ABSTRACT
The protein-protein interaction between the KIX motif of the transcriptional coactivator CBP/p300 and the transcriptional activator Myb is a high-value target due to its established role in certain acute myeloid leukemias (AML) and potential contributions to other cancers. However, the CBP/p300 KIX domain has multiple binding sites, several structural homologues, many binding partners, and substantial conformational plasticity, making it challenging to specifically target using small-molecule inhibitors. Here, we report a picomolar dual-site inhibitor (MybLL-tide) of the Myb-CBP/p300 KIX interaction. MybLL-tide has higher affinity for CBP/p300 KIX than any previously reported compounds while also possessing 5600-fold selectivity for the CBP/p300 KIX domain over other coactivator domains. MybLL-tide blocks the association of CBP and p300 with Myb in the context of the proteome, leading to inhibition of key Myb·KIX-dependent genes in AML cells. These results show that MybLL-tide is an effective, modifiable tool to selectively target the KIX domain and assess transcriptional effects in AML cells and potentially other cancers featuring aberrant Myb behavior. Additionally, the dual-site design has applicability to the other challenging coactivators that bear multiple binding surfaces.
Subject(s)
CREB-Binding Protein/antagonists & inhibitors , E1A-Associated p300 Protein/antagonists & inhibitors , Peptides/pharmacology , Proto-Oncogene Proteins c-myb/metabolism , Amino Acid Sequence , Binding Sites , Cell Line, Tumor , E1A-Associated p300 Protein/genetics , E1A-Associated p300 Protein/metabolism , Gene Expression Regulation/drug effects , Humans , Peptides/chemistry , Protein Binding , Protein Domains , Proto-Oncogene Proteins c-myb/geneticsABSTRACT
Protein-only ribonuclease P (PRORP) is an enzyme responsible for catalyzing the 5' end maturation of precursor transfer ribonucleic acids (pre-tRNAs) encoded by various cellular compartments in many eukaryotes. PRORPs from plants act as single-subunit enzymes and have been used as a model system for analyzing the function of the metazoan PRORP nuclease subunit, which requires two additional proteins for efficient catalysis. There are currently few molecular details known about the PRORP-pre-tRNA complex. Here, we characterize the determinants of substrate recognition by the single subunit Arabidopsis thaliana PRORP1 and PRORP2 using kinetic and thermodynamic experiments. The salt dependence of binding affinity suggests 4-5 contacts with backbone phosphodiester bonds on substrates, including a single phosphodiester contact with the pre-tRNA 5' leader, consistent with prior reports of short leader requirements. PRORPs contain an N-terminal pentatricopeptide repeat (PPR) domain, truncation of which results in a >30-fold decrease in substrate affinity. While most PPR-containing proteins have been implicated in single-stranded sequence-specific RNA recognition, we find that the PPR motifs of PRORPs recognize pre-tRNA substrates differently. Notably, the PPR domain residues most important for substrate binding in PRORPs do not correspond to positions involved in base recognition in other PPR proteins. Several of these residues are highly conserved in PRORPs from algae, plants, and metazoans, suggesting a conserved strategy for substrate recognition by the PRORP PPR domain. Furthermore, there is no evidence for sequence-specific interactions. This work clarifies molecular determinants of PRORP-substrate recognition and provides a new predictive model for the PRORP-substrate complex.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , RNA Precursors/metabolism , RNA, Plant/metabolism , RNA, Transfer/metabolism , Ribonuclease P/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Base Sequence , Nucleic Acid Conformation , RNA Precursors/chemistry , RNA Precursors/genetics , RNA, Plant/chemistry , RNA, Plant/genetics , RNA, Transfer/chemistry , RNA, Transfer/genetics , Ribonuclease P/chemistry , Ribonuclease P/geneticsABSTRACT
A program in which pharmacists help care for cystic fibrosis patients is described. The Egleston Cystic Fibrosis Center at Emory University houses outpatient clinic facilities and a 10-bed inpatient unit and is affiliated with Egleston Children's Hospital. The center provides full-service care for nearly 500 patients. Patients with mild to moderate exacerbations of pulmonary problems can receive their entire course of therapy at the center, and those with severe illness may complete their hospital stay there. A care team consisting of pharmacists, physicians, nurses, and others provides preventive and acute care. Patients can choose a "care partner" who will assist them with their care during any hospitalizations and at home. Both patient and care partner are taught drug administration, nutrition, and physical therapy and meet regularly with the care team. Patients must receive their medication education from a pharmacist before they can administer their own drugs. Pharmacists at the center also evaluate serum drug concentrations, stock the automated dispensing device, monitor for drug interactions, answer drug information questions, and attend multidisciplinary rounds. Pharmacy residents can work with the care team through rotations and clinic experience. Pharmacists at a cystic fibrosis center provide clinical services to patients and promote self-care.
