Enzymes in Synergy: Bacteria Specific Molecular Probe for Locoregional Imaging of Urinary Tract Infection in vivo.

Uropathogenic Escherichia coli (UPECs) is a leading cause for urinary tract infections (UTI), accounting for 70-90 % of community or hospital-acquired bacterial infections owing to high recurrence, imprecision in diagnosis and management, and increasing prevalence of antibiotic resistance. Current methods for clinical UPECs detection still rely on labor-intensive urine cultures that impede rapid and accurate diagnosis for timely UTI therapeutic management. Herein, we developed a first-in-class near-infrared (NIR) UPECs fluorescent probe (NO-AH) capable of specifically targeting UPECs through its collaborative response to bacterial enzymes, enabling locoregional imaging of UTIs both in vitro and in vivo. Our NO-AH probe incorporates a dual protease activatable moiety, which first reacts with OmpT, an endopeptidase abundantly present on the outer membrane of UPECs, releasing an intermediate amino acid residue conjugated with a NIR hemicyanine fluorophore. Such liberated fragment would be subsequently recognized by aminopeptidase (APN) within the periplasm of UPECs, activating localized fluorescence for precise imaging of UTIs in complex living environments. The peculiar specificity and selectivity of NO-AH, facilitated by the collaborative action of bacterial enzymes, features a timely and accurate identification of UPECs-infected UTIs, which could overcome misdiagnosis in conventional urine tests, thus opening new avenues towards reliable UTI diagnosis and personalized antimicrobial therapy management.

Evolution of Streptococcus pyogenes has maximized the efficiency of the Sortase A cleavage motif for cell wall transpeptidation.

Trafficking of M-protein (Mprt) from the cytosol of Group A Streptococcus pyogenes (GAS) occurs via Sec translocase membrane channels that associate with Sortase A (SrtA), an enzyme that catalyzes cleavage of Mprt at the proximal C-terminal [-LPST355∗GEAA-] motif and subsequent transpeptidation of the Mprt-containing product to the cell wall (CW). These steps facilitate stable exposure of the N-terminus of Mprt to the extracellular milieu where it interacts with ligands. Previously, we found that inactivation of SrtA in GAS cells eliminated Mprt CW transpeptidation but effected little reduction in its cell surface exposure, indicating that the C-terminus of Mprt retained in the cytoplasmic membrane (CM) extends its N-terminus to the cell surface. Herein, we assessed the effects of mutating the Thr355 residue in the WT SrtA consensus sequence (LPST355∗GEAA-) in a specific Mprt, PAM. In vitro, we found that synthetic peptides with mutations (LPSX355GEAA) in the SrtA cleavage site displayed slower cleavage activities with rSrtA than the WT peptide. Aromatic residues at X had the lowest activities. Nonetheless, PAM/[Y355G] still transpeptidated the CW in vivo. However, when using isolated CMs from srtA-inactivated GAS cells, rapid cleavage of PAM/[LPSY355GEAA] occurred at E357∗ but transpeptidation did not take place. These results show that another CM-resident enzyme nonproductively cleaved PAM/[LPSYGE357∗AA]. However, SrtA associated with the translocon channel in vivo cleaved and transpeptidated PAM/[LPSX355∗GEAA] variants. These CM features allow diverse cleavage site variants to covalently attach to the CW despite the presence of other potent nonproductive CM proteases.

Identification of the distance between the homologous halves of P-glycoprotein that triggers the high/low ATPase activity switch.

P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. Each homologous half contains a transmembrane domain with six transmembrane segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the transmembrane domain and NBDs, respectively. Drug binding activates ATPase activity by an unknown mechanism. There is no high resolution structure of human P-gp, but homology models based on the crystal structures of bacterial, mouse, and Caenorhabditis elegans ATP-binding cassette drug pumps yield both open (NBDs apart) and closed (NBDs together) conformations. Molecular dynamics simulations predict that the NBDs can be separated over a range of distances (over 20 Å). To determine the distance that show high or low ATPase activity, we cross-linked reporter cysteines L175C (N-half) and N820C (C-half) with cross-linkers of various lengths that separated the halves between 6 and 30 Å (α-carbons). We observed that ATPase activity increased over 10-fold when the cysteines were cross-linked at distances between 6 and 19 Å, although cross-linking at distances greater than 20 Å yielded basal levels of activity. The results suggest that the ATPase activation switch appears to be turned on or off when L175C/N820 are clamped at distances less than or greater than 20 Å, respectively. We predict that the high/low ATPase activity switch may occur at a distance where the NBDs are predicted in molecular dynamic simulations to undergo pronounced twisting as they approach each other (Wise, J. G. (2012) Biochemistry 51, 5125-5141).

