Functional Domains of the Fatty Acid Transport Proteins: Studies Using Protein Chimeras

Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1-FATP4 and FATP6-FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C20:4) and lignocerate (C24:0), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function

Functional Domains of the Fatty Acid Transport Proteins: Studies Using Protein Chimeras

Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1-FATP4 and FATP6-FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C20:4) and lignocerate (C24:0), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function

Cannabinoid biosynthesis using non-canonical enzymes

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Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases

Many eukaryotic organisms encode more than one RNA-dependent RNA polymerase (RdRP) that probably emerged as a result of gene duplication. Such RdRP paralogs often participate in distinct RNA silencing pathways and show characteristic repertoires of enzymatic activities in vitro. However, to what extent members of individual paralogous groups can undergo functional changes during speciation remains an open question. We show that orthologs of QDE-1, an RdRP component of the quelling pathway in Neurospora crassa, have rapidly diverged in evolution at the amino acid sequence level. Analyses of purified QDE-1 polymerases from N. crassa (QDE-1(Ncr)) and related fungi, Thielavia terrestris (QDE-1(Tte)) and Myceliophthora thermophila (QDE-1(Mth)), show that all three enzymes can synthesize RNA, but the precise modes of their action differ considerably. Unlike their QDE-1(Ncr) counterpart favoring processive RNA synthesis, QDE-1(Tte) and QDE-1(Mth) produce predominantly short RNA copies via primer-independent initiation. Surprisingly, a 3.19 Å resolution crystal structure of QDE-1(Tte) reveals a quasisymmetric dimer similar to QDE-1(Ncr). Further electron microscopy analyses confirm that QDE-1(Tte) occurs as a dimer in solution and retains this status upon interaction with a template. We conclude that divergence of orthologous RdRPs can result in functional innovation while retaining overall protein fold and quaternary structure

Extracellular Signal-Regulated Kinase 2 Interacts with and Is Negatively Regulated by the LIM-Only Protein FHL2 in Cardiomyocytes

The mitogen-activated protein kinase (MAPK) signaling pathway regulates diverse biologic functions including cell growth, differentiation, proliferation, and apoptosis. The extracellular signal-regulated kinases (ERKs) constitute one branch of the MAPK pathway that has been implicated in the regulation of cardiac differentiated growth, although the downstream mechanisms whereby ERK signaling affects this process are not well characterized. Here we performed a yeast two-hybrid screen with ERK2 bait and a cardiac cDNA library to identify novel proteins involved in regulating ERK signaling in cardiomyocytes. This screen identified the LIM-only factor FHL2 as an ERK interacting protein in both yeast and mammalian cells. In vivo, FHL2 and ERK2 colocalized in the cytoplasm at the level of the Z-line, and interestingly, FHL2 interacted more efficiently with the activated form of ERK2 than with the dephosphorylated form. ERK2 also interacted with FHL1 and FHL3 but not with the muscle LIM protein. Moreover, at least two LIM domains in FHL2 were required to mediate efficient interaction with ERK2. The interaction between ERK2 and FHL2 did not influence ERK1/2 activation, nor was FHL2 directly phosphorylated by ERK2. However, FHL2 inhibited the ability of activated ERK2 to reside within the nucleus, thus blocking ERK-dependent transcriptional responsiveness of ELK-1, GATA4, and the atrial natriuretic factor promoter. Finally, FHL2 partially antagonized the cardiac hypertrophic response induced by activated MEK-1, GATA4, and phenylephrine agonist stimulation. Collectively, these results suggest that FHL2 serves a repressor function in cardiomyocytes through its ability to inhibit ERK1/2 transcriptional coupling

Crystallization and preliminary crystallographic analysis of human aquaporin 1 at a resolution of 3.28 Å

Aquaporin water channels (AQPs) are found in almost every organism from humans to bacteria. In humans, 13 classes of AQPs control water and glycerol homeostasis. Knockout studies have suggested that modulating the activity of AQPs could be beneficial for the treatment of several pathologies. In particular, aquaporin 1 is a key factor in cell migration and angiogenesis, and constitutes a possible target for anticancer compounds and also for the treatment of glaucoma. Here, a preliminary crystallographic analysis at 3.28 Å resolution of crystals of human aquaporin 1 (hAQP1) obtained from protein expressed in Sf9 insect cells is reported. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 89.28, c = 174.9 Å, and contained one monomer per asymmetric unit. The hAQP1 biological tetramer is generated via the crystallographic fourfold axis. This work extends previous electron crystallo­graphic studies that used material extracted from human red blood cells, in which the resolution was limited to approximately 3.8 Å. It will inform efforts to improve lattice contacts and the diffraction limit for the future structure-based discovery of specific hAQP1 inhibitors.Published versio

