Close Homology Research Articles

Structural comparison of homologous protein-RNA interfaces reveals widespread overall conservation contrasted with versatility in polar contacts.

Protein-RNA interactions play a critical role in many cellular processes and pathologies. However, experimental determination of protein-RNA structures is still challenging, therefore computational tools are needed for the prediction of protein-RNA interfaces. Although evolutionary pressures can be exploited for structural prediction of protein-protein interfaces, and recent deep learning methods using protein multiple sequence alignments have radically improved the performance of protein-protein interface structural prediction, protein-RNA structural prediction is lagging behind, due to the scarcity of structural data and the flexibility involved in these complexes. To study the evolution of protein-RNA interface structures, we first identified a large and diverse dataset of 2,022 pairs of structurally homologous interfaces (termed structural interologs). We leveraged this unique dataset to analyze the conservation of interface contacts among structural interologs based on the properties of involved amino acids and nucleotides. We uncovered that 73% of distance-based contacts and 68% of apolar contacts are conserved on average, and the strong conservation of these contacts occurs even in distant homologs with sequence identity below 20%. Distance-based contacts are also much more conserved compared to what we had found in a previous study of homologous protein-protein interfaces. In contrast, hydrogen bonds, salt bridges, and π-stacking interactions are very versatile in pairs of protein-RNA interologs, even for close homologs with high interface sequence identity. We found that almost half of the non-conserved distance-based contacts are linked to a small proportion of interface residues that no longer make interface contacts in the interolog, a phenomenon we term "interface switching out". We also examined possible recovery mechanisms for non-conserved hydrogen bonds and salt bridges, uncovering diverse scenarios of switching out, change in amino acid chemical nature, intermolecular and intramolecular compensations. Our findings provide insights for integrating evolutionary signals into predictive protein-RNA structural modeling methods.

An Interdisciplinary Approach Provides Insights into the Pronounced Selectivity of Compound 42 for DPP9.

Dipeptidyl peptidase 8 (DPP8) and 9 (DPP9) are proteases gaining significant attention for their role in health and disease. Distinctive studies of these proteases are hampered by their close homology. Furthermore, designing selective compounds is a major challenge due to the highly conserved catalytic site. Here, we provide mechanistic insights underlying the DPP9-over-DPP8 selectivity of the semi-selective inhibitor "Compound 42". We performed enhanced sampling molecular dynamics simulations to investigate the binding pose of "Compound 42", which enabled the design of various DPP9 mutants that were characterized through a combination of biochemical (Ki determinations) and in silico approaches. Our findings show that DPP9 residue F253 is an important selectivity-determining factor. This work marks the discovery and validation of a structural feature that can be exploited for the design of DPP8 or DPP9 selective inhibitors.

ESI MS Studies of T. Brucei Trypanothione Reductase and its Reactions With Selected Metal Compounds

AbstractTrypanothione reductase from Trypanosoma brucei (TbTR) is a key enzyme of the parasite responsible for human African trypanosomiasis. TbTR catalyzes the reduction of trypanothione, an antioxidant dithiol that protects trypanosomatid parasites from oxidative stress induced by the mammalian host as a defense system. TbTR is considered an attractive target for the development of novel anti‐parasitic drugs because it is essential for parasite survival and has no close homologues in humans. Previous studies have shown that metal compounds can act as potent inhibitors of trypanothione reductase and of related enzymes and may be considered for disease treatment. To better understand the molecular basis of such inhibition, we report the study of the interactions of a small group of metal compounds with TbTR, analyzed by ESI MS measurements. This technique allows direct visualization of the enzyme in solution and the detection and characterization of the metal binding process. Accordingly, TbTR was reacted with a panel of metal compounds including thimerosal, auranofin, its halogen analogues, and bismuth acetate. ESI MS results provide a detailed description of the interactions taking place for each metal compound tested. The implications of these results for the development of novel metal‐based compounds as potential anti‐trypanosomal agents are discussed.

Inhibition mechanism of potential antituberculosis compound lansoprazole sulfide

Tuberculosis is one of the most common causes of death worldwide, with a rapid emergence of multi-drug-resistant strains underscoring the need for new antituberculosis drugs. Recent studies indicate that lansoprazole-a known gastric proton pump inhibitor and its intracellular metabolite, lansoprazole sulfide (LPZS)-are potential antituberculosis compounds. Yet, their inhibitory mechanism and site of action still remain unknown. Here, we combine biochemical, computational, and structural approaches to probe the interaction of LPZS with the respiratory chain supercomplex III2IV2 of Mycobacterium smegmatis, a close homolog of Mycobacterium tuberculosis supercomplex. We show that LPZS binds to the Qo cavity of the mycobacterial supercomplex, inhibiting the quinol substrate oxidation process and the activity of the enzyme. We solve high-resolution (2.6 Å) cryo-electron microscopy (cryo-EM) structures of the supercomplex with bound LPZS that together with microsecond molecular dynamics simulations, directed mutagenesis, and functional assays reveal key interactions that stabilize the inhibitor, but also how mutations can lead to the emergence of drug resistance. Our combined findings reveal an inhibitory mechanism of LPZS and provide a structural basis for drug development against tuberculosis.

Structural insights into brassinosteroid export mediated by the Arabidopsis ABC transporter ABCB1

Brassinosteroids (BRs) are steroidal phytohormones indispensable for plant growth, development, and responses to environmental stresses. The export of bioactive BRs to the apoplast is essential for BR signaling initiation, which requires binding of a BR molecule to the extracellular domains of the plasma membrane–localized receptor complex. We have previously shown that the Arabidopsis thaliana ATP-binding cassette (ABC) transporter ABCB19 functions as a BR exporter, and, together with its close homolog ABCB1, positively regulates BR signaling. Here, we demonstrate that ABCB1 is another BR transporter. The ATP hydrolysis activity of ABCB1 can be stimulated by bioactive BRs, and its transport activity was confirmed in proteoliposomes and protoplasts. Structures of ABCB1 were determined in substrate-unbound (apo), brassinolide (BL)-bound, and ATP plus BL-bound states. In the BL-bound structure, BL is bound to the hydrophobic cavity formed by the transmembrane domain and triggers local conformational changes. Together, our data provide additional insights into ABC transporter–mediated BR export.

Soluble amyloid precursor protein-alpha mediates post-ischemic neovascularization after myocardial infarction

Abstract Background Amyloid precursor protein (APP) gives rise to amyloid-β peptides and thus has a key role in the pathogenesis of Alzheimer´s disease. APP is also highly expressed outside the brain including cells of the cardiovascular system, in particular endothelial cells (1). However, the role of APP and its proteolytic fragments in cardiovascular function and disease remains unknown. Purpose The main purpose of this study was to understand the function APP and the close homologue, amyloid precursor-like protein 2 (APLP2) in endothelial cells. Methods To test the potential role of endothelial APP and APLP2 in vivo, we generated inducible endothelium-specific APP/APLP2-double deficient mice (EC-APP/APLP2-KO) by crossing App fl/fl; Aplp2 fl/fl mice with the Cdh5-CreERT2 line. Animals were analyzed after exposure to myocardial infarction (MI). Human umbilical vein endothelial cells were used to study the mechanisms underlying APP/APLP2-mediated endothelial function in vitro. Results APP and APLP2 were found to be upregulated in cardiac endothelial cells after MI both in mice and humans. Loss of APP and APLP2 in endothelial cells had no effect on the basal heart function in mice. But, when subjected to MI, approximately 40 % of the EC-APP/APLP2-KO mice died within 3 weeks after MI in contrast to control animals, where hardly any death occurred. Survival of endothelium-specific APP- or APLP2-single knockouts was indistinguishable from that of control animals after MI. The surviving EC-APP/APLP2-KO mice showed larger infarct scars than the control animals. Magnetic resonance imaging and echocardiography at 3 weeks after MI revealed that EC-APP/APLP2-KO had more severe contractile dysfunction than the control mice (left ventricular ejection fraction 32.8 % vs. 44.9 %, respectively). As the underlying cause of this we identified a strong impairment of post-ischemia neovascularization in the absence of endothelial APP and APLP2. We found that soluble APP-α (APPsα) generated by non-amyloidogenic processing was the predominant form of APP in cardiac endothelial cells after MI. We therefore tested whether the expression of APPsα was able to rescue the observed phenotype of EC-APP/APLP2-KO after MI. Indeed, APPsα knock-in mice that solely express APPsα and no other APP fragments or cell surface APP showed the same phenotype as wild-type animals, indicating that APPsα is sufficient to mimic the function of APP. Under in vitro conditions, we found that APPsα was able to promote tube formation of cultured endothelial cells. APPsα also induced downstream signalling events such as AKT, ERK, and p38 activation which are reminiscent of growth factor induced signaling processes. Conclusions APPsα mediates post-ischemia neovascularization in the heart by exerting growth factor-like activity in endothelial cells, thereby protecting against cardiac injury.