Structural basis for autoinhibition of CTP:phosphocholine cytidylyltransferase (CCT), the regulatory enzyme in phosphatidylcholine synthesis, by its membrane-binding amphipathic helix.

CTP:phosphocholine cytidylyltransferase (CCT) interconverts between an inactive soluble and active membrane-bound form in response to changes in membrane lipid composition. Activation involves disruption of an inhibitory interaction between the αE helices at the base of the active site and an autoinhibitory (AI) segment in the regulatory M domain and membrane insertion of the M domain as an amphipathic helix. We show that in the CCT soluble form the AI segment functions to suppress kcat and elevate the Km for CTP. The crystal structure of a CCT dimer composed of the catalytic and AI segments reveals an AI-αE interaction as a cluster of four amphipathic helices (two αE and two AI helices) at the base of the active sites. This interaction corroborates mutagenesis implicating multiple hydrophobic residues within the AI segment that contribute to its silencing function. The AI-αE interaction directs the turn at the C-terminal end of the AI helix into backbone-to-backbone contact with a loop (L2) at the opening to the active site, which houses the key catalytic residue, lysine 122. Molecular dynamics simulations suggest that lysine 122 side-chain orientations are constrained by contacts with the AI helix-turn, which could obstruct its engagement with substrates. This work deciphers how the CCT regulatory amphipathic helix functions as a silencing device.

A catalytically essential motif in external loop 5 of the bacterial oligosaccharyltransferase PglB.

Asparagine-linked glycosylation is a post-translational protein modification that is conserved in all domains of life. The initial transfer of a lipid-linked oligosaccharide (LLO) onto acceptor asparagines is catalyzed by the integral membrane protein oligosaccharyltransferase (OST). The previously reported structure of a single-subunit OST enzyme, the Campylobacter lari protein PglB, revealed a partially disordered external loop (EL5), whose role in catalysis was unclear. We identified a new and functionally important sequence motif in EL5 containing a conserved tyrosine residue (Tyr293) whose aromatic side chain is essential for catalysis. A synthetic peptide containing the conserved motif can partially but specifically rescue in vitro activity of mutated PglB lacking Tyr293. Using site-directed disulfide cross-linking, we show that disengagement of the structurally ordered part of EL5 is an essential step of the glycosylation reaction, probably by allowing sequon binding or glyco-product release. Our findings define two distinct mechanistic roles of EL5 in OST-catalyzed glycosylation. These functions, exerted by the two halves of EL5, are independent, because the loop can be cleaved by specific proteolysis with only slight reduction in activity.

Rubredoxin-related maturation factor guarantees metal cofactor integrity during aerobic biosynthesis of membrane-bound [NiFe] hydrogenase.

The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H2 as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O2 levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O2-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Niu-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O2. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys6[4Fe-3S] cluster.

NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:Quinone oxidoreductase from Vibrio cholerae.

The sodium ion-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na(+)-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na(+)-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na(+)-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na(+)-NQR.

The vitamin K oxidoreductase is a multimer that efficiently reduces vitamin K epoxide to hydroquinone to allow vitamin K-dependent protein carboxylation.

The vitamin K oxidoreductase (VKORC1) recycles vitamin K to support the activation of vitamin K-dependent (VKD) proteins, which have diverse functions that include hemostasis and calcification. VKD proteins are activated by Glu carboxylation, which depends upon the oxygenation of vitamin K hydroquinone (KH2). The vitamin K epoxide (KO) product is recycled by two reactions, i.e. KO reduction to vitamin K quinone (K) and then to KH2, and recent studies have called into question whether VKORC1 reduces K to KH2. Analysis in insect cells lacking endogenous carboxylation components showed that r-VKORC1 reduces KO to efficiently drive carboxylation, indicating KH2 production. Direct detection of the vitamin K reaction products is confounded by KH2 oxidation, and we therefore developed a new assay that stabilized KH2 and allowed quantitation. Purified VKORC1 analyzed in this assay showed efficient KO to KH2 reduction. Studies in 293 cells expressing tagged r-VKORC1 revealed that VKORC1 is a multimer, most likely a dimer. A monomer can only perform one reaction, and a dimer is therefore interesting in explaining how VKORC1 accomplishes both reactions. An inactive mutant (VKORC1(C132A/C135A)) was dominant negative in heterodimers with wild type VKORC1, resulting in decreased KO reduction in cells and carboxylation in vitro. The results are significant regarding human VKORC1 mutations, as warfarin-resistant patients have mutant and wild type VKORC1 alleles. A VKORC1 dimer indicates a mixed population of homodimers and heterodimers that may have different functional properties, and VKORC1 reduction may therefore be more complex in these patients than appreciated previously.

