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ABSTRACT: Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross‐linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra‐articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein–protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen‐binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen‐binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra‐articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL‐1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL‐1 receptor. In vitro, IL‐1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra‐articular IL‐1‐induced IL‐6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL‐1Ra_M26 and native IL‐1Ra inhibited IL‐6 output when co‐administered with the IL‐1 challenge, only IL‐1Ra_M26 inhibited when administered 1 week prior to IL‐1 challenge. Collagen‐binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1238–1247, 2018. [ABSTRACT FROM AUTHOR]

The Ash1 protein is a daughter cell-specific repressor of HO gene transcription in Saccharomyces cerevisiae. Both ASH1 mRNA and protein are localized to the incipient daughter cell at the end of mitosis; Ash1 then inhibits HO transcription in the daughter cell after cytokinesis. Mother cells, in contrast, contain little or no Ash1 and thus are able to transcribe HO. We show that deletion of PHO85, which encodes a cyclin-dependent protein kinase, causes reduced transcription of HO and that this reduction is dependent on ASH1. In pho85 mutants, Ash1 protein is no longer asymmetrically localized and is present, instead, in both mother and daughter cells. Initially, it appears to be localized properly but then persists as daughter cells mature into mother cells. In contrast, ASH1 mRNA is localized appropriately to daughter cells in pho85 mutants. We observe that Ash1 protein is phosphorylated by Pho85 in vitro and that Ash1 stability increases in a pho85 mutant. These data suggest that phosphorylation of Ash1 by Pho85 governs stability of Ash1 protein. [ABSTRACT FROM AUTHOR]

Pho85 is a cyclin-dependent protein kinase (Cdk) in budding yeast with roles in cell metabolism and cell cycle progression. Activation of Pho85 occurs through association with Pho85 cyclins (Pcls), of which 10 are known. When complexed with the G1 cyclins, Pcl1 and Pcl2, Pho85 is required for cell cycle progression in the absence of the Cdc28-dependent cyclins, Cln1 and Cln2. To identify potential targets of Pcl2-Pho85, we performed a two-hybrid screen using the Pcl2 cyclin as bait and recovered the transcription factor Swi5 as a Pcl2-interacting protein. We performed both biochemical and genetic tests to discover the biological significance of the interaction between Pcl2 and Swi5 seen in the two-hybrid assay. We found that Swi5 interacts in vitro with Pho85 cyclins and is phosphorylated in vitro by the Pho80-Pho85 kinase. We discovered that a subset of genes that are controlled by Swi5 and a homologous transcription factor, Ace2, was misregulated in a pho85 deletion strain; expression of the ASH1 and CTS1 genes was reduced in an ace2 deletion strain, whereas expression of both genes was increased in an ace2Δ pho85Δ double mutant. We also found that overexpression of SWI5 caused cell lethality in a pho85 deletion strain. Our results are consistent with misregulation of Swi5 activity in vivo in the absence of Pho85 and implicate Swi5 as a potential substrate of Pho85 cyclin-dependent kinase complexes. [ABSTRACT FROM AUTHOR]