Unlocking a Key Residue in a Lipase for Efficient Polyethylene Terephthalate (PET) Hydrolysis and Influencing Depolymerization Product Profiles

AbstractPolyethylene terephthalate (PET) pollution is a global challenge. Advancing the bioprospecting of PET‐degrading enzymes through metagenomics and using computational and functional methods to identify key positions influencing the catalytic rate and selectivity are part of the solution. Here, we report PETase activity in the metagenomic lipase LipMRD9, which exhibits peak activity at 30 °C and pH 9.0 and has a denaturation temperature of 42 °C. In addition to acting on long‐chain triglycerides (up to ∼13 units (U)/mg, pH 8.0, 30 °C) and a wide range of 34 other esters (up to ∼228 U/g), LipMRD9 hydrolyses mono(2‐hydroxyethyl) terephthalate (∼57 U/g) and bis(2‐hydroxyethyl) terephthalate (∼131 U/g). It also efficiently deconstructs GoodFellow amorphous submicro‐ and nanosized PET particles (∼984/2238 μM degradation products at 30/40 °C, pH 7.0, 21.5 h) and films (∼112/198 μM degradation products at 30/40 °C, pH 7.0, 7 days). Through molecular modelling and experimental analysis, the active site of LipMRD9 was revealed, identifying a key residue contributing to its PETase activity compared with those of its closest homologues. This residue plays a crucial role in determining the distinct profiles of degradation products from PET hydrolysis and should be studied in other PETases for its influence on the catalytic process.

An Adenosine Analogue Library Reveals Insights into Active Sites of Protein Arginine Methyltransferases and Enables the Discovery of a Selective PRMT4 Inhibitor.

Protein arginine methyltransferases (PRMTs) represent promising drug targets. However, the lack of isoform-selective chemical probes poses a significant hurdle in deciphering their biological roles. To address this issue, we devised a library of 100 diverse adenosine analogues, enabling a detailed exploration of the active site of PRMTs. Despite their close homology, our analysis unveiled specific chemical trends unique to the individual members. Notably, compound YD1130 demonstrated over 1000-fold selectivity for PRMT4 (IC50 < 0.5 nM) over a panel of 38 methyltransferases, including the other PRMTs. Its prodrug YD1342 exhibited potent inhibition on cellular substrate methylation, breast cancer cell colony formation, and tumor growth in the animal model, surpassing or matching known PRMT4-specific inhibitors. In summary, our focused library not only illuminates the intricate active sites of PRMTs to facilitate the discovery of highly potent and isoform-selective probes but also offers a versatile blueprint for identifying chemical probes for other methyltransferases.