Radiolytic mapping of solvent-contact surfaces in Photosystem II of higher plants: experimental identification of putative water channels within the photosystem.

Photosystem II uses water as an enzymatic substrate. It has been hypothesized that this water is vectored to the active site for water oxidation via water channels that lead from the surface of the protein complex to the Mn4O5Ca metal cluster. The radiolysis of water by synchrotron radiation produces amino acid residue-modifying OH(•) and is a powerful technique to identify regions of proteins that are in contact with water. In this study, we have used this technique to oxidatively modify buried amino acid residues in higher plant Photosystem II membranes. Fourier transform ion cyclotron resonance mass spectrometry was then used to identify these oxidized amino acid residues that were located in several core Photosystem II subunits (D1, D2, CP43, and CP47). While, as expected, the majority of the identified oxidized residues (≈75%) are located on the solvent-exposed surface of the complex, a number of buried residues on these proteins were also modified. These residues form groups which appear to lead from the surface of the complex to the Mn4O5Ca cluster. These residues may be in contact with putative water channels in the photosystem. These results are discussed within the context of a number of largely computational studies that have identified putative water channels in Photosystem II.

Ca2+-independent binding of anionic phospholipids by phospholipase C δ1 EF-hand domain.

Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca(2+) that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane.

Diversity of Δ12 fatty acid desaturases in santalaceae and their role in production of seed oil acetylenic fatty acids.

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family.

Membrane binding by prothrombin mediates its constrained presentation to prothrombinase for cleavage.

Long-standing dogma proposes a profound contribution of membrane binding by prothrombin in determining the rate at which it is converted to thrombin by prothrombinase. We have examined the action of prothrombinase on full-length prothrombin variants lacking γ-carboxyglutamate modifications (desGla) with impaired membrane binding. We show an unexpectedly modest decrease in the rate of thrombin formation for desGla prothrombin but with a major effect on the pathway for substrate cleavage. Using desGla prothrombin variants in which the individual cleavage sites have been singly rendered uncleavable, we find that loss of membrane binding and other Gla-dependent functions in the substrate leads to a decrease in the rate of cleavage at Arg(320) and a surprising increase in the rate of cleavage at Arg(271). These compensating effects arise from a loss in the membrane component of exosite-dependent tethering of substrate to prothrombinase and a relaxation in the constrained presentation of the individual cleavage sites for active site docking and catalysis. Loss of constraint is evident as a switch in the pathway for prothrombin cleavage and the intermediate produced but without the expected profound decrease in rate. Extension of these findings to the action of prothrombinase assembled on platelets and endothelial cells on fully carboxylated prothrombin reveals new mechanistic insights into function on physiological membranes. Cell-dependent enzyme function is probably governed by a differential ability to support prothrombin binding and the variable accumulation of intermediates from the two possible pathways of prothrombin activation.

The cation-π box is a specific phosphatidylcholine membrane targeting motif.

Peripheral membrane proteins can be targeted to specific organelles or the plasma membrane by differential recognition of phospholipid headgroups. Although molecular determinants of specificity for several headgroups, including phosphatidylserine and phosphoinositides are well defined, specific recognition of the headgroup of the zwitterionic phosphatidylcholine (PC) is less well understood. In cytosolic proteins the cation-π box provides a suitable receptor for choline recognition and binding through the trimethylammonium moiety. In PC, this moiety might provide a sufficient handle to bind to peripheral proteins via a cation-π cage, where the π systems of two or more aromatic residues are within 4-5 Å of the quaternary amine. We prove this hypothesis by engineering the cation-π box into secreted phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, which lacks specific PC recognition. The N254Y/H258Y variant selectively binds PC-enriched vesicles, and x-ray crystallography reveals N254Y/H258Y binds choline and dibutyroylphosphatidylcholine within the cation-π motif. Such simple PC recognition motifs could be engineered into a wide variety of secondary structures providing a generally applicable method for specific recognition of PC.

