USP16 is an ISG15 cross-reactive deubiquitinase that targets pro-ISG15 and ISGylated proteins involved in metabolism.

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict viral infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for the existence of other ISG15 cross-reactive proteases, these remain to be identified. Here, we identify deubiquitinase USP16 as an ISG15 cross-reactive protease by means of ISG15 activity-based profiling. Recombinant USP16 cleaved pro-ISG15 and ISG15 isopeptide-linked model substrates in vitro, as well as ISGylated substrates from cell lysates. Moreover, interferon-induced stimulation of ISGylation was increased by depletion of USP16. The USP16-dependent ISG15 interactome indicated that the deISGylating function of USP16 may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, and cytoplasmic glutamic-oxaloacetic transaminase 1. USP16 may thus contribute to the regulation of a subset of metabolism-related proteins during type-I interferon responses.

A potent nuclear export mechanism imposes USP16 cytoplasmic localization during interphase.

USP16 (also known as UBP-M) has emerged as a histone H2AK119 deubiquitylase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also present in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has remained elusive. Here, we show that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for maintaining USP16 in the cytoplasm. We found that USP16 is only transiently retained in the nucleus following mitosis and then rapidly exported from this compartment. We also defined a non-canonical nuclear localization signal (NLS) sequence that plays a minimal role in directing USP16 into the nucleus. We further established that this DUB does not accumulate in the nucleus following DNA damage. Instead, only enforced nuclear localization of USP16 abolishes DNA double-strand break (DSB) repair, possibly due to unrestrained DUB activity. Thus, in contrast to the prevailing view, our data indicate that USP16 is actively excluded from the nucleus and that this DUB might indirectly regulate DSB repair.This article has an associated First Person interview with the first author of the paper.

Copy number variation of the USP16 gene and its association with milk traits in Chinese Holstein cattle.

Copy number variations (CNVs) were similar to single nucleotide polymorphisms (SNPs) and insertion-deletion (InDel), regarded as genetic variations in many species. CNV is defined as the variable change of DNA segment length compared with the reference genome, including gains or losses from 50 bp to several mega bases. The functions of USP16 gene are diverse, such as regulating the cell cycle, DNA damage, histone H2A deubiquitination or mitotic nuclear division. To analyze the relationship between CNV of USP16 gene and milk traits in Chinese Holstein, we used qPCR to detect the individuals of Chinese Holstein (n = 180). The results showed that the effect of USP16 gene CNV on daily milk yield and fat percentage had significant difference (p < 0.05). The gain was the advantage type in daily milk yield and the loss was the advantage type in fat percentage. Therefore, CNV of USP16 gene is an important factor of milk traits in Chinese Holstein. Meanwhile, it may be used as a molecular marker for assisted selection of milk traits in Chinese Holstein, which provides a theoretical basis for the genetic improvement of cow breeds in China.

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function.

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

The equilibrium of ubiquitination and deubiquitination at PLK1 regulates sister chromatid separation.

PLK1 regulates almost every aspect of mitotic events, including mitotic entry, spindle assembly, chromosome alignment, sister chromatid segregation, metaphase-anaphase transition, cytokinesis, etc. In regulating the chromosome alignment and sister chromatid segregation, PLK1 has to be localized to and removed from kinetochores at the right times, and the underlying mechanism that regulates PLK1 both spatially and temporally only became clearer recently. It has been found that deubiquitination and ubiquitination of PLK1 are responsible for its localization to and dissociation from the kinetochores, respectively. The equilibrium of this ubiquitination and deubiquitination plays an important role in regulating proper chromosome alignment and timely sister chromatid segregation. Here, we summarize and discuss the recent findings in investigating the spatial and temporal regulation of PLK1 during chromosome alignment and sister chromatid segregation.

Deubiquitinase USP16 induces gouty arthritis via Drp1-dependent mitochondrial fission and NLRP3 inflammasome activation.

BACKGROUND: Gouty arthritis is the most frequently diagnosed inflammatory arthritis worldwide. Dynamin-related protein 1 (Drp1), a regulator of mitochondrial fission, contributes to various inflammatory disorders via activating NLRP3 inflammasome. However, the biological role of Drp1 in gouty arthritis remains undefined. METHODS: A mouse model of monosodium urate (MSU)-induced gouty arthritis and MSU-stimulated macrophages were established as in vivo and in vitro models, respectively. Histological changes were assessed by H&E and IHC analysis. RT-qPCR and western blot were used to detect the expression of Drp1 and the key molecules in joint tissues and macrophages. Cytokine secretion was measured by ELISA assay, and antioxidant enzymes activities and LDH release were monitored using commercial kits. Mitochondrial structure and functions were assessed by transmission electron microscopy (TEM) and MitoSOX staining. Co-IP and GST pull-down assay were used to detect the direct interaction between USP16 and Drp1, as well as the ubiquitination of Drp1. RESULTS: Drp1 was elevated in MSU-induced gouty arthritis model, and it induced gouty arthritis via NF-κB pathway and NLRP3 inflammasome activation. In addition, Drp1 activated NF-κB/NLRP3 signaling via modulating mitochondrial fission. Mechanistically, USP16 mediated deubiquitination and stabilization of Drp1 through its direct interaction with Drp1. Functional studies further showed that USP16 was highly expressed in MSU-stimulated macrophages and induced gouty arthritis via Drp1-dependent NLRP3 inflammasome activation. CONCLUSION: Deubiquitinase USP16 induced gouty arthritis via Drp1-dependent mitochondrial fission and NF-κB/NLRP3 signaling.

