scholarly journals The metalloendopeptidase nardilysin (NRDc) is potently inhibited by heparin-binding epidermal growth factor-like growth factor (HB-EGF)

2002 ◽  
Vol 367 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Véronique HOSPITAL ◽  
Eiichiro NISHI ◽  
Michael KLAGSBRUN ◽  
Paul COHEN ◽  
Nabil G. SEIDAH ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Surface ◽  
Active Site ◽  
Regulatory Element ◽  
Enzymic Activity ◽  
Heparin Binding ◽  
Epidermal Growth

Nardilysin (N-arginine dibasic convertase, or NRDc) is a cytosolic and cell-surface metalloendopeptidase that, in vitro, cleaves substrates upstream of Arg or Lys in basic pairs. NRDc differs from most of the other members of the M16 family of metalloendopeptidases by a 90 amino acid acidic domain (DAC) inserted close to its active site. At the cell surface, NRDc binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) and enhances HB-EGF-induced cell migration. An active-site mutant of NRDc fulfills this function as well as wild-type NRDc, indicating that the enzyme activity is not required for this process. We now demonstrate that NRDc starts at Met49. Furthermore, we show that HB-EGF not only binds to NRDc but also potently inhibits its enzymic activity. NRDc—HB-EGF interaction involves the 21 amino acid heparin-binding domain (P21) of the growth factor, the DAC of NRDc and most probably its active site. Only disulphide-bonded P21 dimers are inhibitory. We also show that Ca2+, via the DAC, regulates both NRDc activity and HB-EGF binding. We conclude that the DAC is thus a key regulatory element for the two distinct functions that NRDc fulfills, i.e. as an HB-EGF modulator and a peptidase.

scholarly journals Fine Molecular Tuning of Chimeric Antigen Receptors through Hinge Length Optimization

2020 ◽  
Author(s):  
Scott McComb ◽  
Tina Nguyen ◽  
Kevin A. Henry ◽  
Darin Bloemberg ◽  
Susanne Maclean ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Specific Activity ◽  
Single Amino Acid ◽  
Single Domain Antibody ◽  
Normal Tissues ◽  
Epidermal Growth

AbstractBackgroundChimeric antigen receptor (CAR) technology has revolutionized the treatment of B-cell malignancies and steady progress is being made towards CAR-immunotherapies for solid tumours. In the context of CARs targeting antigens which are commonly overexpressed in cancer but also expressed at lower levels in normal tissues, such as epidermal growth factor family receptors EGFR or HER2, it is imperative that any targeting strategy consider the potential for on-target off-tumour toxicity. Molecular optimization of the various protein domains of CARs can be used to increase the tumour selectivity.MethodHerein, we utilize high-throughput CAR screening to identify a novel camelid single-domain antibody CAR (sdCAR) targeting human epidermal growth factor (EGFR) with high EGFR-specific activity. To further optimize the target selectivity of this EGFR-sdCAR, we performed progressive N-terminal single amino acid truncations of an extended human CD8 hinge domain [(G4S)3GG-45CD8h] to improve selectivity for EGFR-overexpressing cells. We also make direct comparison of varying hinge domains in scFv-based CARs targeting EGFR-family tumour associated antigens EGFRvIII and HER2.ResultsThrough comparison of various hinge-truncated scFv- and sdAb-based CARs, we show that the CAR hinge/spacer domain plays varying roles in modifying CAR signaling depending upon target epitope location. For membrane-proximal epitopes, hinge truncation by even a single amino acid resulted in fine control of CAR signaling strength. Hinge-modified CARs showed consistent and predictable signaling in Jurkat-CAR cells and primary human CAR-T cells in vitro and in vivo.ConclusionsOverall, these results indicate that membrane-proximal epitope targeting CARs can be optimized through hinge length tuning for improved target selectivity and therapeutic function. Graphical Abstract

scholarly journals Epidermal growth factor receptor regulates pancreatic fibrosis

2009 ◽  
Vol 297 (3) ◽  
pp. G434-G441 ◽  
Author(s):  
Stacy A. Blaine ◽  
Kevin C. Ray ◽  
Kevin M. Branch ◽  
Pamela S. Robinson ◽  
Robert H. Whitehead ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Pancreatic Fibrosis ◽  
Pancreatic Stellate Cells ◽  
Heparin Binding ◽  
Epidermal Growth