Energy transducing roles of antiporter-like subunits in Escherichia coli NDH-1 with main focus on subunit NuoN (ND2).

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) contains a peripheral and a membrane domain. Three antiporter-like subunits in the membrane domain, NuoL, NuoM, and NuoN (ND5, ND4 and ND2, respectively), are structurally similar. We analyzed the role of NuoN in Escherichia coli NDH-1. The lysine residue at position 395 in NuoN (NLys(395)) is conserved in NuoL (LLys(399)) but is replaced by glutamic acid (MGlu(407)) in NuoM. Our mutation study on NLys(395) suggests that this residue participates in the proton translocation. Furthermore, we found that MGlu(407) is also essential and most likely interacts with conserved LArg(175). Glutamic acids, NGlu(133), MGlu(144), and LGlu(144), are corresponding residues. Unlike mutants of MGlu(144) and LGlu(144), mutation of NGlu(133) scarcely affected the energy-transducing activities. However, a double mutant of NGlu(133) and nearby KGlu(72) showed significant inhibition of these activities. This suggests that NGlu(133) bears a functional role similar to LGlu(144) and MGlu(144) but its mutation can be partially compensated by the nearby carboxyl residue. Conserved prolines located at loops of discontinuous transmembrane helices of NuoL, NuoM, and NuoN were shown to play a similar role in the energy-transducing activity. It seems likely that NuoL, NuoM, and NuoN pump protons by a similar mechanism. Our data also revealed that NLys(158) is one of the key interaction points with helix HL in NuoL. A truncation study indicated that the C-terminal amphipathic segments of NTM14 interacts with the Mß sheet located on the opposite side of helix HL. Taken together, the mechanism of H(+) translocation in NDH-1 is discussed.

A novel family of soluble minimal scaffolds provides structural insight into the catalytic domains of integral membrane metallopeptidases.

In the search for structural models of integral-membrane metallopeptidases (MPs), we discovered three related proteins from thermophilic prokaryotes, which we grouped into a novel family called "minigluzincins." We determined the crystal structures of the zymogens of two of these (Pyrococcus abyssi proabylysin and Methanocaldococcus jannaschii projannalysin), which are soluble and, with ∼100 residues, constitute the shortest structurally characterized MPs to date. Despite relevant sequence and structural similarity, the structures revealed two unique mechanisms of latency maintenance through the C-terminal segments previously unseen in MPs as follows: intramolecular, through an extended tail, in proabylysin, and crosswise intermolecular, through a helix swap, in projannalysin. In addition, structural and sequence comparisons revealed large similarity with MPs of the gluzincin tribe such as thermolysin, leukotriene A4 hydrolase relatives, and cowrins. Noteworthy, gluzincins mostly contain a glutamate as third characteristic zinc ligand, whereas minigluzincins have a histidine. Sequence and structural similarity further allowed us to ascertain that minigluzincins are very similar to the catalytic domains of integral membrane MPs of the MEROPS database families M48 and M56, such as FACE1, HtpX, Oma1, and BlaR1/MecR1, which are provided with trans-membrane helices flanking or inserted into a minigluzincin-like catalytic domain. In a time where structural biochemistry of integral-membrane proteins in general still faces formidable challenges, the minigluzincin soluble minimal scaffold may contribute to our understanding of the working mechanisms of these membrane MPs and to the design of novel inhibitors through structure-aided rational drug design approaches.

YidC protein, a molecular chaperone for LacY protein folding via the SecYEG protein machinery.

To understand how YidC and SecYEG function together in membrane protein topogenesis, insertion and folding of the lactose permease of Escherichia coli (LacY), a 12-transmembrane helix protein LacY that catalyzes symport of a galactoside and an H(+), was studied. Although both the SecYEG machinery and signal recognition particle are required for insertion of LacY into the membrane, YidC is not required for translocation of the six periplasmic loops in LacY. Rather, YidC acts as a chaperone, facilitating LacY folding. Upon YidC depletion, the conformation of LacY is perturbed, as judged by monoclonal antibody binding studies and by in vivo cross-linking between introduced Cys pairs. Disulfide cross-linking also demonstrates that YidC interacts with multiple transmembrane segments of LacY during membrane biogenesis. Moreover, YidC is strictly required for insertion of M13 procoat protein fused into the middle cytoplasmic loop of LacY. In contrast, the loops preceding and following the inserted procoat domain are dependent on SecYEG for insertion. These studies demonstrate close cooperation between the two complexes in membrane biogenesis and that YidC functions primarily as a foldase for LacY.

