E3 Ubiquitin Ligase ASB17 Promotes Apoptosis by Ubiquitylating and Degrading BCLW and MCL1

Simple Summary B-cell lymphoma-2 family proteins have been widely accepted as the critical regulators in cell apoptosis, often found to be abnormally expressed in many cancers. Among them, B-cell leukemia/lymphoma w and myeloid cell leukemia-1 are two pro-survival proteins. Here, we reported that the ankyrin repeat and SOCS box protein 17 can degrade the two proteins in a ubiquitylation -dependent way. Furthermore, we generated the first ASB17 knockout C57BL/6J mice line. The results revealed that ASB17 deficiency inhibited apoptosis but did not affect testes development. Moreover, the ASB17-deficient mice were more resistant to the stimuli of etoposide, Altogether, these findings indicate that ASB17 is a novel positive mediator of cell apoptosis. Abstract Apoptosis is a very important process of cell death controlled by multiple genes during which cells undergo certain events before dying. Apoptosis helps to clean the unnecessary cells and has critical physiological significance. Altered apoptosis results in a disorder of cell death and is associated with many diseases such as neurodegenerative diseases and cancers. Here, we reported that the ankyrin repeat and SOCS box protein 17 (ASB17) was mainly expressed in the testis and promoted apoptosis both in vivo and in vitro. Analyzing ASB17-deficient mice generated by using the CRISPR/Cas9 system, we demonstrated that ASB17 deficiency resulted in the reduction of apoptosis in spermatogenic cells, but it did not affect the development of spermatozoa or normal fertility. Next, in an in vivo model, ASB17 deficiency prevented the apoptosis of spermatogonia induced by etoposide in male mice. We noted that ASB17 promoted apoptosis in a caspase-dependent manner in vitro. Moreover, ASB17 interacted with the members of the BCL2 family, including BCL2, BCLX, BCLW, and MCL1. Interestingly, ASB17 specifically degraded the two anti-apoptotic factors, BCLW and MCL1, in a ubiquitylation-dependent fashion. Collectively, our findings suggested that ASB17 acted as a distinct positive regulator of cell apoptosis.


Introduction
Apoptosis is a complicated process during which the regulated destruction of a cell happens, and the activity of many genes influences this process [1]. The typical morphological characteristics of apoptosis include cell shrinkage, nuclear fragmentation, chromatin condensation, and membrane blistering [2][3][4]. There are intrinsic and extrinsic pathways

Animal Studies
C57BL/6J ASB17 +/− mice were generated by Beijing Biocytogen (Beijing, China) using the CRISPR/Cas9 technique. All animal experiments were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Wuhan University Animal Care and Use Committee guidelines. Two pairs of primers were used to examine the genotypes of mice: WT-F: 5 -GGCTGCATCACTCTGCCGCT-3 ; WT-R: 5 -TCCGAAGGCAACAGAAGGGGAA-3 ; Mut-F: 5 -AGGAAGCCCCAGGTCTTCATCCT-3 .

Intratesticular Injections
Male mice of 6 weeks were anesthetized with avertin. The testes were exteriorized through a low midline abdominal incision. Ten microliters of a solution containing 80 mM etoposide in DMSO were injected into the testes via a 30G needle. Following injection, the testes were returned to the peritoneal cavity, and the incisions were sutured. The mice were recovered and raised for 24 h. DMSO was injected into the testes as a control.

Lentiviral Package and Infection
ASB17 was cloned into pLenti-CMV vector, which was derived from the pLenti-CMV-EGFP vector bought from Addgene to generate pLenti-ASB17. Together with pMD2.G (an envelope plasmid) and psPAX2 (a packaging plasmid), pLenti-ASB17 was transfected into HEK293T cells. After 36 h and 48 h post-transfection, the cell supernatants were collected and then passed through a 0.45 µm filter to remove the cells. Hela cells were infected with harvested supernatants plus 8 µg/mL of Polybrene for 24 h. Hela stably expressing ASB17 cells were identified by Q-PCR.

Immunofluorescence Microscopy
The total cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. And then they were permeabilized with PBS containing 0.2% Triton X-100 for 5 min and blocked with PBS containing 5% BSA for 1 h at 24°C. After being incubated with the indicated antibody at 4°C for 12-16 h, the cells were washed with PBS and then incubated with anti-Mouse IgG Dylight 649 and anti-Rabbit IgG FITC at room temperature for 45-60 min. Cells were incubated with DAPI for 5 min at 37°C after being washed with PBS. Eventually, the cells were analyzed using FluoView FV 1000 (Olympus, Tokyo, Japan).

