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MCPIP1, also known as Regnase-1, is a ribonuclease crucial for regulation of stability of transcripts related to inflammatory processes. Here, we report that MCPIP1 acts as an endonuclease by degrading several stem-loop RNA structures and single-stranded RNAs. Our studies revealed cleavage sites present in the stem-loops derived from the 3' untranslated region of the interleukin-6 transcript. Furthermore, MCPIP1 induced endonuclease cleavage at the loop motif of stem-loop structures. Additionally, we observed that MCPIP1 could cleave single-stranded RNA fragments. However, RNA substrates shorter than 6 nucleotides were not further affected by MCPIP1 nucleolytic activity. In this study, we also determined the dissociation constants of full-length MCPIP1D141N and its ribonuclease domain PIN D141N with twelve oligonucleotides substrates. The equilibrium binding constants (Kd) for MCPIP1D141N and the RNA targets were approximately 10 nM. Interestingly, we observed that the presence of a zinc finger in the PIN domain increases the affinity of this protein fragment to 25-nucleotide-long stem-loop RNA but not to shorter ones. Furthermore, size exclusion chromatography of the MCPIP1 and PIN proteins suggested that MCPIP1 undergoes homooligomerization during interaction with RNA substrates. Our results provide insight into the mechanism of MCPIP1 substrate recognition and its affinity towards various oligonucleotides.
Tulane virus (TV) is a cultivable calicivirus isolated from rhesus monkeys. In this study, we characterized the substrate specificity of TV protease in trans using recombinant proteases and TV polyprotein fragments containing the predicted proteolytic cleavage sites. Cleavage products have been obtained from 4 of the 5 fragments containing (573)Q-S(574) between the helicase and 3A-like protein, (712)E-A(713) between the 3A-like protein and Vpg, (802)E-G(803) between Vpg and the protease, and (976)E-G(977) between the protease and RdRp. We also characterized the enzymatic activities of the recombinant proteases of TV and Norwalk virus using synthetic fluorogenic peptide substrates. Under optimal conditions for enzymatic assays, partial cross-reactivities on reciprocal substrates were observed between TV and Norwalk virus proteases. The apparently shared substrate specificities between TV and Norwalk virus proteases suggested that the cultivable TV could be used as a model for in vivo evaluation of lead candidates of protease inhibitors for human norovirus.
Yersinia bacteria cause a range of human diseases, including yersiniosis, Far East scarlet-like fever and the plague. Yersiniae modulate and evade host immune defences through injection of Yersinia outer proteins (Yops) into phagocytic cells. One of the Yops, YopO (also known as YpkA) obstructs phagocytosis through disrupting actin filament regulation processes - inhibiting polymerization-promoting signaling through sequestration of Rac/Rho family GTPases and by using monomeric actin as bait to recruit and phosphorylate host actin-regulating proteins. Here we set out to identify mechanisms of specificity in protein phosphorylation by YopO that would clarify its effects on cytoskeleton disruption. We report the MgADP structure of Yersinia enterocolitica YopO in complex with actin, which reveals its active site architecture. Using a proteome-wide kinase-interacting substrate screening (KISS) method, we identified that YopO phosphorylates a wide range of actin-modulating proteins and located their phosphorylation sites by mass spectrometry. Using artificial substrates we clarified YopO's substrate length requirements and its phosphorylation consensus sequence. These findings provide fresh insight into the mechanism of the YopO kinase and demonstrate that YopO executes a specific strategy targeting actin-modulating proteins, across multiple functionalities, to compete for control of their native phospho-signaling, thus hampering the cytoskeletal processes required for macrophage phagocytosis.