Elongator protein 3 (Elp3) lysine acetyltransferase is a tail-anchored mitochondrial protein in Toxoplasma gondii.

Lysine acetylation has recently emerged as an important, widespread post-translational modification occurring on proteins that reside in multiple cellular compartments, including the mitochondria. However, no lysine acetyltransferase (KAT) has been definitively localized to this organelle to date. Here we describe the identification of an unusual homologue of Elp3 in early-branching protozoa in the phylum Apicomplexa. Elp3 is the catalytic subunit of the well-conserved transcription Elongator complex; however, Apicomplexa lack all other Elongator subunits, suggesting that the Elp3 in these organisms plays a role independent of transcription. Surprisingly, Elp3 in the parasites of this phylum, including Toxoplasma gondii (TgElp3), possesses a unique C-terminal transmembrane domain (TMD) that localizes the protein to the mitochondrion. As TgElp3 is devoid of known mitochondrial targeting signals, we used selective permeabilization studies to reveal that this KAT is oriented with its catalytic components facing the cytosol and its C-terminal TMD inserted into the outer mitochondrial membrane, consistent with a tail-anchored membrane protein. Elp3 trafficking to mitochondria is not exclusive to Toxoplasma as we also present evidence that a form of Elp3 localizes to these organelles in mammalian cells, supporting the idea that Elp3 performs novel functions across eukaryotes that are independent of transcriptional elongation. Importantly, we also present genetic studies that suggest TgElp3 is essential in Toxoplasma and must be positioned at the mitochondrial surface for parasite viability.

Oligomerization of DHHC protein S-acyltransferases.

The formation of dimers or higher-order oligomers is a property of numerous integral membrane proteins, including ion channels, transporters, and receptors. In this study, we examined whether members of the DHHC-S-acyltransferase family oligomerize in intact cells and in vitro. DHHC-S-acyltransferases are integral membrane proteins that catalyze the addition of palmitate to cysteine residues on proteins at the cytoplasmic face of cell membranes. Bioluminescence resonance energy transfer (BRET) experiments revealed that DHHC2 or DHHC3 (Golgi-specific DHHC zinc finger protein (GODZ)) self-associate when expressed in HEK-293 cells. Homomultimer formation was confirmed by coimmunoprecipitation. Purified DHHC3 resolved predominately as a monomer and dimer on blue native polyacrylamide gels. In intact cells and in vitro, catalytically inactive DHHC proteins displayed a greater propensity to form dimers. BRET signals were higher for the catalytically inactive DHHC2 or DHHC3 than their wild-type counterparts. DHHC3 BRET in cell membranes was decreased by the addition of its lipid substrate palmitoyl-CoA, a treatment that results in autoacylation of the enzyme. Enzyme activity of a covalently linked DHHC3 dimer was less than that of the monomeric form, suggesting that enzyme activity may be modulated by the oligomerization status of the protein.

Squeezing at entrance of proton transport pathway in proton-translocating pyrophosphatase upon substrate binding.

Homodimeric proton-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H(+)-PPase consists of 14-16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H(+)-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H(+)-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H(+)-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H(+)-PPase upon substrate binding.

Comparative Proteomics Inspired Self-Stimulated Release Hydrogel Reinforces the Therapeutic Effects of MSC-EVs on Alzheimer's Disease.

The clinical application of extracellular vesicles (EVs)-based therapeutics continues to be challenging due to their rapid clearance, restricted retention, and low yields. Although hydrogel possesses the ability to impede physiological clearance and increase regional retention, it typically fails to effectively release the incorporated EVs, resulting in reduced accessibility and bioavailability. Here an intelligent hydrogel in which the release of EVs is regulated by the proteins on the EVs membrane is proposed. By utilizing the EVs membrane enzyme to facilitate hydrogel degradation, sustained retention and self-stimulated EVs release can be achieved at the administration site. To achieve this goal, the membrane proteins with matrix degrading activity in the mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are identified using comparative proteomics. After that, a hydrogel comprised of self-assembled peptides that are susceptible to degradation by the membrane enzymes present in MSC-EVs is designed and synthesized. After intranasal administration, this peptide hydrogel facilitates sustained and thermo-sensitive release of MSC-EVs, thereby extending the retention of the MSC-EVs and substantially enhancing their potential for treating Alzheimer's disease. This research presents a comparative proteomics-driven approach to intelligent hydrogel design, which holds the capacity to significantly enhance the applicability of EVs in clinical settings.