Screen for ISG15-crossreactive Deubiquitinases

Background: The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15. Results: We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome. Conclusions: By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice

Screen for ISG15-crossreactive deubiquitinases

Background. The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15. Results. We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome. Conclusions. By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice. Citation: Catic A, Fiebiger E, Korbel GA, Blom D, Galardy PJ, et al (2007) Screen for ISG15-crossreactive Deubiquitinases. PLoS ONE 2(7): e679

Excess Usp44 leads to increased cyclin B in early mitosis.

<p>(a) Cells were synchronized in G1/G0 by serum starvation and were then released. Nocodazole was added 23 hours after release. Samples were collected at the indicated times and were immunoblotted with the indicated antibodies. Results are representative of at least 3 independent experiments. (b) Immunofluorescence imaging of cells transduced with empty lentivirus or Usp44-HA using the indicated antibodies. The stage of mitosis was determined by DNA morphology. (c) Quantitation of cyclin B levels from (a) using imageJ. Ten cells from each of three independent MEF lines (total 30 cells per condition per stage) were analyzed. (d) Cyclin B1 mRNA was measured using qRT-PCR. Cells were synchronized as in (a) and harvested at the indicated times. (e) MEFs were stably transduced with the indicated construct and were then transfected with a construct encoding a fusion between cyclin B1 and Cerulean (CyclinB1^Cerulean). Cells were monitored by live-cell microscopy. Images were obtained every 4 minutes and quantified with imageJ. Values were normalized such that the level at nuclear envelope breakdown (NEBD) was set at 100%. The arrow with “A:” refers to the average time of anaphase observed in the cells in each condition. (f) MEFs were transduced with the indicated constructs and were fixed and stained to detect Usp44^Cherry, cyclin B1, and DNA. The levels of Usp44^Cherry and cyclin B1 were quantitated in G2 cells (n = 11–19 cells each) using imageJ. (g) MEFs transduced with empty vector were treated with MG132 for 1 hour prior to fixation. The amount of cyclin B1 was determined in G2 or early prophase using imageJ in comparison to untreated, or Usp44^Cherry transduced MEFs. Graph represents the average of 20 cells in each group from three independent experiments. * p<0.05 calculated with an unpaired t-test.</p

Expression of Usp44 leads to chromosome missegregation and aneuploidy.

<p>(a) Expression of Usp44-HA in MEFs as seen by immunoblotting (upper) or quantitative real-time (qRT)-PCR (lower). Four independent MEF lines were transduced with Usp44-HA or Usp44Cherry followed by TaqMan qRT-PCR. Expression was normalized to GAPDH, and fold-change was calculated using the ΔCt method. (b) MEFs stably transduced with either empty vector (n = 78 cells), Usp44-HA (n = 106 cells), or Usp44^Cherry (n = 53 total) were analyzed by live-cell microscopy through the indicated numbers of un-perturbed cell divisions. Chromosomes were visualized by transduction with lentivirus encoding histone H2B fused with yellow fluorescent protein (H2B-YFP). The results depict the average and standard error from three-independent MEF lines. (c) Example of a cell with a lagging chromosome (upper) and anaphase bridge (lower). (d) Karyotype analysis of low passage MEFs. Stable transduction with the indicated lentivirus was performed at passage 2. N (metaphases)  = 175 (Empty vector control) and 223 (Usp44-HA) from a total of four independent MEF lines per genotype. (e) Representative spectral karyotype (SKY) of a cell expressing Usp44-HA. Note this cell has a normal chromosome number (40) but has a trisomy of chromosome 12 and monosomy of chromosome 7. * p<0.05 calculated with the unpairted t-test. Error bars represent the SEM.</p

USP44 is over-expressed in T-cell acute lymphoblastic leukemia.

<p>(a) Relative USP44 mRNA expression in various cancer types versus matched normal tissue was collected from publicly available microarray studies (<a href="http://www.oncomine.org" target="_blank">www.oncomine.org</a>). For all studies shown, p<0.05 for alterations in USP44 mRNA. (b) USP44 mRNA was measured by TaqMan quantitative real-time PCR in a series of 24 samples of T-cell acute lymphoblastic leukemia compared with peripheral T-cells isolated from 10 healthy volunteers. The p value was calculated with an unpaired t-test comparing the mean fold-change of T-ALL to controls. (c) Human foreskin fibroblasts were transduced with the indicated constructs (two independent lines for each), cultured for 5-passages, and then analyzed by chromosome counting. The p value was calculated using the unpaired t test.</p