TUNEL Assay
Testes and epididymis were isolated from male mice and fixed in Bouin's solution or 4% paraformaldehyde at 4 • C. TUNEL assay was done by using the In Situ Cell Death Detection Kit, POD (No. 11684817910; Roche Applied Science, Indianapolis, IN, USA), in accordance with the manufacturer's instructions.

Fertility Evaluation
Male ASB17 +/+ or ASB17 −/− mice were mated to wild-type females at a male/female ratio of 1/2. The litter sizes were analyzed as a measure of fertility.

ASB17 Is Mainly Expressed in the Testis and Epididymis and Slightly Elevated in Spleen and Lung
To investigate the function of the ASB17 gene in vivo, we generated ASB17 +/− mice by using the CRISPR-Cas 9 technique to delete exon 1 of the whole genome of the ASB17 gene ( Figure 1A). After the breeding of ASB17 +/− mice, the mutant, heterozygous, and wild-type (WT) mice were bred. Their offspring were then identified by PCR analysis using DNA isolated from tail snips ( Figure 1B). The expression of ASB17 mRNA in the testis from WT and ASB17 −/− mice was checked by quantitative PCR (Q-PCR) ( Figure 1C). RNA isolated from the heart, liver, spleen, lung, kidney, large intestine, small intestine, testis, and Biology 2021, 10, 234 5 of 16 epididymis was used for Q-PCR analysis. The results told us ASB17 was mainly expressed in the testis and epididymis and slightly expressed in the spleen and lung ( Figure 1D).

ASB17 Deficiency in the Testes Attenuated Apoptosis with No Effect on Testes Development
To determine the effect of ASB17 on testis development, the testes from mice of different ages were harvested. The testes morphology in six-week ASB17 −/− male mice showed no significant difference, compared to those from wild-type mice (Figure 2A). Testes isolated from male ASB17 −/− mice weighed almost as much as those from the agematched WT mice ( Figure 2B). To further understand the function of ASB17 in the testis, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was used. Surprisingly, testes isolated from three-week and six-week ASB17 −/− male mice had significantly fewer apoptotic cells than those from the control group, whereas the apoptosis in the testes did not change much between two-week KO mice and WT mice (Figure 2C,E,G). Further analyses showed that apoptosis in three-week and six-week ASB17 −/− testes decreased by two-fold (p = 0.0044) and eight-fold (p = 0.0001), respectively ( Figure 2F,H), whereas no significant change can be found between two-week KO and WT mice ( Figure  2D). Furthermore, ASB17 deficiency was associated with decreased cleaved-Caspase3 protein expression in the testes from six-week mice ( Figure 2I). The role of ASB17 in regulating Next, we examined the expression pattern of ASB17 in mice testis. The Q-PCR analysis revealed that ASB17 was expressed from three weeks onwards and reached the top level in around four weeks ( Figure 1E). We also checked the ASB17 mRNA expression in four mice testis-derived cell lines, i.e., spermatogonium GC1-spg, spermatocyte GC2-spd, Leydig (TM3), and Sertoli (TM4) cells. The results indicated that ASB17 was barely expressed ( Figure 1F). HepG2 cells, Huh7 cells, RD cells, U251 cells, A549 cells, Hela cells, THP1 cells, 293T cells, and Hela cells infected by lentivirus-carrying ASB17 vectors were used to examine the mRNA level of ASB17. The results indicated that ASB17 was barely expressed in these cells ( Figure 1G). Furthermore, the expression status of ASB17 was determined in different human testicular cells using the Human Atlas Browser https://humantestisatlas.shinyapps.io/humantestisatlas1/ (accessed on 10 December 2020) [22]. It was concluded that the highest level of ASB17 was expressed in the round and elongated spermatids and differentiating germ cells. Taken together, Biology 2021, 10, 234 6 of 16 these data suggested that ASB17 −/− mice were successfully generated, and ASB17 expression was in a tissue-specific and temporospatial pattern. The high expression pattern of ASB17 in the testis suggested that ASB17 might play roles in testis development and spermatogenesis.

ASB17 Deficiency in the Testes Attenuated Apoptosis with No Effect on Testes Development
To determine the effect of ASB17 on testis development, the testes from mice of different ages were harvested. The testes morphology in six-week ASB17 −/− male mice showed no significant difference, compared to those from wild-type mice ( Figure 2A). Testes isolated from male ASB17 −/− mice weighed almost as much as those from the age-matched WT mice ( Figure 2B). To further understand the function of ASB17 in the testis, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was used. Surprisingly, testes isolated from three-week and six-week ASB17 −/− male mice had significantly fewer apoptotic cells than those from the control group, whereas the apoptosis in the testes did not change much between two-week KO mice and WT mice ( Figure 2C,E,G). Further analyses showed that apoptosis in three-week and six-week ASB17 −/− testes decreased by two-fold (p = 0.0044) and eight-fold (p = 0.0001), respectively ( Figure 2F,H), whereas no significant change can be found between two-week KO and WT mice ( Figure 2D). Furthermore, ASB17 deficiency was associated with decreased cleaved-Caspase3 protein expression in the testes from six-week mice ( Figure 2I). The role of ASB17 in regulating the apoptosis of testis was strongly related to the expression pattern of ASB17 since it was expressed from three weeks and reached the top level by four weeks. Thus, these results indicated that ASB17 promoted apoptosis in the testis when it was expressed from three weeks of age, and the deletion of ASB17 did not affect testes development.
Biology 2021, 10, x FOR PEER REVIEW 7 of 16 the apoptosis of testis was strongly related to the expression pattern of ASB17 since it was expressed from three weeks and reached the top level by four weeks. Thus, these results indicated that ASB17 promoted apoptosis in the testis when it was expressed from three weeks of age, and the deletion of ASB17 did not affect testes development. GAPDH was used as a loading control. For each mouse, 100 random seminiferous tubules sections were analyzed to count the apoptotic cells, and per 10 random tubules, random tubules cross-sections were presented as a number. Data shown are the mean ± SEM. **, p < 0.01; ****, p < 0.0001, and no significance (ns).

ASB17 −/− Mice Display Normal Spermatogenesis
To find out if ASB17 deficiency affected spermatogenesis, hematoxylin and eosin (H&E) staining and periodic acid-Schiff (PAS) staining were used to examine the histology of testes from two, three, and six weeks. Histology analysis revealed that ASB17 −/− testes had no obvious structural change and contained a complete lineage of germ cells GAPDH was used as a loading control. For each mouse, 100 random seminiferous tubules sections were analyzed to count the apoptotic cells, and per 10 random tubules, random tubules cross-sections were presented as a number. Data shown are the mean ± SEM. **, p < 0.01; ****, p < 0.0001, and no significance (ns).

ASB17 −/− Mice Display Normal Spermatogenesis
To find out if ASB17 deficiency affected spermatogenesis, hematoxylin and eosin (H&E) staining and periodic acid-Schiff (PAS) staining were used to examine the histology of testes from two, three, and six weeks. Histology analysis revealed that ASB17 −/− testes had no obvious structural change and contained a complete lineage of germ cells compared to WT testes ( Figure 3A-F). Furthermore, acrosome morphogenesis did not change in ASB17 −/− testes, indicating normal spermiogenesis ( Figure 3G,H). Almost the same amounts of spermatid cells could be found in the epididymis cauda of six-week WT and ASB17 −/− male mice ( Figure 3G,H). Next, the fertility of adult ASB17 −/− mice was checked. Unsurprisingly, ASB17 −/− male mice were fertile, and the size of the litter showed no significant change with the litter farther from the WT group ( Figure 3I). In a word, although ASB17 was highly expressed in the testis, it did not seem to have a role in spermatogenesis under normal conditions.

ASB17 Deficiency Prevents the Apoptosis of Spermatogonia Induced by Etoposide in Male Mice
A previous study showed that the intratesticular injection of etoposide promoted germ cell apoptosis [13]. Etoposide dissolved in DMSO was intratesticular injected into both WT and ASB17 −/− male mice at six weeks. The results revealed that testes isolated from ASB17 −/− male mice had significantly fewer apoptotic cells than those from WT mice

