K48- and K63-linked polyubiquitination of deubiquitinating enzyme USP44.
Ubiquitination and deubiquitination have a critical role in protein homoeostasis in the cell. Here, we have characterized a novel USP44 (ubiquitin-specific protease 44), which has a ZnF-UBP (zinc-finger ubiquitin-specific protease) domain and conserved cysteine, histidine and asparagine/aspartic acid residues characteristic of deubiquitinating enzymes. The biochemical assay revealed that USP44 can cleave ubiquitin from ubiquitinated substrates both in vitro and in vivo. Further, USP44 undergoes both lysine 48- and lysine 63-linked polyubiquitination. In situ hybridization using mouse tissues showed a basal detection level in all organs tested, with strong detection in lung, pancreas, skin, liver, stomach and intestine. RT-PCR (reverse-transcription PCR) analysis showed high levels of detection of USP44 mRNA in testis, spleen, lung, stomach and ovary. Furthermore, we raised a polyclonal antibody against USP44 and checked its endogenous protein expression in different cell lines. A localization study of USP44 showed its predominant expression in the nucleus.
Suresh
Bharathi
B
CHA Stem Cell Institute, College of Medicine, CHA University, CHA General Hospital, Seoul 135081, South Korea.
Ramakrishna
Suresh
S
Lee
Hey-Jin
HJ
Choi
Jong-Ho
JH
Kim
Jin-Young
JY
Ahn
Woong-Shick
WS
Baek
Kwang-Hyun
KH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D010450:Endopeptidases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D017404:In Situ Hybridization, Fluorescence; D008239:Lysine / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase; D064570:Ubiquitin-Specific Proteases; D054875:Ubiquitination; D016335:Zinc Fingers
D000906:Antibodies; D012333:RNA, Messenger; D025521:Tumor Suppressor Proteins; D010450:Endopeptidases; C577772:USP44 protein, mouse; D043222:Ubiquitin Thiolesterase; D064570:Ubiquitin-Specific Proteases; D008239:Lysine