Universitéde Montreal

Luc Desgroseillers

Guy Boileau

Eric A Cohen

Louise Wickham

Andrew J Mouland

Ming Luo

Thomas Duchaîne

In this paper, we describe RT-PCR strategies that allowed us to identify and clone members of the NEP-like family. Degenerate oligoncleotide primers corresponding to consensus sequences located on either side of the HEXXH consensus sequence for zincins were designed and used in RT-PCR with mouse and human testis cDNAs. DNA fragments with lengths expected from the sequence of this class of enzympes were obtained. These DNA fragments were cloned and sequenced. Using this PCR strategy and the PCR fragments as probes to screen cDNA libraries, three zincin-like peptidases were identified in addition of known members of the family. The cDNA sequences allowed to derive specific probes for Northern and in situ hybridization, and probe human chromosomes to localize the gene and establish potential links to genetic diseases. Furthermore, these cDNA sequences were used to produce recombinant fusion proteins inin order to raise specific antibodies. Finally, the cDNA sequences were cloned in mammalian expression vectors and transfected in various mammalian cell lines to produce active recombinant enzymes suitable for testing specific inhibitors.

Metalloproteases of the neprilysin family

The present invention relates to mammalian staufen, a double-stranded RNA-binding protein involved in mRNA transport and localization. The invention further relates to the demonstration of the association of a RNA-binding protein with the rough endoplasmic reticulum (RER), implicating staufen and related proteins in the transport of RNA to its site of translation. Broadly, the invention therefore relates to transport and translation of RNA. More specifically, the present invention relates to human and mouse staufen proteins and to the modulation of transport of RNA to the RER by these proteins. The present invention also relates to isolated nucleic acid molecules encoding mammalian staufen, as well as vectors and host cells harboring same. In addition, the present invention relates to screening assays for identifying modulators of staufen activity and to the identification of mutants thereof which abrogate their interaction with RER. Furthermore, the present invention relates to the use of the double-stranded RNA binding activity of staufen as a means to target proteins into virions. The invention in addition relates to the incorporation of staufen into RNA viruses and the use of overexpression of staufen to significantly decrease the infectivity thereof. More particularly, the present invention relates to a novel and broad class of molecules which can be used as carriers to target molecules into virions of RNA viruses and to decrease infectivity of a wide variety of RNA viruses including retroviruses.

Mammalian stauffen and use thereof

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St-Basile-le-Grand, Canada

Luc Desgroseillers