ASB17 Deficiency Prevents the Apoptosis of Spermatogonia Induced by Etoposide in Male Mice
A previous study showed that the intratesticular injection of etoposide promoted germ cell apoptosis [13]. Etoposide dissolved in DMSO was intratesticular injected into both WT and ASB17 −/− male mice at six weeks. The results revealed that testes isolated from ASB17 −/− male mice had significantly fewer apoptotic cells than those from WT mice in both the control and experiment groups ( Figure 4A,B). Furthermore, the intratesticular injection of etoposide significantly promoted germ cell apoptosis in both WT and ASB17 −/− testes ( Figure 4A,B). The data showed that the apoptosis of ASB17 −/− testes decreased by 2.8-fold (p = 0.0001) in the control group and 2.4-fold (p = 0.0002) in the experiment group, respectively ( Figure 4C). Moreover, much less cleaved-Caspase-3 protein expression could be detected in ASB17 −/− testes from both the control and experiment groups ( Figure 4D). Overall, etoposide can promote germ cell apoptosis in both WT and ASB17 −/− mice, whereas ASB17 −/− mice were much more resistant in response to etoposide (ETO) treatment. Thus, it indicated that ASB17 deficiency prevented apoptosis of spermatogonia induced by etoposide in male mice.
Biology 2021, 10, x FOR PEER REVIEW 9 of 16 testes ( Figure 4A,B). The data showed that the apoptosis of ASB17 −/− testes decreased by 2.8-fold (p = 0.0001) in the control group and 2.4-fold (p = 0.0002) in the experiment group, respectively ( Figure 4C). Moreover, much less cleaved-Caspase-3 protein expression could be detected in ASB17 −/− testes from both the control and experiment groups ( Figure  4D). Overall, etoposide can promote germ cell apoptosis in both WT and ASB17 −/− mice, whereas ASB17 −/− mice were much more resistant in response to etoposide (ETO) treatment. Thus, it indicated that ASB17 deficiency prevented apoptosis of spermatogonia induced by etoposide in male mice.

ASB17 Promotes Apoptosis In Vitro
To further invest ASB17′s role in apoptosis, Hela cells were transfected with Flagtagged ASB17. We chose cleaved (activated) PARP, cleaved Caspase-9, and cleaved Caspase-3 as the indicators of apoptosis [23]. The results showed that the cleaved PARP, cleaved Caspase-9, and cleaved Caspase-3 ( Figure 5A) were elevated in the presence of ASB17. Since ASB17 was of low expression in Hela cells, we next engineered Hela cells stably expressed ASB17 and detected the mRNA level of ASB17 ( Figure 5B). Staurosporine (STS) and etoposide (ETO) were used to induce apoptosis in Hela cells [24,25]. Firstly, a cell counting kit-8 (CCK8) assay was used to check the effect of ASB17 on the cell viability of Hela cells with or without STS and ETO. The results showed the number of cell survival in the control group was significantly higher than that in the experiment group ( Figure 5C). Western blot analysis of cleaved-Caspase-3 protein expression in the testes from WT and ASB17 −/− mice of the control and experiment groups. GAPDH was used as a loading control. For each mouse, 100 random seminiferous tubules sections were analyzed to count the apoptotic cells, and per 10 random tubules, random tubules cross-sections were presented as a number. Data shown are the mean ± SEM. ***, p < 0.01; ****, p < 0.0001.

ASB17 Promotes Apoptosis In Vitro
To further invest ASB17 s role in apoptosis, Hela cells were transfected with Flagtagged ASB17. We chose cleaved (activated) PARP, cleaved Caspase-9, and cleaved Caspase-3 as the indicators of apoptosis [23]. The results showed that the cleaved PARP, cleaved Caspase-9, and cleaved Caspase-3 ( Figure 5A) were elevated in the presence of ASB17.
Since ASB17 was of low expression in Hela cells, we next engineered Hela cells stably expressed ASB17 and detected the mRNA level of ASB17 ( Figure 5B). Staurosporine (STS) and etoposide (ETO) were used to induce apoptosis in Hela cells [24,25]. Firstly, a cell counting kit-8 (CCK8) assay was used to check the effect of ASB17 on the cell viability of Hela cells with or without STS and ETO. The results showed the number of cell survival in the control group was significantly higher than that in the experiment group ( Figure 5C). The Western blot results showed the cleaved PARP, cleaved Caspase-9, and cleaved Caspase-3 were significantly elevated in ASB17 stably expressed Hela cells compared to the control group ( Figure 5D,E). Furthermore, STS was added in a time-dependent manner to induce apoptosis. Following exposure to STS, the ectopic expression of ASB17 increased the cleaved PARP, cleaved Caspase-9, and cleaved Caspase-3 ( Figure 5F). Wandering whether ASB17 promoted apoptosis in a caspase-dependent way, Z-VAD-FMK, an inhibitor of caspase, was used [26]. Following exposure to ETO and Z-VAD-FMK, ASB17 could no longer promote apoptosis ( Figure 5G). Collectively, these results indicated that ASB17 promoted apoptosis in Hela cells in a caspase-dependent manner. The Western blot results showed the cleaved PARP, cleaved Caspase-9, and cleaved Caspase-3 were significantly elevated in ASB17 stably expressed Hela cells compared to the control group ( Figure 5D,E). Furthermore, STS was added in a time-dependent manner to induce apoptosis. Following exposure to STS, the ectopic expression of ASB17 increased the cleaved PARP, cleaved Caspase-9, and cleaved Caspase-3 ( Figure 5F). Wandering whether ASB17 promoted apoptosis in a caspase-dependent way, Z-VAD-FMK, an inhibitor of caspase, was used [26]. Following exposure to ETO and Z-VAD-FMK, ASB17 could no longer promote apoptosis ( Figure 5G). Collectively, these results indicated that ASB17 promoted apoptosis in Hela cells in a caspase-dependent manner.

