[Frontiers in Bioscience E3, 1493-1499, June 1, 2011]

Identification of protein-protein interactions of human HtrA1

mara Campioni1, Anna Severino2, Lucrezia Manente3, Antonio De Luca3, Raffaele La Porta1, Antonio Vitiello1, Paola Fiore1, Stefano Toldo4, Enrico P. Spugnini5, Marco G. Paggi6, Alfonso Baldi1

1Department of Biochemistry, Section of Pathology, Second University of Naples, Italy, 2Institute of Cardiology, Catholic University, Rome, Italy,3Department of Medicine and Public Health, Second University of Naples, Naples, Italy,4Victoria Johnson Research Center and VCU Pauley Heart Center, Virginia Commonwealth University, Richmond, VA, USA,5Department SAFU, National Cancer Institute,Regina Elena, Rome, Italy, 6Department of Development of Therapeutic Programs, National Cancer Institute, Regina Elena, Rome, Italy

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Reagents and plasmids
3.2. Cell line and proliferation assay
3.3. Yeast transformation
3.4. Identification, sequencing and BLAST analysis of putative positive clones
4. Results
4.1. Effect of Htra1 and Htra1-S328A on human melanoma cell growth
4.2. Identification of Expression Bait Plasmid
4.3. Screening of the Human Fetal Brain cDNA Library
4.4. Identification of Putative Positive Clones and BLAST Analysis
5. Discussion
6. Acknowledgment
7. References

1. ABSTRACT

The human heat shock protein HtrA1, a member of the HtrA family of serine proteases, is a evolutionarily highly conserved factor which displays a widespread pattern of expression. The yeast two-hybrid technique was employed to identify new cellular proteins physically interacting with HtrA1, and thus potential targets of this serine protease. An enzymatically inactive HtrA1 point mutant, HtrA1-S328A, was generated and used as bait in a yeast two-hybrid system. Fifty-two plasmids were isolated from primary positive yeast clones. Subsequent sequencing and BLAST analysis revealed cDNAs encoding for 13 different proteins. These putative binding partners of HtrA1 appeared to be a) components of extracellular matrix; b) factors related to signal pathways, and c) unknown proteins. Among the 13 positive clones identified and reported here, it is worth of note that the interaction of HtrA1 with tubulin and collagen (extracellular matrix proteins) and with tuberin (cytoplasmic protein) is confirmed by other studies, and this further supports previous findings in which HtrA1 can be found active as an intracytoplasmic protein or as secreted protein as well.