USP16 regulates castration-resistant prostate cancer cell proliferation by deubiquitinating and stablizing c-Myc.

BACKGROUND: c-Myc, a well-established oncogene, plays an important role in the initiation and progression of various cancers, including prostate cancer. However, its mechanism in cancer cell remains largely unknown and whether there exist a deubiquitinase targeting c-Myc also remains elusive. METHODS: Bioinformatic analysis and shRNA screening methods were used to identify potential deubiquitinases that correlate with c-Myc gene signature. Cell proliferation and viability were measured by Cell-Counting-Kit 8 and colony formation assays. A mouse xenograft model of PC3 cells was established to confirm the function of USP16 in vivo. The interaction between USP16 and c-Myc protein was assessed by co-immunoprecipitation and protein co-localization assays. Immunohistochemistry staining was performed to detect the expression of USP16, Ki67, and c-Myc in xenograft tissues and clinical tumour tissues. Furthermore, the correlation between USP16 and c-Myc was confirmed by RNA sequencing. RESULTS: Functional analyses identified USP16, known as a deubiquitinase, was strongly correlated with the c-Myc gene signature. Depletion of USP16 was shown to significantly suppress the growth of PCa cells both in vitro and in vivo. Co-immunoprecipitation and ubiquitination assays confirmed that USP16 served as a novel deubiquitinase of c-Myc and overexpression of c-Myc significantly rescued the effects of USP16 disruption. Immunohistochemistry staining and RNA-seq tactics were further used to confirm the positive correlation between USP16 and c-Myc expression. Expression of USP16 in human PCa tissues was higher than that seen in normal prostate tissues and its high expression was found associated with poor prognosis. CONCLUSIONS: USP16 serves as a novel deubiquitinase of c-Myc. Downregulation of USP16 markedly suppressed PCa cell growth both in vitro and in vivo. USP16 regulates PCa cell proliferation by deubiquitinating and stabilizing c-Myc, making it a potential therapeutic candidate for the treatment of PCa.

CRISPR-Cas9 Editing of Human Histone Deubiquitinase Gene USP16 in Human Monocytic Leukemia Cell Line THP-1.

USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the USP16 gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (USP12, BAP1, and MYSM1). However, three homozygotes failed to fully induce USP3 during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors.

USP16 counteracts mono-ubiquitination of RPS27a and promotes maturation of the 40S ribosomal subunit.

Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors. We demonstrate that USP16 constitutes a component of late cytoplasmic pre-40S subunits that promotes the removal of ubiquitin from an internal lysine of ribosomal protein RPS27a/eS31. USP16 deletion leads to late 40S subunit maturation defects, manifesting in incomplete processing of 18S rRNA and retarded recycling of late-acting ribosome biogenesis factors, revealing an unexpected contribution of USP16 to the ultimate step of 40S synthesis. Finally, ubiquitination of RPS27a appears to depend on active translation, pointing at a potential connection between 40S maturation and protein synthesis.

Transcriptional activation of USP16 gene expression by NFκB signaling.

Ubiquitin Specific Peptidase 16 (USP16) has been reported to contribute to somatic stem-cell defects in Down syndrome. However, how this gene being regulated is largely unknown. To study the mechanism underlying USP16 gene expression, USP16 gene promoter was cloned and analyzed by luciferase assay. We identified that the 5' flanking region (- 1856 bp ~ + 468 bp) of the human USP16 gene contained the functional promotor to control its transcription. Three bona fide NFκB binding sites were found in USP16 promoter. We showed that p65 overexpression enhanced endogenous USP16 mRNA level. Furthermore, LPS and TNFα, strong activators of the NFκB pathway, upregulated the USP16 transcription. Our data demonstrate that USP16 gene expression is tightly regulated at transcription level. NFκB signaling regulates the human USP16 gene expression through three cis-acting elements. The results provide novel insights into a potential role of dysregulation of USP16 expression in Alzheimer's dementia in Down Syndrome.