Turning an asparaginyl endopeptidase into a peptide ligase

Butelase-1 is a peptide asparaginyl ligase (PAL) that efficiently catalyzes peptide bond formation after Asn/Asp (Asx), whereas its homologue butelase-2 is an asparaginyl endopeptidase (AEP) that catalyzes the reverse reaction, hydrolyzing Asx-peptide bonds. Since PALs and AEPs share essentially similar overall structures, we surmised that the S2 and S1′ substrate-binding pockets immediately flanking the catalytic S1 site constitute the major ligase activity determinants (LADs) that control the catalytic directionality of these enzymes. Here, we report the successful conversion of butelase-2 into butelase-1-like ligases based on the LAD hypothesis. We prepared 23 LAD mutants by mutating residues of the S2 pocket (LAD1) and/or the S1′ pocket (LAD2) of butelase-2 into homologous residues in PALs. These LAD mutants markedly diminished protease activity and increased ligase activity. In contrast, substituting 12 non-LAD residues to the corresponding residues in butelase-1 did not change their protease profiles. At physiological pH, mutations targeting both LADs resulted in shifting the catalytic directionality from 95% hydrolysis to >95% peptide ligation. This results in the engineering of efficient recombinant peptide ligases that were demonstrated to be useful for macrocyclization and site-specific labeling of bioactive peptides and proteins. Five high-resolution crystal structures of butelase-2 and its engineered mutants reveal subtle changes proximal to the catalytic S1 site that account for the reversal in enzymatic activity. Computational simulations of enzyme-substrate complexes suggest how the S-acyl intermediate is positioned at the S2 site and the substrate orientated, controlling accessibility of either water or incoming nucleophiles from the prime-side (S1′ site) of the catalytic center. Together, these features determine the catalytic directionality between hydrolysis and ligation in AEPs and PALs. Overall, this work validates the LAD hypothesis as a central guide for making peptide ligases from their corresponding widely available protease counterparts, to produce precision tools for exquisite site-specific conjugation and the biomanufacturing of biologics.Ministry of Education (MOE)National Research Foundation (NRF)Accepted versionThis research was supported by the Ministry of Education, Singapore, under its MOE AcRF Tier 3 Award No. MOE2016- T3-1-003, NMRC Grant Nos.CBRG/0028/2014, NRF2016NRF-CRP001-063, and Synzymes and Natural Products Center (SYNC), Nanyang Technological Universit

Assessing Opportunities to Increase Yield and Profit in Rainfed Lowland Rice Systems in Indonesia

In this study, we aimed to improve rice farmers’ productivity and profitability in rainfed lowlands through appropriate crop and nutrient management by closing the rice yield gap during the dry season in the rainfed lowlands of Indonesia. The Integrated Crop Management package, involving recommended practices (RP) from the Indonesian Agency for Agricultural Research and Development (IAARD), were compared to the farmers’ current practices at ten farmer-participatory demonstration plots across ten provinces of Indonesia in 2019. The farmers’ practices (FP) usually involved using old varieties in their remaining land and following their existing fertilizer management methods. The results indicate that improved varieties and nutrient best management practices in rice production, along with water reservoir infrastructure and information access, contribute to increasing the productivity and profitability of rice farming. The mean rice yield increased significantly with RP compared with FP by 1.9 t ha−1 (ranges between 1.476 to 2.344 t ha−1), and net returns increased, after deducting the cost of fertilizers and machinery used for irrigation supplements, by USD 656 ha−1 (ranges between USD 266.1 to 867.9 ha−1) per crop cycle. This represents an exploitable yield gap of 37%. Disaggregated by the wet climate of western Indonesia and eastern Indonesia’s dry climate, the RP increased rice productivity by 1.8 and 2.0 t ha−1, with an additional net return gain per cycle of USD 600 and 712 ha−1, respectively. These results suggest that there is considerable potential to increase the rice production output from lowland rainfed rice systems by increasing cropping intensity and productivity. Here, we lay out the potential for site-specific variety and nutrient management with appropriate crop and supplemental irrigation as an ICM package, reducing the yield gap and increasing farmers’ yield and income during the dry season in Indonesia’s rainfed-prone areas

Structural determinants for peptide-bond formation by asparaginyl ligases

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)-Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx-Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1' sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1-3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1-3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.Ministry of Education (MOE)Accepted versionThis research was supported by Academic Research Grant Tier 3 (MOE2016-T3-1-003) from the Singapore Ministry of Education (MOE) to the J.P.T., J.L., and C.-F.L. laboratories, and NMRC Grants CBRG/0028/2014 and NRF2016NRF-CRP001-063