A Whole Genome-Wide Arrayed CRISPR Screen in Primary Organ Fibroblasts to Identify Regulators of Kidney Fibrosis.

Kidney fibrosis presents a hallmark of chronic kidney disease. With ever-increasing patient numbers and limited treatment options available, novel strategies for therapeutic intervention in kidney disease are warranted. Fibrosis commonly results from a wound healing response to repeated or chronic tissue damage, irrespective of the underlying etiology, and can occur in virtually any solid organ or tissue. In order to identify targets relevant for kidney fibrosis, we aimed to employ CRISPR screening in primary human kidney fibroblasts. We demonstrate that CRISPR technology can be applied in primary kidney fibroblasts and can furthermore be used to conduct arrayed CRISPR screening using a high-content imaging readout in a whole genome-wide manner. Hits coming out of this screen were validated using orthogonal approaches and present starting points for validation of novel targets relevant to kidney disease.

Development of Potent and Metabolically Stable APJ Ligands with High Therapeutic Potential.

The apelin ligand receptor system is an important target to develop treatment strategies for cardiovascular diseases. Although apelin exhibits strong inotropic effects, its pharmaceutical application is limited because no agonist with suitable properties is available. On the one hand, peptide ligands are too instable, and on the other hand, small-molecule agonists show only low potency. This study describes the development of apelin (APJ) receptor agonists with not only high activity but also metabolic stability. Several strategies including capping of termini, insertion of unnatural amino acids, cyclization, and lipidation were analyzed. Peptide activity was tested using a Ca2+ -mobilization assay and the degradation of selected analogues was analyzed in rat plasma. The best results were obtained by N-terminal lipidation of a 13-mer apelin derivative. This analogue displayed a half-life of 29 h in rat plasma, compared with 0.025 h for the wild-type peptide. Furthermore, in vivo pharmacokinetics revealed a clearance of 0.049 L h-1 kg-1 and a half-life of 0.36 h. In summary, amino acid substitution and fatty acid modification resulted in a potent and 1000-fold more stable peptide that exhibits high pharmaceutical potential.

Novel analgesic triglycerides from cultures of Agaricus macrosporus and other basidiomycetes as selective inhibitors of neurolysin.

The agaricoglycerides are a new class of fungal secondary metabolites that constitute esters of chlorinated 4-hydroxy benzoic acid and glycerol. They are produced in cultures of the edible mushroom, Agaricus macrosporus, and several other basidiomycetes of the genera Agaricus, Hypholoma, Psathyrella and Stropharia. The main active principle, agaricoglyceride A, showed strong activities against neurolysin, a protease involved in the regulation of dynorphin and neurotensin metabolism (IC50 = 200 nM), and even exhibited moderate analgesic in vivo activities in an in vivo model. Agaricoglyceride monoacetates (IC50 = 50 nM) showed even stronger in vitro activities. Several further co-metabolites with weaker or lacking bioactivities were also obtained and characterized. Among those were further agaricoglyceride derivatives, as well as further chlorinated phenol derivatives such as the new compound, agaricic ester. The characteristics of the producer organisms, the isolation of bioactive metabolites from cultures of A. macrosporus, their biological activities, and preliminary results on their occurrence in basidiomycetes, are described.

Fusion of Aequorea victoria GFP and aequorin provides their Ca(2+)-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer.

The bioluminescence emitted by Aequorea victoria jellyfish is greenish while its single bioluminescent photoprotein aequorin emits blue light. This phenomenon may be explained by a bioluminescence resonance energy transfer (BRET) from aequorin chromophore to green fluorescent protein (GFP) co-localized with it. However, a slight overlapping of the aequorin bioluminescence spectrum with the GFP absorption spectrum and the absence of marked interaction between these proteins in vitro pose a question on the mechanism providing the efficient BRET in A. victoria. Here we report the in vitro study of BRET between homologous Ca(2+)-activated photoproteins, aequorin or obelin (Obelia longissima), as bioluminescence energy donors, and GFP, as an acceptor. The fusions containing donor and acceptor proteins linked by a 19 aa peptide were purified after expressing their genes in Escherichia coli cells. It was shown that the GFP-aequorin fusion has a significantly greater BRET efficiency, compared to the GFP-obelin fusion. Two main factors responsible for the difference in BRET efficiency of these fusions were revealed. First, it is the presence of Ca(2+)-induced interaction between the donor and acceptor in the aequorin-containing fusion and the absence of the interaction in the obelin-containing fusion. Second, it is a red shift of GFP absorption toward better overlapping with aequorin bioluminescence induced by the interaction of aequorin with GFP. Since the connection of the two proteins in vitro mimics their proximity in vivo, Ca(2+)-induced interaction between aequorin and GFP may occur in A. victoria jellyfish providing efficient BRET in this organism.

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.

Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process.

During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo- electron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon-anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.

Formation of 70S ribosomes: large activation energy is required for the adaptation of exclusively the small ribosomal subunit.

Association of ribosomal subunits is an essential reaction during the initiation phase of protein synthesis. Optimal conditions for 70S formation in vitro were determined to 20 mM Mg2+ and 30 mM K+. Under these conditions, the association reaction proceeds with first order kinetics, suggesting a conformational change to be the rate-limiting step. 70S formation separates into two sub-reactions, the adaptation of the ribosomal subunits to the association conditions and the association step itself. The activation energy of the process was determined to 78 kJ/mol and revealed to be required exclusively for the adaptation of the small subunit, rather than the large subunit or the association step. The presence of mRNA [poly(U)] together with cognate AcPhe-tRNA, accelerates the association rate significantly, forming a well-defined 70S peak in sucrose gradient profiles. mRNA alone provokes an equivalent acceleration, however, the resulting 70S couple impresses as an ill-defined, broad peak, probably indicating the readiness of the ribosome for tRNA binding, upon which the ribosome flips into a defined state.