In the present studies, we focused on substrate specificity of tocopherol cyclase, the key enzyme in the biosynthesis of the tocopherols and plastochromanol-8, the main plant lipid antioxidants, with special emphasis on the preference for tocopherols and plastochromanol-8 precursors, taking advantage of the recombinant enzyme originating from Arabidopsis thaliana and isolated plastoglobules, thylakoids and various model systems like micelles and thylakoids. Plastoglobules and triacylglycerol micelles were the most efficient reaction environment for the cyclase. In various investigated systems, synthesis of γ-tocopherol proceeded considerably faster than that of plastochromanol-8, probably mainly due to different localization of the corresponding substrates in the analyzed lipid structures. Moreover, our study was complemented by bioinformatics analysis of the phylogenetic relations of the cyclases and sequence motifs, crucial for the enzyme activity, were proposed. The analysis revealed also a group of tocopherol cyclase-like proteins in a number of heterotrophic bacterial species, with a conserved region common with photosynthetic organisms, that might be engaged in the catalytic activity of both groups of organisms.
Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2' and P11', for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13-VWF exosite interactions outside of VWF73.
The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated L-olivosyl-8.8a-deoxyoleandolide (L-O-DEO) intermediates of oleandomycin biosynthesis. We investigated the substrate versatility of the enzyme. X-ray and equilibrium binding data show that the aglycone DEO loosely fits the OleP active site, triggering the closure that prepares it for catalysis only on a minor population of enzyme. The open-to-closed state transition allows solvent molecules to accumulate in a cavity that forms upon closure, mediating protein-substrate interactions. In silico docking of the monoglycosylated L-O-DEO in the closed OleP-DEO structure shows that the L-olivosyl moiety can be hosted in the same cavity, replacing solvent molecules and directly contacting structural elements involved in the transition. X-ray structures of aglycone-bound OleP in the presence of L-rhamnose confirm the cavity as a potential site for sugar binding. All considered, we propose L-O-DEO as the optimal substrate of OleP, the L-olivosyl moiety possibly representing the molecular wedge that triggers a more efficient structural response upon substrate binding, favoring and stabilizing the enzyme closure before catalysis. OleP substrate versatility is supported by structural solvent molecules that compensate for the absence of a glycosyl unit when the aglycone is bound.
Previously, ways to adapt docking programs that were developed for modelling inhibitor-receptor interaction have been explored. Two main issues were discussed. First, when trying to model catalysis a reaction intermediate of the substrate is expected to provide more valid information than the ground state of the substrate. Second, the incorporation of protein flexibility is essential for reliable predictions.
Protein tyrosine phosphatases dephosphorylate tyrosine residues of proteins, whereas, dual specificity phosphatases (DUSPs) are a subgroup of protein tyrosine phosphatases that dephosphorylate not only Tyr(P) residue, but also the Ser(P) and Thr(P) residues of proteins. The DUSPs are linked to the regulation of many cellular functions and signaling pathways. Though many cellular targets of DUSPs are known, the relationship between catalytic activity and substrate specificity is poorly defined. We investigated the interactions of peptide substrates with select DUSPs of four types: MAP kinases (DUSP1 and DUSP7), atypical (DUSP3, DUSP14, DUSP22 and DUSP27), viral (variola VH1), and Cdc25 (A-C). Phosphatase recognition sites were experimentally determined by measuring dephosphorylation of 6,218 microarrayed Tyr(P) peptides representing confirmed and theoretical phosphorylation motifs from the cellular proteome. A broad continuum of dephosphorylation was observed across the microarrayed peptide substrates for all phosphatases, suggesting a complex relationship between substrate sequence recognition and optimal activity. Further analysis of peptide dephosphorylation by hierarchical clustering indicated that DUSPs could be organized by substrate sequence motifs, and peptide-specificities by phylogenetic relationships among the catalytic domains. The most highly dephosphorylated peptides represented proteins from 29 cell-signaling pathways, greatly expanding the list of potential targets of DUSPs. These newly identified DUSP substrates will be important for examining structure-activity relationships with physiologically relevant targets.
