ABSTRACT
Mid-infrared spectroscopy is one of the major analytical techniques employed for measurements of protein structure in solution. Traditional Fourier Transform-Infrared (FT-IR) measurement is limited by its blackbody light source that is inherently spatially incoherent and has low optical power output. This limitation is pronounced when working with proteins in aqueous solutions. Strong absorbance of water in protein amide I region 1600-1700 cm-1 restricts light path length to <10 µm and imposes significant experimental challenges in sample and flow cell handling. Emerging laser spectroscopic techniques use high-power coherent laser as light source that overcomes the limitation in FT-IR measurement. In this study, we employed an innovative infrared spectrometer that uses quantum cascade laser (QCL) as light source. Continuous infrared radiation from this laser source can be swiftly swept within the amide I region (1600-1700 cm-1) and amide II region (1500-1600 cm-1), which makes this technique ideal for protein secondary structure study. Protein solutions as low as 0.5 mg/mL were measured rapidly without any sample preparation. Infrared spectra of model proteins were thus collected, and a chemometric model based on partial least squares regression was developed to quantify α-helix and ß-strand motifs in protein secondary structure. The model was applied to measurement of the native secondary structure of commercial therapeutic proteins and bovine serum albumin (BSA) and in thermal degradation studies.
Subject(s)
Lasers, Semiconductor , Serum Albumin, Bovine , Spectroscopy, Fourier Transform Infrared/methods , Spectrophotometry, Infrared/methods , Water/chemistry , AmidesABSTRACT
The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.
Subject(s)
Histone Acetyltransferases/metabolism , Lysine/genetics , Acetylation , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Histone Acetyltransferases/genetics , Histones/metabolism , Humans , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Cervical cancer is the sixth most common cancer in women worldwide and the leading cause of women's death in developing countries. Nearly all cervical cancers are associated with infection of the human papillomavirus (HPV). This sexually transmitted pathogen disrupts the cell cycle via two oncoproteins: E6 and E7. Cells respond to E7-mediated degradation of pRB by upregulating the p53 tumor suppressor pathway. However, E6 thwarts this response by binding to the cellular E6-Associating Protein (E6AP) and targeting p53 for degradation. These two virus-facilitated processes pave the way for cellular transformation. Prophylactic HPV vaccines are available, but individuals already infected with HPV lack drug-based therapeutic options. To fill this void, we sought to identify small molecule inhibitors of the E6-E6AP interaction. We designed an ELISA-based high throughput assay to rapidly screen compound libraries, and hits were confirmed in several orthogonal biochemical and cell-based assays. Over 88,000 compounds were screened; 30 had in vitro potencies in the mid-nanomolar to mid-micromolar range and were classified as validated hits. Seven of these hits inhibited p53 degradation in cell lines with HPV-integrated genomes. Two compounds of similar scaffold successfully blocked p53 degradation and inhibited cell proliferation in cells stably transfected with E6. Together, these studies suggest that small molecules can successfully block E6-dependent p53 degradation and restore p53 activity. The compounds identified here constitute attractive starting points for further medicinal chemistry efforts and development into beneficial therapeutics.
Subject(s)
Alphapapillomavirus/physiology , Anticarcinogenic Agents/pharmacology , Antiviral Agents/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , Host-Pathogen Interactions/drug effects , Oncogene Proteins, Viral/antagonists & inhibitors , Repressor Proteins/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Alphapapillomavirus/drug effects , Anticarcinogenic Agents/chemistry , Antiviral Agents/chemistry , Cell Cycle/drug effects , Cell Line, Tumor , DNA-Binding Proteins/metabolism , Female , High-Throughput Screening Assays/methods , Human papillomavirus 16/drug effects , Human papillomavirus 16/physiology , Human papillomavirus 18/drug effects , Human papillomavirus 18/physiology , Humans , Oncogene Proteins, Viral/metabolism , Papillomaviridae , Papillomavirus Infections/metabolism , Papillomavirus Infections/virology , Proteolysis/drug effects , Repressor Proteins/metabolism , Small Molecule Libraries/chemistry , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/metabolism , Uterine Cervical Neoplasms/virologyABSTRACT
To gain insight into the functional antibody repertoire of rabbits, the VH and VL repertoires of bone marrow (BM) and spleen (SP) of a naïve New Zealand White rabbit (NZW; Oryctolagus cuniculus) and that of lymphocytes collected from a NZW rabbit immunized (IM) with a 16-mer peptide were deep-sequenced. Two closely related genes, IGHV1S40 (VH1a3) and IGHV1S45 (VH4), were found to dominate (~90%) the VH repertoire of BM and SP, whereas, IGHV1S69 (VH1a1) contributed significantly (~40%) to IM. BM and SP antibodies recombined predominantly with IGHJ4. A significant proportion (~30%) of IM sequences recombined with IGHJ2. The VK repertoire was encoded by nine IGKV genes recombined with one IGKJ gene, IGKJ1. No significant bias in the VK repertoire of the BM, SP and IM samples was observed. The complementarity-determining region (CDR)-H3 and -L3 length distributions were similar in the three samples following a Gaussian curve with average length of 12.2 ± 2.4 and 11.1 ± 1.1 amino acids, respectively. The amino acid composition of the predominant CDR-H3 and -L3 loop lengths was similar to that of humans and mice, rich in Tyr, Gly, Ser and, in some specific positions, Asp. The average number of mutations along the IGHV/KV genes was similar in BM, SP and IM; close to 12 and 15 mutations for VH and VL, respectively. A monoclonal antibody specific for the peptide used as immunogen was obtained from the IM rabbit. The CDR-H3 sequence was found in 1,559 of 61,728 (2.5%) sequences, at position 10, in the rank order of the CDR-H3 frequencies. The CDR-L3 was found in 24 of 11,215 (0.2%) sequences, ranking 102. No match was found in the BM and SP samples, indicating positive selection for the hybridoma sequence. Altogether, these findings lay foundations for engineering of rabbit V regions to enhance their potential as therapeutics, i.e., design of strategies for selection of specific rabbit V regions from NGS data mining, humanization and design of libraries for affinity maturation campaigns.