The development of pancreatic fibrosis has been shown to be a major component in several diseases of the pancreas including pancreatic cancer, chronic pancreatitis, and type 2 diabetes mellitus, but its actual role in the progression of these disorders is still unknown. This fibrosis is characterized by stromal expansion and the excessive deposition of extracellular matrix (ECM) that replaces pancreatic tissue. This eventually leads to dysregulation of ECM turnover, production of cytokines, restriction of blood flow, and often exocrine and endocrine insufficiencies. Activated pancreatic stellate cells (PSCs) have been identified as key mediators in the progression of pancreatic fibrosis, serving as the predominant source of excess ECM proteins. Previously, we found that overexpression of the growth factor heparin-binding epidermal growth factor-like growth factor (HB-EGF) in pancreatic islets led to intraislet fibrosis. HB-EGF binds to and activates two receptors, epidermal growth factor receptor (EGFR) and ErbB4, as well as heparin moieties and CD9/DRAP27. To understand the mechanism underlying the induction of fibrogenesis by HB-EGF, we utilized a hypomorphic allele of Egfr, the Waved-2 allele, to demonstrate that EGFR signaling regulates fibrogenesis in vivo. Using an in vitro cell migration assay, we show that HB-EGF regulates both chemoattraction and stimulation of proliferation of PSCs via EGFR activation.

Insulin-Induced Ectodomain Shedding of Heparin-Binding Epidermal Growth Factor-Like Growth Factor in Adipocytes In Vitro

Obesity ◽  
2010 ◽  
Vol 18 (10) ◽  
pp. 1888-1894 ◽  
Author(s):  
Takanobu Yamamoto ◽  
Takayoshi Suganami ◽  
Minako Kiso-Narita ◽  
Peggy A. Scherle ◽  
Yasutomi Kamei ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Ectodomain Shedding ◽  
Heparin Binding ◽  
Epidermal Growth

scholarly journals Heparin-Binding Epidermal Growth Factor-Like Growth Factor Stimulates Androgen-Independent Prostate Tumor Growth and Antagonizes Androgen Receptor Function

Endocrinology ◽  
2002 ◽  
Vol 143 (12) ◽  
pp. 4599-4608 ◽  
Author(s):  
Rosalyn M. Adam ◽  
Jayoung Kim ◽  
Jianqing Lin ◽  
Anna Orsola ◽  
Liyan Zhuang ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Neuron Specific Enolase ◽  
Continuous Exposure ◽  
Heparin Binding ◽  
Independent State ◽  
Epidermal Growth ◽  
Androgen Independent

Abstract Peptide growth factors have been implicated in progression of prostate cancer (PCa) to the androgen-independent state; however, much of the evidence linking diffusible mitogens and survival factors to this process remains circumstantial. Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a prostate stroma-derived factor, promotes survival, proliferation, and neuroendocrine differentiation of androgen-dependent LNCaP PCa cells in vitro. To test whether sustained exposure to HB-EGF can confer an androgen-independent phenotype, we generated stable populations of LNCaP cells that express constitutively a secreted form of HB-EGF (LNCaP/sHB). LNCaP/sHB cells proliferated more rapidly under androgen-depleted conditions in vitro and formed larger tumors with higher frequency in intact and castrated severe combined immunodeficient mice, in comparison to control cells. LNCaP/sHB tumors also expressed higher levels of the neuroendocrine marker, neuron-specific enolase, compared with control tumors. In castrates, increased neuron-specific enolase expression in LNCaP/sHB tumors was associated with reduced androgen receptor (AR) levels. In vitro, AR protein levels were reduced in LNCaP/sHB cells, and in transient transfection assays using an androgen-responsive promoter (mouse mammary tumor virus-long terminal repeat), LNCaP/sHB cells showed reduced sensitivity to dihydrotestosterone compared with controls. This is the first demonstration that continuous exposure of AR-positive PCa cells to a single growth factor can promote an androgen-independent phenotype in vivo. These findings also emphasize the potential role of pathways other than the AR axis in acquisition of androgen independence.

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