Comparative Genomics of Carbohydrate Utilization in Bacteria of the Family Sphaerochaetaceae: Evolutionary Origin of the Genes Encoding Galacturonidase and Unsaturated Rhamnogalacturonyl Hydrolase

Abstract A comparative analysis of carbohydrate degradation proteins encoded in the currently available genomic sequences of bacteria of the family Sphaerochaetaceae, namely Sphaerochaeta associata GLS2T, S. globosa BuddyT, S. pleomorpha GrapesT, S. halotolerans 4-11T, S. halotolerans 585, Sphaerochaeta sp. S2, Sphaerochaeta sp. PS, and Parasphaerochaetacoccoides SPN1T was carried out. The genomes of Sphaerochaeta spp. encode a medium-sized and diverse set of proteins potentially involved in the degradation of different classes of carbohydrates, mainly oligosaccharides. All studied genomes encode glycoside hydrolases of the GH1, GH2, GH3, GH4, GH13, GH20, GH28, GH36, GH43, GH57, GH63, GH77 and GH105 families, as well as carbohydrate esterases of the CE8 and CE9 families. All studied bacteria, with the exception of P. coccoides SPN1T, have many genes of the GH31 family proteins. The studied members of Sphaerochaetaceae do not have genes coding for endo-β-acetylmuramidase (lysozyme) of the GH23 family, which participates in peptidoglycan turnover. However, the genomes of S. associata, S. globosa, Sphaerochaeta sp. PS, and S. pleomorpha contain exo-β-acetylmuramidase genes (GH171 family). A significant part of the genes encoding carbohydrate degradation enzymes have the closest homologues among representatives of the phyla Bacillota, Bacteroidota, and Pseudomonadota. The genomes of the studied bacteria encode proteins that could potentially be involved in the degradation of pectin. The ability of the Sphaerochaetaceae members to use pectin for growth, as well as the evolutionary origin of the genes encoding potential α-galacturonidase (GH4 family) and unsaturated glucuronyl/rhamnogalacturonyl hydrolase (GH105 family), which participate in the degradation of pectin components, were studied.

In silico structural studies on the vesicular neutral amino acid transporter NTT4 (SLC6A17)

NTT4 is one of the neutral amino acid transporters that regulate neural concentration of precursors for glutamate biosynthesis. Here, we provide insight into the structure of NTT4 and rationalize substrate selectivity. Furthermore, we demonstrate how the mutations associated with mental disabilities imply malfunction of the transporter at the molecular level. We also compared the structures of NTT4 and B0AT2 (SLC6A15), which is a close homolog, sharing 66 % of the common amino acids. Our analyses may be useful in the search for compounds that inhibit substrate transport. Moreover, they allow a better understanding of the function of these transporters.

Selectivity analysis of diaminopyrimidine-based inhibitors of MTHFD1, MTHFD2 and MTHFD2L

The mitochondrial enzyme methylenetetrahydrofolate dehydrogenase (MTHFD2) is involved in purine and thymidine synthesis via 1C metabolism. MTHFD2 is exclusively overexpressed in cancer cells but absent in most healthy adult human tissues. However, the two close homologs of MTHFD2 known as MTHFD1 and MTHFD2L are expressed in healthy adult human tissues and share a great structural resemblance to MTHFD2 with 54% and 89% sequence similarity, respectively. It is therefore notably challenging to find selective inhibitors of MTHFD2 due to the structural similarity, in particular protein binding site similarity with MTHFD1 and MTHFD2L. Tricyclic coumarin-based compounds (substrate site binders) and xanthine derivatives (allosteric site binders) are the only selective inhibitors of MTHFD2 reported till date. Nanomolar potent diaminopyrimidine-based inhibitors of MTHFD2 have been reported recently, however, they also demonstrate significant inhibitory activities against MTHFD1 and MTHFD2L. In this study, we have employed extensive computational modeling involving molecular docking and molecular dynamics simulations in order to investigate the binding modes and key interactions of diaminopyrimidine-based inhibitors at the substrate binding sites of MTHFD1, MTHFD2 and MTHFD2L, and compare with the tricyclic coumarin-based selective MTHFD2 inhibitor. The outcomes of our study provide significant insights into desirable and undesirable structural elements for rational structure-based design of new and selective inhibitors of MTHFD2 against cancer.

Evolving Archetypes: Learning from Pathogen Emergence on a Nonmodel Host.

Research initiatives undertaken in response to disease outbreaks accelerate our understanding of microbial evolution, mechanisms of virulence and resistance, and plant-pathogen coevolutionary interactions. The emergence and global spread of Pseudomonas syringae pv. actinidiae (Psa) on kiwifruit (Actinidia chinensis) showed that there are parallel paths to host adaptation and antimicrobial resistance evolution, accelerated by the movement of mobile elements. Significant progress has been made in identifying type 3 effectors required for virulence and recognition in A. chinensis and Actinidia arguta, broadening our understanding of how host-mediated selection shapes virulence. The rapid development of Actinidia genomics after the Psa3 pandemic began has also generated new insight into molecular mechanisms of immunity and resistance gene evolution in this recently domesticated, nonmodel host. These findings include the presence of close homologs of known resistance genes RPM1 and RPS2 as well as the novel expansion of CCG10-NLRs (nucleotide-binding leucine-rich repeats) in Actinidia spp. The advances and approaches developed during the pandemic response can be applied to new pathosystems and new outbreak events.

Identification of clerodane diterpene modifying cytochrome P450 (CYP728D26) in Salvia divinorum - en route to psychotropic salvinorin A biosynthesis.

Salvia divinorum is a hallucinogenic plant native to the Oaxaca in Mexico. The active ingredient for psychotropic effects in this plant is salvinorin A, a potent and highly selective κ-opioid receptor agonist. Salvinorin A is distinct from other well-known opioids, such as morphine and codeine, in that it is a non-nitrogenous diterpenoid with no affinity for μ-opioid receptor, the prime receptor of alkaloidal opioids. A terpene opioid that selectively targets a new opioid receptor (κ-opioid receptor) can be instrumental in developing alternative analgesics. Elucidation of the salvinorin A biosynthetic pathway can help bio-manufacture diverse semi-synthetic derivatives of salvinorin A but, to date, only two enzymes in the Salvinorin A pathway have been identified. Here, we identify CYP728D26 that catalyzes a C18 oxygenation on crotonolide G, which bears a clerodane backbone. Biochemical identity of CYP728D26 was validated by in vivo reconstitution in yeast, 1H- and 13C-NMR analyses of the purified product, and kinetic analysis of CYP728D26 with a Km value of 13.9 μM. Beyond the single oxygenation on C18, collision-induced dissociation analysis suggested two additional oxygenations are catalyzed by CYP728D26 to form crotonoldie G acid, although this carboxylic acid form is a minor product. Its close homologue CYP728D25 exhibited a C1-hydroxylation on the clerodane backbone in a reconstituted yeast system. However, CYP728D25 showed no activity in in vitro assays. This result implies that catalytic activities observed from overexpression systems should be interpreted cautiously. This work identified a new CYP catalyst and advanced our knowledge of salvinorin A biosynthesis.

Structural and functional analysis of plant ELO-like elongase for fatty acid elongation.

ELO-like elongase is a condensing enzyme elongating long chain fatty acids in eukaryotes. Eranthis hyemalis ELO-like elongase (EhELO1) is the first higher plant ELO-type elongase that is highly active in elongating a wide range of polyunsaturated fatty acids (PUFAs) and some monounsaturated fatty acids (MUFAs). This study attempted using domain swapping and site-directed mutagenesis of EhELO1 and EhELO2, a close homologue of EhELO1 but with no apparent elongase activity, to elucidate the structural determinants critical for catalytic activity and substrate specificity. Domain swapping analysis of the two showed that subdomain B in the C-terminal half of EhELO1 is essential for MUFA elongation while subdomain C in the C-terminal half of EhELO1 is essential for both PUFA and MUFA elongations, implying these regions are critical in defining the architecture of the substrate tunnel for substrate specificity. Site-directed mutagenesis showed that the glycine at position 220 in the subdomain C plays a key role in differentiating the function of the two elongases. In addition, valine at 161 and cysteine at 165 in subdomain A also play critical roles in defining the architecture of the deep substrate tunnel, thereby contributing significantly to the acceptance of, and interaction with primer substrates.

