.

Mevalonate diphosphate decarboxylases (MDDs) catalyze the ATP-dependent-Mg2+-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. Initial binding of MVAPP modulates the ATP binding pocket with a large loop movement. Upon ATP binding, a phosphate binding loop bends over the active site to recognize ATP and bring the molecules to their catalytically favored configuration. Positioned substrates then can chelate two Mg2+ ions for the two steps of the reaction. Closure of the active site entrance brings a conserved lysine to trigger dissociative phosphoryl transfer of γ-phosphate from ATP to MVAPP, followed by the production of IPP.

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

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 Enterococcus faecalis 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 E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF-ATP) revealed that the phosphate-binding loop (amino acids 97-105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

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. To investigate transmission-related processes in response to Wolbachia infection, ovarian proteomes were analyzed from Wolbachia-infected Drosophila melanogaster and D. simulans. Endogenous and variant host-strain combinations were investigated. Significant and differentially abundant ovarian proteins were detected, indicating substantial regulatory changes in response to Wolbachia Variant Wolbachia strains were associated with a broader impact on the ovary proteome than endogenous Wolbachia strains. The D. melanogaster ovarian environment also exhibited a higher level of diversity of proteomic responses to Wolbachia than D. simulans. Overall, many Wolbachia-responsive ovarian proteins detected in this study were consistent with expectations from the experimental literature. This suggests that context-specific changes in protein abundance contribute to Wolbachia manipulation of transmission-related mechanisms in oogenesis. IMPORTANCE: Millions of insect species naturally carry bacterial endosymbionts called Wolbachia. Wolbachia bacteria are transmitted by females to their offspring through a robust egg-loading mechanism. The molecular basis for Wolbachia transmission remains poorly understood at this time, however. This proteomic study identified specific fruit fly ovarian proteins as being upregulated or downregulated in response to Wolbachia infection. The majority of these protein responses correlated specifically with the type of host and Wolbachia strain involved. This work corroborates previously identified factors and mechanisms while also framing the broader context of ovarian manipulation by Wolbachia.

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. The present study demonstrates auranofin's antibacterial activity is a complex process that involves inhibition of multiple biosynthetic pathways including cell wall, DNA, and bacterial protein synthesis. We also confirmed that the lack of activity of auranofin observed against Gram-negative bacteria is due to the permeability barrier conferred by the outer membrane. Auranofin's ability to suppress bacterial protein synthesis leads to significant reduction in the production of key methicillin-resistant Staphylococcus aureus (MRSA) toxins. Additionally, auranofin is capable of eradicating intracellular MRSA present inside infected macrophage cells. Furthermore, auranofin is efficacious in a mouse model of MRSA systemic infection and significantly reduces the bacterial load in murine organs including the spleen and liver. Collectively, this study provides valuable evidence that auranofin has significant promise to be repurposed as a novel antibacterial for treatment of invasive bacterial infections.

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). Proteomics and macromolecular synthesis analyses revealed that simvastatin inhibits multiple biosynthetic pathways and cellular processes in bacteria, including selective interference of bacterial protein synthesis. This property appears to assist in simvastatin's ability to suppress production of key MRSA toxins (α-hemolysin and Panton-Valentine leucocidin) that impair healing of infected skin wounds. A murine MRSA skin infection experiment confirmed that simvastatin significantly reduces the bacterial burden and inflammatory cytokines in the infected wounds. Additionally, simvastatin exhibits excellent anti-biofilm activity against established staphylococcal biofilms and demonstrates the ability to be combined with topical antimicrobials currently used to treat MRSA skin infections. Collectively the present study lays the foundation for further investigation of repurposing simvastatin as a topical antibacterial agent to treat skin infections.

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. In this report, we found that members of the Legionella pneumophila SidE effector family harbor a DUB module important for ubiquitin dynamics on the bacterial phagosome. Structural analysis of this domain alone and in complex with ubiquitin vinyl methyl ester (Ub-VME) reveals unique molecular contacts used in ubiquitin recognition. Instead of relying on the Ile44 patch of ubiquitin, as commonly used in eukaryotic counterparts, the SdeADub module engages Gln40 of ubiquitin. The architecture of the active-site cleft presents an open arrangement with conformational plasticity, permitting deubiquitination of three of the most abundant polyubiquitin chains, with a distinct preference for Lys63 linkages. We have shown that this preference enables efficient removal of Lys63 linkages from the phagosomal surface. Remarkably, the structure reveals by far the most parsimonious use of molecular contacts to achieve deubiquitination, with less than 1,000 Å(2) of accessible surface area buried upon complex formation with ubiquitin. This type of molecular recognition appears to enable dual specificity toward ubiquitin and the ubiquitin-like modifier NEDD8.

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. Emphasis on sample preparation for LC-MS/MS analyses is essential to acquisition of high-quality data for proteomic research. Certain classes of reagents, materials, and contaminants that can be introduced during sample processing may limit the effectiveness of LC-MS/MS analysis. These protocols outline methods for proteolytic digestion of proteins that are compatible with LC-MS/MS, along with procedures that allow for simplification of complex protein matrices.

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. To gain insight into the contribution of this flap in substrate recognition and catalysis, we generated mutants of Sst2 and characterized them using a combination of enzyme kinetics, X-ray crystallography, molecular dynamics simulations, and isothermal titration calorimetry (ITC). Our analysis shows that the Phe residue in the flap contributes key interactions during the rate-limiting step but not to substrate binding, since mutants of Phe403 exhibit a defect only in kcat but not in KM. Moreover, ITC studies show Phe403 mutants have similar KD for ubiquitin compared to the wild-type enzyme. The X-ray structures of both Phe403Ala and the Phe403Trp, in both the free and ubiquitin bound form, reveal no appreciable structural change that might impair substrate or alter product binding. We observed that the side chain of the Trp residue is oriented identically with respect to the isopeptide moiety of the substrate as the Phe residue in the wild-type enzyme, so the loss of activity seen in this mutant cannot be explained by the absence of a group with the ability to provide van der Waals interactions that facilitate the hyrdolysis of the Lys63-linked diubiquitin. Molecular dynamics simulations indicate that the flap in the Trp mutant is quite flexible, allowing almost free rotation of the indole side chain. Therefore, it is possible that these different dynamic properties of the flap in the Trp mutant, compared to the wild-type enzyme, manifest as a defect in interactions that facilitate the rate-limiting step. Consistent with this notion, the Trp mutant was able to cleave Lys48-linked and Lys11-linked diubiquitin better than the wild-type enzyme, indicating altered mobility and hence reduced selectivity.

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. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization or aggregation of αSyn, citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells were rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H2O2, in the diauxic phase of normal growth conditions, and in cells under αSyn-mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents the formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins.

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. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency.

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 ß-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 ß-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. Analytical ultracentrifugation studies support that MERS-CoV 3CL(pro) is a weakly associated dimer (Kd ∼52 µm) with a slow off-rate. Peptidomimetic inhibitors of MERS-CoV 3CL(pro) were synthesized and utilized in analytical ultracentrifugation experiments and demonstrate that MERS-CoV 3CL(pro) undergoes significant ligand-induced dimerization. Kinetic studies also revealed that designed reversible inhibitors act as activators at a low compound concentration as a result of induced dimerization. Primary sequence comparisons and x-ray structural analyses of two MERS-CoV 3CLpro and inhibitor complexes, determined to 1.6 Å, reveal remarkable structural similarity of the dimer interface with 3CL(pro) from HKU4-CoV and HKU5-CoV. Despite this structural similarity, substantial differences in the dimerization ability suggest that long range interactions by the nonconserved amino acids distant from the dimer interface may control MERS-CoV 3CL(pro) dimerization. Activation of MERS-CoV 3CL(pro) through ligand-induced dimerization appears to be unique within the genogroup 2c and may potentially increase the complexity in the development of MERS-CoV 3CL(pro) inhibitors as antiviral agents.

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. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption.

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. Our isothermal titration calorimetry (ITC) analysis of iron binding implies that polarity at position 139 is not the sole criterion for metal affinity, as binding studies with D139E suggest that the size of the amino acid side chain also appears to be important. High-resolution crystal structures of the mutants reveal that Asp139 may not be essential for holding the bridging water molecules together, because many of these waters are retained even in the Ala mutant. However, interactions via the bridging waters contribute to cofactor binding at the active site, interactions for which charge of the residue is important, as the D139N mutant shows a 5-fold decrease in its affinity for pterin as revealed by ITC (compared to a 16-fold loss of affinity in the case of the Ala mutant). The Asn and Ala mutants show a much more pronounced defect in their kcat values, with nearly 16- and 100-fold changes relative to that of the wild type, respectively, indicating a substantial role of this residue in stabilization of the transition state by aligning the cofactor in a productive orientation, most likely through direct binding with the cofactor, supported by data from molecular dynamics simulations of the complexes. Our results indicate that the intervening water structure between the cofactor and the acidic residue masks direct interaction between the two, possibly to prevent uncoupled hydroxylation of the cofactor before the arrival of phenylalanine. It thus appears that the second-coordination sphere Asp residue in cPAH, and, by extrapolation, the equivalent residue in other AAAHs, plays a role in fine-tuning pterin affinity in the ground state via deformable interactions with bridging waters and assumes a more significant role in the transition state by aligning the cofactor through direct hydrogen bonding.

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)-ß-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.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.

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. To investigate this possibility in BFDC, we sought to shift the equilibrium toward dimer formation. Point mutations were made in the noncatalytic monomer-monomer interfaces, but these had a minimal effect on both tetramer formation and catalytic activity. Subsequently, the R141E/Y288A/A306F variant was shown by analytical ultracentrifugation to be partially dimeric. It was also found to be catalytically inactive. Further experiments revealed that just two mutations, R141E and A306F, were sufficient to markedly alter the dimer-tetramer equilibrium and to provide an ~450-fold decrease in kcat. Equilibrium denaturation studies suggested that the residual activity was possibly due to the presence of residual tetramer. The structures of the R141E and A306F variants, determined to <1.5 Å resolution, hinted that disruption of the monomer interfaces will be accompanied by movement of a loop containing Leu109 and Leu110. As these residues contribute to the hydrophobicity of the active site and the correct positioning of the substrate, it seems that tetramer formation may well be critical to the catalytic activity of BFDC.

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 1.45, 1.7, and 2.3 Å, respectively. The structures reveal that the P-side product fragment maintains nearly all the contacts with the enzyme as seen with the P portion (distal ubiquitin) of the Lys63-linked diubiquitin substrate, with additional coordination of the Gly76 carboxylate group of the product with the active-site Zn(2+). One of the product-bound structures described herein is the result of an attempt to cocrystallize the diubiquitin substrate bound to an active site mutant presumed to render the enzyme inactive, instead yielding a cocrystal structure of the enzyme bound to the P-side ubiquitin fragment of the substrate (distal ubiquitin). This fragment was generated in situ from the residual activity of the mutant enzyme. In this structure, the catalytic water is seen placed between the active-site Zn(2+) and the carboxylate group of Gly76 of ubiquitin, providing what appears to be a snapshot of the active site when the product is about to depart. Comparison of this structure with that of the substrate-bound form suggests the importance of dynamics of a flexible flap near the active site in catalysis. The crystal structure of the Thr319Ile mutant of the catalytic domain of Sst2 provides insight into structural basis of microcephaly capillary malformation syndrome. Isothermal titration calorimetry yields a dissociation constant (KD) of 10.2 ± 0.6 µM for the binding of ubiquitin to the enzyme, a value comparable to the KM of the enzyme catalyzing hydrolysis of the Lys63-linked diubiquitin substrate (~20 µM). These results, together with the previously reported observation that the intracellular concentration of free ubiquitin (~20 µM) exceeds that of Lys63-linked polyubiquitin chains, imply that the free, cytosolic form of the enzyme remains inhibited by being tightly bound to free ubiquitin. We propose that when AMSH associates with endosomes, inhibition would be relieved because of ubiquitin binding domains present on its endosomal binding partners that would shift the balance toward better recognition of polyubiquitin chains via the avidity effect.

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 11.2 S for 0 kpsi/0.1 MPa to 18 S for 20 kpsi/137.9 MPa). Moderate dosage of transglutaminase at 0.5-1.0 U/mL emulsion enhanced intrinsic emulsion oxidative stability further, despite a contradictory reduction in the antioxidant property of cross-linked caseinate as tested by the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. This implied the prominent role of cross-linked interfacial caseinate as a physical barrier for oxygen transfer, hence its efficacy in retarding oil oxidation.