ABSTRACT
Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.
Subject(s)
Anemia, Sickle Cell , Antisickling Agents , Fetal Hemoglobin , Kruppel-Like Transcription Factors , Nerve Tissue Proteins , Animals , Humans , Mice , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/genetics , Anemia, Sickle Cell/drug therapy , Anemia, Sickle Cell/metabolism , Antisickling Agents/chemistry , Antisickling Agents/pharmacology , Antisickling Agents/therapeutic use , Crystallography, X-Ray , Drug Discovery , Fetal Hemoglobin/genetics , Fetal Hemoglobin/metabolism , Kruppel-Like Transcription Factors/metabolism , Macaca fascicularis , Nerve Tissue Proteins/metabolism , Proteolysis/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Small Molecule Libraries/therapeutic use , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/geneticsABSTRACT
Helicobacter pylori is the strongest risk factor for gastric cancer. Initial interactions between H. pylori and its host originate at the microbial-gastric epithelial cell interface, and contact between H. pylori and gastric epithelium activates signaling pathways that drive oncogenesis. One microbial constituent that increases gastric cancer risk is the cag pathogenicity island, which encodes a type IV secretion system that translocates the effector protein, CagA, into host cells. We previously demonstrated that infection of Mongolian gerbils with a carcinogenic cag+H. pylori strain, 7.13, recapitulates many features of H. pylori-induced gastric cancer in humans. Therefore, we sought to define gastric proteomic changes induced by H. pylori that are critical for initiation of the gastric carcinogenic cascade. Gastric cell scrapings were harvested from H. pylori-infected and uninfected gerbils for quantitative proteomic analyses using isobaric tags for relative and absolute quantitation (iTRAQ). Quantitative proteomic analysis of samples from two biological replicate experiments quantified a total of 2764 proteins, 166 of which were significantly altered in abundance by H. pylori infection. Pathway mapping identified significantly altered inflammatory and cancer-signaling pathways that included Rab/Ras signaling proteins. Consistent with the iTRAQ results, RABEP2 and G3BP2 were significantly up-regulated in vitro, ex vivo in primary human gastric monolayers, and in vivo in gerbil gastric epithelium following infection with H. pylori strain 7.13 in a cag-dependent manner. Within human stomachs, RABEP2 and G3BP2 expression in gastric epithelium increased in parallel with the severity of premalignant and malignant lesions and was significantly elevated in intestinal metaplasia and dysplasia, as well as gastric adenocarcinoma, compared with gastritis alone. These results indicate that carcinogenic strains of H. pylori induce dramatic and specific changes within the gastric proteome in vivo and that a subset of altered proteins within pathways with oncogenic potential may facilitate the progression of gastric carcinogenesis in humans.
Subject(s)
Carrier Proteins/metabolism , Helicobacter Infections/complications , Helicobacter pylori/pathogenicity , Stomach Neoplasms/microbiology , Vesicular Transport Proteins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Cell Line , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Gerbillinae , Helicobacter Infections/microbiology , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/microbiology , Male , Protein Interaction Maps , Proteomics , RNA-Binding Proteins , Stomach Neoplasms/metabolism , Up-RegulationABSTRACT
MALDI imaging mass spectrometry (IMS) has been applied to whole animal tissue sections of Pacific White Shrimp, Litopenaeus vannamei, in an effort to identify and spatially localize proteins in specific organ systems. Frozen shrimp were sectioned along the ventral-dorsal axis and methods were optimized for matrix application. In addition, tissue microextraction and homogenization was conducted followed by top-down LC-MS/MS analysis of intact proteins and searches of shrimp EST databases to identify imaged proteins. IMS images revealed organ system specific protein signals that highlighted the hepatopancreas, heart, nervous system, musculature, and cuticle. Top-down proteomics identification of abdominal muscle proteins revealed the sequence of the most abundant muscle protein that has no sequence homology to known proteins. Additional identifications of abdominal muscle proteins included titin, troponin-I, ubiquitin, as well as intact and multiple truncated forms of flightin; a protein known to function in high frequency contraction of insect wing muscles. The combined use of imaging mass spectrometry and top-down proteomics allowed for identification of novel proteins from the sparsely populated shrimp protein databases.