Claudin-4 polymerizes after a small extracellular claudin-3-like substitution

Tight junctions play a pivotal role in the functional integrity of the human body by forming barriers that compartmentalize tissues and protect the body from external threats. Essential components of tight junctions are the transmembrane claudin proteins, which can polymerize into tight junction strands and meshworks. This study delves into the structural determinants of claudin polymerization, utilizing the close homology yet strong difference in polymerization capacity between claudin-3 and claudin-4. Through a combination of sequence alignment and structural modeling, critical residues in the second extracellular segment are pinpointed. Molecular dynamics simulations provide insights into the interactions of and the conformational changes induced by the identified extracellular segment 2 residues. Live-STED imaging demonstrates that introduction of these residues from claudin-3 into claudin-4 significantly enhances polymerization in non-epithelial cells. In tight junction-deficient epithelial cells, mutated claudin-4 not only influences tight junction morphology but also partially restores barrier function. Understanding the structural basis of claudin polymerization is crucial, as it offers insights into the dynamic nature of tight junctions. This knowledge could be applied to targeted therapeutic interventions, offering insight to repair or prevent barrier defects associated with pathological conditions, or introduce temporary barrier openings during drug delivery.

Identification of glycosyltransferases mediating 2-O-arabinopyranosyl and 2-O-galactosyl substitutions ofglucuronosyl side chains of xylan.

Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O-2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2-O-arabinopyranose (Arap) or 2-O-galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3-clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Arap from UDP-Arap onto O-2 of GlcA side chains of xylan, and thus it was named xylan 2-O-arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T-DNA knockout mutation showing a loss of the Arap substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus, lemon-scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2-O-Arap and 2-O-Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site-directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2-O-arabinopyranosylation and 2-O-galactosylation of GlcA side chains of xylan.

Abstract Tu121: Investigating Mechanisms Of MCUB Inhibition Of Mitochondrial Calcium Uptake

The mitochondrial calcium uniporter mediates Ca 2+ entry into the matrix, and matches energetic supply to demand during cardiac activity. However, many cardiac diseases are characterized by mitochondrial Ca 2+ overload, such as ischemia/reperfusion (I/R) injury. The uniporter channel is typically formed by a tetramer of MCU subunits. These subunits have two transmembrane domains and create a pore lined with negative charges to allow rapid and selective Ca 2+ permeation. Intriguingly, many studies have shown that a close homolog, MCUB can potentially oligomerize with MCU subunits to alter the pore. When the tetramer is made of MCU Ca 2+ transmission is facilitated, but the presence of MCUB inhibits Ca 2+ intake through the channel, suggesting that it is a dominant negative subunit. Moreover, recent studies in animals subject to ischemic cardiovascular injury has revealed endogenous upregulation of the MCUB subunit, proposed as an endogenous mechanism to prevent Ca 2+ overload. Deletion of MCUB enhances activation and uptake when exposed to high Ca 2+ , making MCUB −/− cells prone to mitochondrial Ca 2+ overload and pore opening. Here, we investigate the mechanisms by which MCUB regulates the behavior of the channel, focusing on how MCUB may influence complex composition and regulate its targeting. For this study, we performed structure-function studies of reconstituted MCUB in MCU -/- MCUB -/- double knockout HEK 293T cells. Using modified MCUB constructs and a range of MCU to MCUB transfection ratios, we queried how many MCUB subunits can successfully incorporate into uniporter complex. We subsequently created a range of tetrameric concatemers featuring different ratios of MCU to MCUB, allowing us to observe that increasing amounts of MCUB incorporation lead to greater inhibition of mitochondrial Ca 2+ uptake. Finally, we examined whether replacing portions of MCUB with the corresponding MCU sequence was able to rescue Ca 2+ uptake, finding that these substitutions failed to significantly increase mitochondrial Ca 2+ uptake. Our findings clarify how MCUB incorporation into the uniporter complex leads to dominant-negative inhibition of mitochondrial Ca 2+ uptake.