The removal of sialic acid (Sia) residues from glycoconjugates in vertebrates is mediated by a family of neuraminidases (sialidases) consisting of Neu1, Neu2, Neu3 and Neu4 enzymes. The enzymes play distinct physiological roles, but their ability to discriminate between the types of linkages connecting Sia and adjacent residues and between the identity and arrangement of the underlying sugars has never been systematically studied. Here we analyzed the specificity of neuraminidases by studying the kinetics of hydrolysis of BODIPY-labeled substrates containing common mammalian sialylated oligosaccharides: 3'Sia-LacNAc, 3'SiaLac, SiaLex, SiaLea, SiaLec, 6'SiaLac, and 6'SiaLacNAc. We found significant differences in substrate specificity of the enzymes towards the substrates containing α2,6-linked Sia, which were readily cleaved by Neu3 and Neu1 but not by Neu4 and Neu2. The presence of a branching 2-Fuc inhibited Neu2 and Neu4, but had almost no effect on Neu1 or Neu3. The nature of the sugar residue at the reducing end, either glucose (Glc) or N-acetyl-D-glucosamine (GlcNAc) had only a minor effect on all neuraminidases, whereas core structure (1,3 or 1,4 bond between D-galactose (Gal) and GlcNAc) was found to be important for Neu4 strongly preferring β3 (core 1) to β4 (core 2) isomer. Neu3 and Neu4 were in general more active than Neu1 and Neu2, likely due to their preference for hydrophobic substrates. Neu2 and Neu3 were examined by molecular dynamics to identify favorable substrate orientations in the binding sites and interpret the differences in their specificities. Finally, using knockout mouse models, we confirmed that the substrate specificities observed in vitro were recapitulated in enzymes found in mouse brain tissues. Our data for the first time provide evidence for the characteristic substrate preferences of neuraminidases and their ability to discriminate between distinct sialoside targets.
The very long chain fatty acids (VLCFA) incorporated into plant lipids are derived from the iterative addition of C2 units provided by malonyl-CoA to an acyl-CoA by the 3-ketoacyl-CoA synthase (KCS) component of a fatty acid elongase (FAE) complex. Mining of the Arabidopsis genome sequence database revealed 20 genes with homology to seed-specific FAE1 KCS. Eight of the 20 putative KCSs were cloned, expressed in yeast, and isolated as (His)6 fusion proteins. Five of the eight (At1g71160, At1g19440, At1g07720, At5g04530, and At4g34250) had little or no activity with C16 to C20 substrates while three demonstrated activity with C16, C18, and C20 saturated acyl-CoA substrates. At1g01120 KCS (KCS1) and At2g26640 KCS had broad substrate specificities when assayed with saturated and mono-unsaturated C16 to C24 acyl-CoAs while At4g34510 KCS was specific for saturated fatty acyl-CoA substrates.
During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity is apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association shows co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduces Mtr4 recruitment and increases RNA abundance for mRNAs specifically showing high Trf5 binding.
Amino acid antiporters mediate the 1:1 exchange of groups of amino acids. Whether substrate specificity can be different for the inward and outward facing conformation has not been investigated systematically, although examples of asymmetric transport have been reported. Here we used LC-MS to detect the movement of 12C- and 13C-labelled amino acid mixtures across the plasma membrane of Xenopus laevis oocytes expressing a variety of amino acid antiporters. Differences of substrate specificity between transporter paralogs were readily observed using this method. Our results suggest that antiporters are largely symmetric, equalizing the pools of their substrate amino acids. Exceptions are the antiporters y+LAT1 and y+LAT2 where neutral amino acids are co-transported with Na+ ions, favouring their import. For the antiporters ASCT1 and ASCT2 glycine acted as a selective influx substrate, while proline was a selective influx substrate of ASCT1. These data show that antiporters can display non-canonical modes of transport.