ASB17 Promotes the Ubiquitylation and Degradation of BCLW and MCL1
The influence of ASB17 on the protein expression of BCL2, BCLX, BCLW, and MCL1 was checked. Notably, ASB17 was found to reduce the BCLW and MCL1 protein levels ( Figure 8A). A Hela cell line stably expressing ASB17 was successfully generated. ASB17, BCLW, and MCL1 mRNA levels were detected ( Figure 8B,D,F). Next, Flag-tagged BCLW or MCL1 were transfected into the control group and ASB17 stably expressed group of Hela cells. The results indicated ASB17 decreased the protein level of both BCLW and MCL1 but did not affect their mRNA level. (Figure 8C,E). Furthermore, ASB17 overexpression greatly decreased BCLW and MCL1 protein expression in a dose-dependent manner ( Figure 8G,H). Cycloheximide (CHX) chase assay was used to compare the stability of HA-BCLW ( Figure 8I) and HA-MCL1 ( Figure 8J) with or without Flag-tagged ASB17. As expected, both HA-BLW ( Figure 8I) and HA-MCL1 ( Figure 8J) were destabilized by the ectopic expression of ASB17. The ASB17-mediated repression of BCLW ( Figure 8K) and MCL1 ( Figure 8L) was reversed by the proteasome inhibitor MG132. Moreover, mutant ASB17 lacking SOCS box (251-296aa) failed to decrease the protein level of BCLW ( Figure 8M) and MCL1 ( Figure 8N). ASB17 is a member of ASB family proteins, which show E3 ligase enzymatic activity [15]. We next examined the degradation of BCLW and MCL1 mediated by ASB17. Myc-tagged ASB17 and HA-ubiquitin were transfected into Hela cells together with Flag-tagged BCLW or MCL1. The results revealed that ASB17 promoted the ubiquitylation of BCLW and MCL1 ( Figure 8O,P). Furthermore, the deletion of the SOCS box abolished the capability of ASB17 to promote the ubiquitylation of BCLW ( Figure 8Q) and MCL1 ( Figure 8R). Altogether, these data indicated that ASB17 promoted the ubiquitylation and degradation of BCLW and MCL1.

ASB17 Promotes the Ubiquitylation and Degradation of BCLW and MCL1
The influence of ASB17 on the protein expression of BCL2, BCLX, BCLW, and MCL1 was checked. Notably, ASB17 was found to reduce the BCLW and MCL1 protein levels (Figure 8A). A Hela cell line stably expressing ASB17 was successfully generated. ASB17, BCLW, and MCL1 mRNA levels were detected ( Figure 8B,D,F). Next, Flag-tagged BCLW or MCL1 were transfected into the control group and ASB17 stably expressed group of Hela cells. The results indicated ASB17 decreased the protein level of both BCLW and MCL1 but did not affect their mRNA level. (Figure 8C,E). Furthermore, ASB17 overexpression greatly decreased BCLW and MCL1 protein expression in a dose-dependent manner ( Figure 8G,H). Cycloheximide (CHX) chase assay was used to compare the stability of HA-BCLW ( Figure 8I) and HA-MCL1 ( Figure 8J) with or without Flag-tagged show E3 ligase enzymatic activity [15]. We next examined the degradation of BCLW and MCL1 mediated by ASB17. Myc-tagged ASB17 and HA-ubiquitin were transfected into Hela cells together with Flag-tagged BCLW or MCL1. The results revealed that ASB17 promoted the ubiquitylation of BCLW and MCL1 ( Figure 8O,P). Furthermore, the deletion of the SOCS box abolished the capability of ASB17 to promote the ubiquitylation of BCLW ( Figure 8Q) and MCL1 ( Figure 8R). Altogether, these data indicated that ASB17 promoted the ubiquitylation and degradation of BCLW and MCL1.

