Poly-ADP ribosylation of p21 by tankyrases promotes p21 degradation and regulates cell cycle progression.

p21WAF1/Cip1 acts as a key negative regulator of cell cycle progression, which can form complexes with cyclin-dependent kinases together with specific cyclins to induce cell cycle arrest at specific stages. p21 protein levels have been shown to be regulated primarily through phosphorylation and ubiquitination during various stages of the cell cycle. Although phosphorylation and ubiquitin-dependent proteasomal degradation of p21 have been well established, other post-translational modifications that contribute to regulation of p21 stability and function remain to be further elucidated. Here, we show that p21 degradation and its function are controlled by tankyrases, which are members of the poly(ADP-ribose) polymerase (PARP) protein family. p21 interacts with tankyrases via newly defined tankyrase-binding motifs and is PARylated by tankyrases in vitro and in vivo, suggesting that PARylation is a new post-translational modification of p21. Up-regulation of tankyrases induces ubiquitin-dependent proteasomal degradation of p21 through an E3 ligase RNF146, thus promoting cell cycle progression in the G1/S phase transition. On the contrary, inhibition of tankyrases by knockdown or inhibitor treatment stabilizes p21 protein and leads to cell cycle arrest in the G1 phase. Together, our data demonstrate that tankyrase may function as a new molecular regulator that controls the protein levels of p21 through PARylation-dependent proteasomal degradation. Hence, a novel function of the tankyrase-p21 axis may represent a new avenue for regulating cell cycle progression.

Cataract Surgery Practice in the Republic of Korea: A Survey of the Korean Society of Cataract and Refractive Surgery 2020.

PURPOSE: To describe current cataract surgery practice patterns and trends among Korean ophthalmologists. METHODS: A survey was conducted among members of the Korean Society of Cataract and Refractive Surgery in October 2020. Of the 998 questionnaires, 262 (26.3%) were received for analysis. Data were analyzed using descriptive statistics and compared with those of previous surveys. RESULTS: The largest percentage of respondents (39%) had <5 years of practical experience, and 40% had >11 years of practical experience. The average, median, and mode monthly volumes of cataract surgeries performed by the Korean Society of Cataract and Refractive Surgery members were 31, 20, and 10 cases, respectively. Topical anesthesia was administered by 85% of the respondents. For intraocular lens (IOL) calculations, 96% of the respondents used optical biometry. The proportion of surgeons providing femtosecond laser-assisted cataract surgery increased significantly from 5% in 2018 to 29% in 2020. This increase was accompanied by an increase in the multifocal IOLs. Those who implant multifocal IOL for >10% of their cases increased from 16% (2018) to 29% (2020). Topical nonsteroidal anti-inflammatory drugs were prescribed postoperatively by 76% of the respondents. Most respondents (70%) prescribed these anti-inflammatory drugs for 4 weeks. CONCLUSIONS: This survey provided a comprehensive update on current cataract surgery practice in the Republic of Korea. The results highlighted the increasing use of premium IOLs, femtosecond laser-assisted cataract surgery, optical biometry, and topical anesthesia to better meet the patients' needs.

RING-finger protein 166 plays a novel pro-apoptotic role in neurotoxin-induced neurodegeneration via ubiquitination of XIAP.

The dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), has been widely utilized to establish experimental models of Parkinson disease and to reveal the critical molecules and pathway underlying neuronal death. The profile of gene expression changes following 6-OHDA treatment of MN9D dopaminergic neuronal cells was investigated using a TwinChip Mouse-7.4K microarray. Functional clustering of altered sets of genes identified RING-finger protein 166 (RNF166). RNF166 is composed of an N-terminal RING domain and C-terminal ubiquitin interaction motif. RNF166 localized in the cytosol and nucleus. At the tissue level, RNF166 was widely expressed in the central nervous system and peripheral organs. In the cerebral cortex, its expression decreased over time. In certain conditions, overexpression of RNF166 accelerates the naturally occurring neuronal death and 6-OHDA-induced MN9D cell death as determined by TUNEL and annexin-V staining, and caspase activation. Consequently, 6-OHDA-induced apoptotic cell death was attenuated in RNF166-knockdown cells. In an attempt to elucidate the mechanism underlying this pro-apoptotic activity, binding protein profiles were assessed using the yeast two-hybrid system. Among several potential binding candidates, RNF166 was shown to interact with the cytoplasmic X-linked inhibitor of apoptosis (XIAP), inducing ubiquitin-dependent degradation of XIAP and eventually accelerating caspase activation following 6-OHDA treatment. RNF166's interaction with and resulting inhibition of the XIAP anti-caspase activity was further enhanced by XIAP-associated factor-1 (XAF-1). Consequently, depletion of RNF166 suppressed 6-OHDA-induced caspase activation and apoptotic cell death, which was reversed by XIAP knockdown. In summary, our data suggest that RNF166, a novel E3 ligase, plays a pro-apoptotic role via caspase activation in neuronal cells.

Corrigendum to "Impact of peripheral optical properties induced by orthokeratology lens use on myopia progression" [Heliyon 6 (4), (April 2020) Article e03642].

[This corrects the article DOI: 10.1016/j.heliyon.2020.e03642.].

Impact of peripheral optical properties induced by orthokeratology lens use on myopia progression.

The objective of the present retrospective comparative cohort study was to compare the impact of wearing glasses versus an orthokeratology (Ortho-K) lens on peripheral optical properties and myopia progression in a population of South Korean children. Participants included children with myopia, between 8 and 12 years of age (n = 22 eyes), and divided into two groups: those who used glasses (Group I, n = 9) and those who used an Ortho-K lens (Group II, n = 13). Myopia progression over one year was quantified by changes in the central axial length of the eye. Keratometry and corneal aberrations on both the anterior and posterior surfaces of the eye were obtained using a Scheimpflug camera. A custom-developed Shack-Hartmann aberrometer was also used to measure peripheral aberrations across the horizontal visual field, up to 30°, and along the nasal-temporal meridian in 10-degree steps. Central axial elongation was larger in Group I (0.59 ± 0.21 mm) than in Group II (0.34 ± 0.18 mm) (P = .01). Relative peripheral spherical refractions at 10 and 20° nasally and at 10° temporally (P = 0.04, 0.049, and 0.042, respectively) relative to the fovea were positively correlated with central axial elongation in Group II. Group II exhibited an increase in peripheral ocular high order aberrations, such as horizontal coma and asymmetric trefoil. The use of Ortho-K lenses was found to slow the rate of central axis elongation in children with myopia. This effect might be related to an increase in both peripheral spherical refraction and peripheral ocular higher order aberrations with Ortho-K lens use.

HIGHER-ORDER ABERRATIONS IN EYES WITH SILICONE OIL TAMPONADE.

PURPOSE: To investigate the changes in higher-order aberrations (HOAs) after silicone oil removal and to evaluate their associations with visual acuity. METHODS: Fifty-nine eyes of 58 patients who underwent SO removal were included. Total, corneal, and internal optic HOAs, and best-corrected visual acuity were measured before and 1 month after SO removal, and changes were compared between phakic and pseudophakic eyes. RESULTS: Total ocular and internal optic HOAs decreased significantly after SO removal both in pseudophakic (n = 40, all P < 0.001) and phakic eyes (n = 19, P = 0.017, P = 0.004). Preoperative HOAs (P < 0.001) and changes in HOAs (P = 0.006) were greater in pseudophakic eyes than in phakic eyes. Best-corrected visual acuity was significantly improved after SO removal, from 20/105 to 20/78 (P < 0.001) in pseudophakic eyes, whereas there was no difference in phakic eyes (P = 0.714). Preoperative HOAs and the reduction in HOAs after SO removal were greater in best-corrected visual acuity-improved eyes than best-corrected visual acuity-unchanged eyes (P < 0.001). CONCLUSION: Silicone oil tamponade induced an increase in HOAs, and these increases were greater in pseudophakic eyes than in phakic eyes. Silicone oil may cause additional visual impairments because of HOAs, beyond those caused by retinal diseases, particularly in pseudophakic eyes.

Loss of RNA-binding protein HuR facilitates cellular senescence through posttranscriptional regulation of TIN2 mRNA.

Cellular senescence can be induced by high levels of reactive oxygen species (ROS) produced by mitochondria. However, the mechanism by which elevated mitochondrial ROS levels are produced during replicative senescence is not yet fully understood. Here, we report that loss of the RNA-binding protein, human antigen R (HuR), during replicative senescence leads to an increase in ROS levels through enhanced mitochondrial localization of the telomeric protein TIN2. HuR binds to the 3' untranslated region of TIN2 mRNA. This association decreases TIN2 protein levels by both destabilizing TIN2 mRNA and reducing its translation. Conversely, depletion of HuR levels enhances TIN2 expression, leading to increased mitochondrial targeting of TIN2. Mitochondrial localization of TIN2 increases ROS levels, which contributes to induction and maintenance of cellular senescence. Our findings provide compelling evidence for a novel role of HuR in controlling the process of cellular senescence by regulating TIN2-mediated mitochondrial ROS production, and for a useful therapeutic route for modulating intracellular ROS levels in treating both aging-related complications and cancer.

Hsp90-binding immunophilin FKBP52 modulates telomerase activity by promoting the cytoplasmic retrotransport of hTERT.