Subject(s)
Antibodies/genetics , Antibodies/immunology , Rabbits/genetics , Rabbits/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Bone Marrow/immunology , Complementarity Determining Regions/genetics , High-Throughput Nucleotide Sequencing , Humans , Hybridomas/immunology , Immunization , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/genetics , Mice , Molecular Sequence Data , Mutation , Peptides/immunology , Protein Engineering , Sequence Homology, Amino Acid , Spleen/immunologyABSTRACT
The retinoblastoma protein pRb is essential for regulating many cellular activities through its binding and inhibition of E2F transcription activators, and pRb inactivation leads to many cancers. pRb activity can be perturbed by viral oncoproteins including human papillomavirus (HPV) that share an LxCxE motif. Because there are no treatments for existing HPV infection leading to nearly all cervical cancers and other cancers to a lesser extent, we screened for compounds that inhibit the ability of HPV-E7 to disrupt pRb/E2F complexes. This lead to the identification of thiadiazolidinedione compounds that bind to pRb with mid-high nanomolar dissociation constants, are competitive with the binding of viral oncoproteins containing an LxCxE motif, and are selectively cytotoxic in HPV-positive cells alone and in mice. These inhibitors provide a promising scaffold for the development of therapies to treat HPV-mediated pathologies.
Subject(s)
Papillomavirus E7 Proteins/metabolism , Retinoblastoma Protein/antagonists & inhibitors , Amino Acid Motifs , Amino Acid Sequence , Animals , Apoptosis/drug effects , Cell Line, Tumor , E2F Transcription Factors/metabolism , G1 Phase Cell Cycle Checkpoints/drug effects , HeLa Cells , Humans , Mice , Molecular Sequence Data , Papillomaviridae/drug effects , Papillomaviridae/metabolism , Papillomavirus E7 Proteins/antagonists & inhibitors , Retinoblastoma Protein/metabolism , Sequence Alignment , Thiadiazoles/chemistry , Thiadiazoles/pharmacologyABSTRACT
The human monocytic leukemia zinc finger (MOZ) protein is an essential transcriptional coactivator and histone acetyltransferase (HAT) that plays a primary role in the differentiation of erythroid and myeloid cells and is required to maintain hematopoietic stem cells. Chromosomal translocations involving the HAT-encoded region are also associated with acute myeloid leukemia. Here we present the x-ray crystal structure of the MOZ HAT domain and related biochemical studies. We find that the HAT domain contains a central region that is structurally and functionally conserved with the yeast MYST HAT protein Esa1, but contains more divergent N- and C-terminal regions harboring a TFIIIA-type zinc finger and helix-turn-helix DNA-binding motifs. Solution DNA-binding and acetyltransferase activity assays, in concert with mutagenesis, confirm that the MOZ HAT domain binds strongly to DNA through the zinc finger and helix-turn-helix motifs and that DNA binding and catalysis are not mutually exclusive. Consistent with the DNA-binding properties of MOZ, we also show that MOZ is able to acetylate nucleosomes and free histones equally well, whereas other HATs prefer free histones. Our results reveal, for the first time, that enzymatic and DNA-targeting activities can be contained within the same chromatin regulatory domain.
Subject(s)
Histone Acetyltransferases/chemistry , Acetylation , Amino Acid Motifs/physiology , Animals , Cell Differentiation/physiology , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , Erythroid Precursor Cells/cytology , Erythroid Precursor Cells/metabolism , Histone Acetyltransferases/metabolism , Histones/metabolism , Humans , Myeloid Progenitor Cells/cytology , Myeloid Progenitor Cells/metabolism , Nucleosomes/metabolism , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Structure-Activity Relationship , Translocation, Genetic/physiology , Xenopus laevisABSTRACT
The histidine triad superfamily of nucleotide hydrolases and nucleotide transferases consists of a branch of proteins related to Hint and Aprataxin, a branch of Fhit-related hydrolases, and a branch of galactose-1-phosphate uridylyltransferase (GalT)-related transferases. Although substrates of Fhit and GalT are known and consequences of mutations in Aprataxin, Fhit, and GalT are known, good substrates had not been reported for any member of the Hint branch, and mutational consequences were unknown for Hint orthologs, which are the most ancient and widespread proteins in the Hint branch and in the histidine triad superfamily. Here we show that rabbit and yeast Hint hydrolyze the natural product adenosine-5'-monophosphoramidate (AMPNH(2)) in an active-site-dependent manner at second order rates exceeding 1,000,000 m(-1) s(-1). Yeast strains constructed with specific loss of the Hnt1 active site fail to grow on galactose at elevated temperatures. Loss of Hnt1 enzyme activity also leads to hypersensitivity to mutations in Ccl1, Tfb3, and Kin28, which constitute the TFIIK kinase subcomplex of general transcription factor TFIIH and to mutations in Cak1, which phosphorylates Kin28. The target of Hnt1 regulation in this pathway was shown to be downstream of Cak1 and not to affect stability of Kin28 monomers. Functional complementation of all Hnt1 phenotypes was provided by rabbit Hint, which is only 22% identical to yeast Hnt1 but has very similar adenosine monophosphoramidase activity.