Discussion
E3 ubiquitin ligases have been found to play an important role in regulating testis development and spermatogenesis [28,29]. As a member of ASB family proteins, which shows E3 ubiquitin ligase enzymatic activity, ASB17, which is highly expressed in the testis, remains poorly understood. Here, we characterized the expression pattern and function of ASB17 to our best for the first time, helping to extend the knowledge in this field.
Unlike the previous study in which ASB17 was exclusively expressed in the testis, we found ASB17 was also expressed in the epididymis, spleen, and lung. The temporospatial expression pattern of ASB17 in mice was in line with what was reported before [17]. Compared with wild-type mice, ASB17 −/− mice had fewer apoptotic cells in the testes by using TUNNEL assays. We expected something different in testis development and spermatogenesis between WT and ASB17 −/− mice. Notably, no significant difference was detected in the testes' weight and histology. ASB17 −/− mice at an early age remained normal in testis development and spermatogenesis. We thought that ASB17 might be not so important in the testis under normal conditions. However, under certain stimuli-like testicular toxins, heat stress, or chemotherapeutic agents that triggered germ cell apoptosis, ASB17 might help to activate apoptosis in the testis [30]. We then found that ASB17 −/− mice were much less sensitive to the stimuli of etoposide. It came to the conclusion that ASB17 did help activate apoptosis in the testis under etoposide stimuli.
We wondered how ASB17 promoted apoptosis and the mechanism inside it and then screened some apoptosis-associated proteins that might interact with ASB17. Among them, four BCL2 family proteins attracted our interest. We confirmed their interaction by Co-IP and immunofluorescence microscopy. Next, we checked if ASB17 affected the protein level of the four proteins and found that ASB17 degraded BCLW and MCL1 in a ubiquitylationdependent manner. These results confirmed that ASB17 could promote apoptosis. Since ASB17 can degrade BCLW and MCL1, the function of the two proteins can broaden our knowledge about ASB17. Even though the mice that lacked BCLW were normal, male BCLW-deficient mice were infertile and had much more apoptotic cells in the testes than those from the control group [31]. Since BCLW was located in the round and elongating spermatids where ASB17 was located. This might explain why male ASB17 −/− mice had fewer apoptotic cells than those from WT mice. BCLW was also found to be elevated in Alzheimer's disease (AD) and had a protective role in neurons [32,33]. It meant ASB17 could have a role in AD too. MCL1 is highly expressed in a variety of human cancer and is often related to chemotherapeutic resistance and relapse [34]. E3 ubiquitin ligases (Mule, SCF β-TrCP , SCF FBW7 , TRIM17, APC/C Cdc20 , and FBXO4) and deubiquitinases (USP9X, Ku70, USP13, JOSD1, and DUB3) work together to balance MCL1 stability, which has an important role in the chemoresistance of cancer cells [35,36]. Our finding that that ASB17 promoted the ubiquitylation of MCL1 and degraded it might provide a potential therapeutic option. Because no antibody of good quality for ASB17 can be used, a lot of endogenous experiments between ASB17 and BCLW or MCL1 were unable to be undertaken. Overall, our findings suggested that E3 ubiquitin ligase ASB17 was a positive regulator of cell apoptosis by promoting ubiquitylation and degradation of BCLW and MCL1. Furthermore, we provided a potential target for BCLW-and MCL1-related diseases.

Conclusions
In this study, we elaborated the function of ASB17 in apoptosis pathway. ASB17 was mainly expressed in testis and promoted apoptosis both in vivo and in vitro. Furthermore, ASB17 deficiency resulted in the reduction of apoptosis in spermatogenic cells, but it did not affect the development of spermatozoa or the normal fertility. And the ASB17 −/− mice were more resistant to the stimuli of etoposide. Next, overexpression of ASB17 could promote apoptosis in HELA cells. Moreover, BCLW and MCL1 were found to interact with ASB17. And, ASB17 can promote ubiquitylation and degradation of them. Overall, ASB17 was a novel positive regulator of cell apoptosis.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data needed to evaluate the conclusions in the paper are present in the paper.