Guanine monophosphate (GMP) synthetase is a bifunctional two-domain enzyme. The N-terminal glutaminase domain generates ammonia from glutamine and the C-terminal synthetase domain aminates xanthine monophosphate (XMP) to form GMP. Mammalian GMP synthetases (GMPSs) contain a 130-residue-long insert in the synthetase domain in comparison to bacterial proteins. We report here the structure of a eukaryotic GMPS. Substrate XMP was bound in the crystal structure of the human GMPS enzyme. XMP is bound to the synthetase domain and covered by a LID motif. The enzyme forms a dimer in the crystal structure with subunit orientations entirely different from the bacterial counterparts. The inserted sub-domain is shown to be involved in substrate binding and dimerization. Furthermore, the structural basis for XMP recognition is revealed as well as a potential allosteric site. Enzymes in the nucleotide metabolism typically display an increased activity in proliferating cells due to the increased need for nucleotides. Many drugs used as immunosuppressants and for treatment of cancer and viral diseases are indeed nucleobase- and nucleoside-based compounds, which are acting on or are activated by enzymes in this pathway. The information obtained from the crystal structure of human GMPS might therefore aid in understanding interactions of nucleoside-based drugs with GMPS and in structure-based design of GMPS-specific inhibitors.
The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the 'water retention site', suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.
CK1 kinases participate in many signaling pathways; how these enzymes are regulated is therefore of significant biological consequence. CK1s autophosphorylate their C-terminal non-catalytic tails, and eliminating these modifications increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and phosphoablating mutations increased Hhp1 and CK1ε activity towards substrates. Interestingly, substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. The presence or absence of tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, indicating that tails contribute to substrate specificity. Combining this mechanism with autophosphorylation of the T220 site in the catalytic domain, we propose a displacement specificity model to describe how autophosphorylation regulates substrate specificity for the CK1 family.
A bifurcation of the mevalonate (MVA) pathway was recently discovered in bacteria of the Chloroflexi phylum. In this alternative route for the biosynthesis of isopentenylpyrophosphate (IPP), the penultimate step is the decarboxylation of (R)-mevalonate 5-phosphate ((R)-MVAP) to isopentenyl phosphate (IP), which is followed by the ATP-dependent phosphorylation of IP to IPP catalyzed by isopentenyl phosphate kinase (IPK). Notably, the decarboxylation reaction is catalyzed by mevalonate 5-phosphate decarboxylase (MPD), which shares considerable sequence similarity with mevalonate diphosphate decarboxylase (MDD) of the classical MVA pathway. We show that an enzyme originally annotated as an MDD from the Chloroflexi bacterium Anaerolinea thermophila possesses equal catalytic efficiency for (R)-MVAP and (R)-mevalonate 5-diphosphate ((R)-MVAPP). Further, the molecular basis for this dual specificity is revealed by near atomic-resolution X-ray crystal structures of A. thermophila MPD/MDD bound to (R)-MVAP or (R)-MVAPP. These findings, when combined with sequence and structural comparisons of this bacterial enzyme, functional MDDs, and several putative MPDs, delineate key active-site residues that confer substrate specificity and functionally distinguish MPD and MDD enzyme classes. Extensive sequence analyses identified functional MPDs in the halobacteria class of archaea that had been annotated as MDDs. Finally, no eukaryotic MPD candidates were identified, suggesting the absence of the alternative MVA (altMVA) pathway in all eukaryotes, including, paradoxically, plants, which universally encode a structural and functional homologue of IPK. Additionally, we have developed a viable engineered strain of Saccharomyces cerevisiae as an in vivo metabolic model and a synthetic biology platform for enzyme engineering and terpene biosynthesis in which the classical MVA pathway has been replaced with the altMVA pathway.
The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2'-O-MTase.
