title: A tandem repeat of a fragment of Listeria monocytogenes internalin B protein induces cell survival and proliferation.
abstract: Hepatocyte growth factor (HGF) is critical for tissue homeostasis and repair in many organs including the lung, heart, kidney, liver, nervous system, and skin. HGF is a heterodimeric protein containing 20 disulfide bonds distributed among an amino-terminal hairpin, four kringle domains, and a serine protease-like domain. Due to its complex structure, recombinant production of HGF in prokaryotes requires denaturation and refolding, processes that are impractical for large-scale manufacture. Thus, pharmaceutical quantities of HGF are not available despite its potential applications. A fragment of the Listeria monocytogenes internalin B protein from amino acids 36-321 (InlB₃₆₋₃₂₁) was demonstrated to bind to and partially activate the HGF receptor Met. InlB₃₆₋₃₂₁ has a stable β-sheet structure and is easily produced in its native conformation by Escherichia coli. We cloned InlB₃₆₋₃₂₁ (1×InlB₃₆₋₃₂₁) and engineered a head-to-tail repeat of InlB₃₆₋₃₂₁ with a linker peptide (2×InlB₃₆₋₃₂₁); 1×InlB₃₆₋₃₂₁ and 2×InlB₃₆₋₃₂₁ were purified from E. coli. Both 1× and 2×InlB₃₆₋₃₂₁ activated the Met tyrosine kinase. We subsequently compared signal transduction of the two proteins in primary lung endothelial cells. 2×InlB₃₆₋₃₂₁ activated ERK1/2, STAT3, and phosphatidylinositol 3-kinase/Akt pathways, whereas 1×InlB₃₆₋₃₂₁ activated only STAT3 and ERK1/2. The 2×InlB₃₆₋₃₂₁ promoted improved motility compared with 1×InlB₃₆₋₃₂₁ and additionally stimulated proliferation equivalent to full-length HGF. Both the 1× and 2×InlB₃₆₋₃₂₁ prevented apoptosis by the profibrotic peptide angiotensin II in cell culture and ex vivo lung slice cultures. The ease of large-scale production and capacity of 2×InlB₃₆₋₃₂₁ to mimic HGF make it a potential candidate as a pharmaceutical agent for tissue repair.
authors.affiliation: Dept. of Pharmacology, Uniformed Services University of the Health Sciences, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA. rday@usuhs.mil
authors.forename: Ognoon
authors.initials: O
authors.lastname: Mungunsukh
authors.forename: Young H
authors.initials: YH
authors.lastname: Lee
authors.forename: Ana P
authors.initials: AP
authors.lastname: Marquez
authors.forename: Fabiola
authors.initials: F
authors.lastname: Cecchi
authors.forename: Donald P
authors.initials: DP
authors.lastname: Bottaro
authors.forename: Regina M
authors.initials: RM
authors.lastname: Day
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D002465:Cell Movement; D049109:Cell Proliferation; D002470:Cell Survival; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D017228:Hepatocyte Growth Factor / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012091:Repetitive Sequences, Nucleic Acid; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D050796:STAT3 Transcription Factor; C095768:inlB protein, Listeria monocytogenes; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases