Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization–Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.
Chemokine release promotes cross-talk between opioid and chemokine receptors that in part leads to reduced efficacy of morphine in the treatment of chronic pain. On the basis of the possibility that a MOR-CCR5 heteromer is involved in such cross-talk, we have synthesized bivalent ligands (MCC series) that contain mu opioid agonist and CCR5 antagonist pharmacophores linked through homologous spacers (14–24 atoms). When tested on lipopolysaccharide-inflamed mice, a member of the series (MCC22; 3e) with a 22-atom spacer exhibited profound antinociception (i.t. ED50 = 0.0146 pmol/mouse) that was 2000× greater than morphine. Moreover, MCC22 was ∼3500× more potent than a mixture of mu agonist and CCR5 antagonist monovalent ligands. These data strongly suggest that MCC22 acts by bridging the protomers of a MOR-CCR5 heteromer having a TM5,6 interface. Molecular simulation studies are consistent with such bridging. This study supports the MOR-CCR5 heteromer as a novel target for the treatment of chronic pain.
A precise balance of DNA methylation and demethylation is required for epigenetic control of cell identity, development, and growth. DNA methylation marks are introduced by de novo DNA methyltransferases DNMT3a/b and are maintained throughout cell divisions by DNA methyltransferase 1 (DNMT1), which adds methyl groups to hemimethylated CpG dinucleotides generated during DNA replication. Ten eleven translocation (TET) dioxygenases oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC), a process known to induce DNA demethylation and gene reactivation. In this study, we investigated the catalytic activity of human DNMT1 in the presence of oxidized forms of mC. A mass spectrometry-based assay was employed to study the kinetics of DNMT1-mediated cytosine methylation in CG dinucleotides containing C, mC, hmC, fC, or caC across from the target cytosine. Homology modeling, coupled with molecular dynamics simulations, was used to explore the structural consequences of mC oxidation with regard to the geometry of protein–DNA complexes. The DNMT1 enzymatic activity was strongly affected by the oxidation status of mC, with the catalytic efficiency decreasing in the following order: mC > hmC > fC > caC. Molecular dynamics simulations revealed that DNMT1 forms an unproductive complex with DNA duplexes containing oxidized forms of mC as a consequence of altered interactions of the target recognition domain of the protein with the C-5 substituent on cytosine. Our results provide new structural and mechanistic insight into TET-mediated DNA demethylation.
Abstract Purpose: Advances in immunotherapy have revolutionized care for some patients with cancer. However, current checkpoint inhibitors are associated with significant toxicity and yield poor responses for patients with central nervous system tumors, calling into question whether cancer immunotherapy can be applied to glioblastoma multiforme. We determined that targeting the CD200 activation receptors (CD200AR) of the CD200 checkpoint with a peptide inhibitor (CD200AR-L) overcomes tumor-induced immunosuppression. We have shown the clinical efficacy of the CD200AR-L in a trial in companion dogs with spontaneous high-grade glioma. Addition of the peptide to autologous tumor lysate vaccines significantly increased the median overall survival to 12.7 months relative to tumor lysate vaccines alone, 6.36 months. Experimental Design: This study was developed to elucidate the mechanism of the CD200ARs and develop a humanized peptide inhibitor. We developed macrophage cell lines with each of four CD200ARs knocked out to determine their binding specificity and functional response. Using proteomics, we developed humanized CD200AR-L to explore their effects on cytokine/chemokine response, dendritic cell maturation and CMV pp65 antigen response in human CD14+ cells. GMP-grade peptide was further validated for activity. Results: We demonstrated that the CD200AR-L specifically targets a CD200AR complex. Moreover, we developed and validated a humanized CD200AR-L for inducing chemokine response, stimulating immature dendritic cell differentiation and significantly enhanced an antigen-specific response, and determined that the use of the CD200AR-L downregulated the expression of CD200 inhibitory and PD-1 receptors. Conclusions: These results support consideration of a CD200AR-L as a novel platform for immunotherapy against multiple cancers including glioblastoma multiforme.
Abstract Understanding the dynamical motions and ligand recognition motifs of specific glycosyltransferase enzymes, like Heptosyltransferase I (HepI), is critical to discerning the behavior of other carbohydrate binding enzymes. Prior studies in our lab demonstrated that glycosyltransferases in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conservation of both structure and dynamical motions, despite low sequence conservation, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through a series of 100 nanosecond Molecular Dynamics simulations of HepI in apo enzyme state, and also in the binary and ternary complexes with the native substrates/products. Ligand free energy analysis allowed determination of an anticipated enzymatic path for ligand binding and release. Principle component, dynamic cross correlation and network analyses of the simulation trajectories revealed that there are not only correlated motions between the N- and C-termini, but also that residues within the N-terminal domain communicate via a path that includes substrate proximal residues of the C-terminal domain. Analysis of structural changes, energetics of substrate/products binding and changes in pK a have elucidated a variety of inter- and intradomain interactions that are critical for catalysis. These data corroborate and allow visualization of previous experimental observations of protein conformational changes of HepI. This study has provided valuable insights into the regions involved in HepI conformational rearrangement upon ligand binding, and are likely to enhance efforts to develop new dynamics disrupting enzyme inhibitors for GT-B structural enzymes in the future.