Palmitoylation is a post-translational modification involving the thioesterification of cysteine residues with a 16-carbon-saturated fatty acid. Little is known about rates of depalmitoylation or the parameters that dictate these rates. Here we report a modular strategy to synthesize quenched fluorogenic substrates for the specific detection of depalmitoylase activity and for mapping the substrate specificity of individual depalmitoylases. We demonstrate that human depalmitoylases APT1 and APT2, and TgPPT1 from the parasite Toxoplasma gondii, have distinct specificities that depend on amino acid residues distal to the palmitoyl cysteine. This information informs the design of optimal and non-optimal substrates as well as isoform-selective substrates to detect the activity of a specific depalmitoylase in complex proteomes. In addition to providing tools for studying depalmitoylases, our findings identify a previously unrecognized mechanism for regulating steady-state levels of distinct palmitoylation sites by sequence-dependent control of depalmitoylation rates.
MUS81 plays important cellular roles in the restart of stalled replication forks, the resolution of recombination intermediates and in telomere length maintenance. Although the actions of MUS81-EME1 have been extensively investigated, MUS81 is the catalytic subunit of two human structure-selective endonucleases, MUS81-EME1 and MUS81-EME2. Little is presently known about the activities of MUS81-EME2. Here, we have purified MUS81-EME2 and compared its activities with MUS81-EME1. We find that MUS81-EME2 is a more active endonuclease than MUS81-EME1 and exhibits broader substrate specificity. Like MUS81-EME1, MUS81-EME2 cleaves 3'-flaps, replication forks and nicked Holliday junctions, and exhibits limited endonuclease activity with intact Holliday junctions. In contrast to MUS81-EME1, however, MUS81-EME2 cuts D-loop recombination intermediates and in so doing disengages the D-loop structure by cleaving the 3'-invading strand. Additionally, MUS81-EME2 acts on 5'-flap structures to cleave off a duplex arm, in reactions that cannot be promoted by MUS81-EME1. These studies suggest that MUS81-EME1 and MUS81-EME2 exhibit similar and yet distinct DNA structure selectivity, indicating that the two MUS81 complexes may promote different nucleolytic cleavage reactions in vivo.
Lysoplasmalogen-specific phospholipase D (LyPls-PLD) catalyzes reactions in a manner similar to those catalyzed by glycerophosphodiester phosphodiesterase (GDPD) and other well-known PLDs. Although these enzymes hydrolyze the glycerophosphodiester bond, their substrate specificities are completely different. Previously, we reported that LyPls-PLD from Thermocrispum sp. RD004668 shows only 53% activity with 1-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (LysoPAF) relative to the 100% activity it shows with choline lysoplasmalogen (LyPlsCho). Lipoprotein-associated phospholipase A2 (Lp-PLA2 ) activity can be used to evaluate for cardiovascular disease. Hence, development of a point-of-care testing kit requires a LysoPAF-specific PLD (LysoPAF-PLD) to measure Lp-PLA2 activity. Rational site-directed mutagenesis and kinetic analysis were applied to generate LysoPAF-PLD from LyPls-PLD and to clarify the mechanisms underlying the substrate-recognition ability of LyPls-PLD. Our results suggest that LyPls-PLD variants A47, M71, N173, F211, and W282 are possibly involved in substrate recognition and that F211L may substantially alter substrate preference. Moreover, the specific activity ratio LysoPAF/LyPlsCho corresponding to F211L was up to 25-fold higher than that corresponding to the wild-type enzyme. Thus, we succeeded in switching from LyPlsCho- to LysoPAF-PLD. These results suggest that the F211L variant may be utilized to measure Lp-PLA2 activity. Kinetic analyses demonstrated that product release was the rate-limiting step of the reaction, with flexibility of the sn-1 ether-linked vinyl/alkyl chain of the substrate being essential for substrate binding and product release. Our findings may lead to a better understanding of the differences between homologous enzymes (such as PLD, sphingomyelinase D, and GDPD of the phosphatidylinositol-phosphodiesterase superfamily) in relation to substrate recognition. ENZYME: EC 3.1.4.2 (currently assigned).
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