Functional Analysis of the Channelrhodopsin Genes from the Green Algae of the White Sea Basin

Optogenetics, the method of light-controlled regulation of cellular processes is based on the use of the channelrhodopsins that directly generate photoinduced currents. Most of the channelrhodopsin genes have been identified in the green microalgae Chlorophyta, and the demand for increasing the number of functionally characterized channelrhodopsins and the diversity of their photochemical parameters keeps growing. We performed the expression analysis of cation channelrhodopsin (CCR) genes in natural isolates of microalgae of the genera Haematococcus and Bracteacoccus from the unique Arctic Circle region. The identified full-length CCR transcript of H. lacustris is the product of alternative splicing and encodes the Hl98CCR2 protein with no photochemical activity. The 5′-partial fragment of the B. aggregatus CCR transcript encodes the Ba34CCR protein containing a conserved TM1-TM7 membrane domain and a short cytosolic fragment. Upon heterologous expression of the TM1-TM7 fragment in CHO-K1 cell culture, light-dependent current generation was observed with the parameters corresponding to those of the CCR. The first discovered functional channelrhodopsin of Bracteacoccus has no close CCR homologues and may be of interest as a candidate for optogenetics.

1,4-Pyrazolyl-Containing SAFit-Analogues are Selective FKBP51 Inhibitors With Improved Ligand Efficiency and Drug-Like Profile.

The FK506 binding protein 51 (FKBP51) is an appealing drug target due to its role in several diseases such as depression, anxiety, chronic pain and obesity. Towards this, selectivity versus the close homolog FKBP52 is essential. However, currently available FKBP51-selective ligands such as SAFit2 are too large and lack drug-like properties. Here, we present a structure activity relationship (SAR) analysis of the pipecolic ester moiety of SAFit1 and SAFit2, which culminated in the discovery of the 1,4-pyrazolyl derivative 23 d, displaying a binding affinity of 0.077 μM for FKBP51, reduced molecular weight (541.7 g/mol), lower hydrophobicity (cLogP=3.72) and higher ligand efficiency (LE=0.25). Cocrystal structures revealed the importance of the 1,4- and 1,3,4- substitution patterns of the pyrazole ring versus the 1,4,5 arrangement.

Investigating a role for PUB17 and PUB16 in the self-incompatibility signaling pathway in transgenic Arabidopsis thaliana.

In Brassicaceae self-incompatibility (SI), self-pollen rejection is initiated by the S-haplotype specific interactions between the pollen S cysteine-rich/S-locus protein 11 (SCR/SP11) ligands and the stigma S receptor kinases (SRK). In Brassica SI, a member of the Plant U-Box (PUB) E3 ubiquitin ligases, ARM-repeat containing 1 (ARC1), is then activated by SRK in this stigma and cellular events downstream of this cause SI pollen rejection by inhibiting pollen hydration and pollen tube growth. During the transition to selfing, Arabidopsis thaliana lost the SI components, SCR, SRK, and ARC1. However, this trait can be reintroduced into A.thaliana by adding back functional copies of these genes from closely related SI species. Both SCR and SRK are required for this, though the degree of SI pollen rejection varies between A.thaliana accessions, and ARC1 is not always needed to produce a strong SI response. For the A.thaliana C24 accession, only transforming with Arabidopsis lyrata SCR and SRK confers a strong SI trait (SI-C24), and so here, we investigated if ARC1-related PUBs were involved in the SI pathway in the transgenic A.thaliana SI-C24 line. Two close ARC1 homologs, PUB17 and PUB16, were selected, and (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)technology was used to generate pub17 and pub16 mutations in the C24 accession. These mutants were then crossed into the transgenic A.thaliana SI-C24 line and their potential impact on SI pollen rejection was investigated. Overall, we did not observe any significant differences in SI responses to implicate PUB17 and PUB16 functioning in the transgenic A.thaliana SI-C24 stigma to reject SI pollen.