Proteome changes induced by knock-down of the deubiquitylating enzyme HAUSP/USP7.
Modification of proteins by ubiquitin plays a major role in a broad array of biological processes. Reversal of this process through deubiquitylation likely represents an important regulatory step in the maintenance of cellular homeostasis. However, the biological functions of deubiquitylating enzymes still remain poorly characterized. To investigate the biological role of the herpes virus-associated ubiquitin-specific protease HAUSP/USP7, we have generated stably transfected cells carrying inducible shRNA expression plasmids. USP7 mRNA and protein were strongly down-regulated 48-72 h after shRNA induction. We used a selected clone to compare whole-cell proteomes by 2D-SDS-PAGE before and after knockdown of USP7. Alterations in 36 proteins were detected and their identities were revealed by mass spectrometry analysis. Components of the replication machinery, DNA/RNA binding proteins, enzymes involved in apoptosis and metabolism were found to be down-regulated upon USP7 removal, representing proteins that are either more rapidly turned over or synthesized less efficiently in the absence of USP7-mediated deubiquitylation. Alix/HP95, a protein implicated in endosomal organization and virus budding, was confirmed by immunoblotting to become down-regulated when USP7 levels were reduced. Our results extend the current list of USP7-dependent biological processes and suggest a role for this enzyme not only in transcriptional regulation but also in DNA replication, apoptosis, and possibly endosomal organization.
Kessler
Benedikt M
BM
Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Fortunati
Elisabetta
E
Melis
Monique
M
Pals
Cornelieke E G M
CE
Clevers
Hans
H
Maurice
Madelon M
MM
D000818:Animals; D017209:Apoptosis; D019556:COS Cells; D002277:Carcinoma / Q000378:metabolism; D002522:Chlorocebus aethiops; D003110:Colonic Neoplasms / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D011992:Endosomes / Q000378:metabolism; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D040901:Proteomics / Q000379:methods*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D043222:Ubiquitin Thiolesterase / Q000096:biosynthesis*; D000076084:Ubiquitin-Specific Peptidase 7
D034741:RNA, Small Interfering; D025801:Ubiquitin; C464569:USP7 protein, human; D043222:Ubiquitin Thiolesterase; D000076084:Ubiquitin-Specific Peptidase 7