Telomerase is a unique ribonucleoprotein enzyme that is required for continued cell proliferation. To generate catalytically active telomerase, human telomerase reverse transcriptase (hTERT) must translocate to the nucleus and assemble with the RNA component of telomerase. The molecular chaperones heat shock protein 90 (Hsp90) and p23 maintain hTERT in a conformation that enables nuclear translocation. However, the regulatory role of chaperones in nuclear transport of hTERT remains unclear. In this work, we demonstrate that immunophilin FK506-binding protein (FKBP)52 linked the hTERT-Hsp90 complex to the dynein-dynactin motor, thereby promoting the transport of hTERT to the nucleus along microtubules. FKBP52 interacted with the hTERT-Hsp90 complex through binding of the tetratricopeptide repeat domain to Hsp90 and binding of the dynamitin (Dyt) component of the dynein-associated dynactin complex to the peptidyl prolyl isomerase domain. The depletion of FKBP52 inhibited nuclear transport of hTERT, resulting in cytoplasmic accumulation. Cytoplasmic hTERT was rapidly degraded through ubiquitin (Ub)-dependent proteolysis, thereby abrogating telomerase activity. In addition, overexpression of dynamitin, which is known to dissociate the dynein-dynactin motor from its cargoes, reduced telomerase activity. Collectively, these results provide a molecular mechanism by which FKBP52 modulates telomerase activity by promoting dynein-dynactin-dependent nuclear import of hTERT.

NEDD8 ultimate buster-1 regulates the abundance of TRF1 at telomeres by promoting its proteasomal degradation.

The human telomeric protein TRF1 negatively regulates telomere length by inhibiting the access of telomerase to telomeres. Here, we describe a novel function of NEDD8 ultimate buster-1 (NUB1) for regulating the levels of TRF1 at telomeres. NUB1 is a NEDD8-interacting protein, which down-regulates the NEDD8 conjugation system. We showed that NUB1 physically interacts with TRF1 and promotes its degradation by the proteasome in the absence of NEDD8 conjugation. We also demonstrated that TRF1 is conjugated to NEDD8, and that neddylated TRF1 is targeted to the proteasome for degradation in a NUB1-dependent manner. These data suggest that NUB1 participates in telomere maintenance by regulating the levels of TRF1 at telomeres through both NEDD8-dependent and NEDD8-independent pathways.

Telomerase activates transcription of cyclin D1 gene through an interaction with NOL1.

Telomerase is a ribonucleoprotein enzyme that is required for the maintenance of telomere repeats. Although overexpression of telomerase in normal human somatic cells is sufficient to overcome replicative senescence, the ability of telomerase to promote tumorigenesis requires additional activities that are independent of its role in telomere extension. Here, we identify proliferation-associated nucleolar antigen 120 (NOL1, also known as NOP2) as a telomerase RNA component (TERC)-binding protein that is found in association with catalytically active telomerase. Although NOL1 is highly expressed in the majority of human tumor cells, the molecular mechanism by which NOL1 contributes to tumorigenesis remained unclear. We show that NOL1 binds to the T-cell factor (TCF)-binding element of the cyclin D1 promoter and activates its transcription. Interestingly, telomerase is also recruited to the cyclin D1 promoter in a TERC-dependent manner through the interaction with NOL1, further enhancing transcription of the cyclin D1 gene. Depletion of NOL1 suppresses cyclin D1 promoter activity, thereby leading to induction of growth arrest and altered cell cycle distributions. Collectively, our findings suggest that NOL1 represents a new route by which telomerase activates transcription of cyclin D1 gene, thus maintaining cell proliferation capacity.

PIAS1-mediated sumoylation promotes STUbL-dependent proteasomal degradation of the human telomeric protein TRF2.

The human telomeric protein TRF2 protects chromosome ends by facilitating their organization into the protective capping structure. Here we show that the stability of TRF2 is regulated via modification by the small ubiquitin-like modifiers (SUMO). TRF2 specifically interacts with and is sumoylated by PIAS1 in mammalian cells. The proteasome inhibitor stabilizes SUMO-conjugated TRF2 without affecting the level of unmodified TRF2, suggesting that SUMO conjugation is required for proteasomal degradation of TRF2. We also show that RNF4, a mammalian SUMO-targeted ubiquitin ligase, interacts with TRF2 in a SUMO-dependent manner and preferentially targets SUMO-conjugated TRF2 for ubiquitination. Collectively, our data demonstrate that the PIAS1-mediated sumoylation status of TRF2 serves as a molecular switch that controls the level of TRF2 at telomeres.

Involvement of SRSF11 in cell cycle-specific recruitment of telomerase to telomeres at nuclear speckles.

Telomerase, a unique ribonucleoprotein complex that contains the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC) and the TERC-binding protein dyskerin, is required for continued cell proliferation in stem cells and cancer cells. Here we identify SRSF11 as a novel TERC-binding protein that localizes to nuclear speckles, subnuclear structures that are enriched in pre-messenger RNA splicing factors. SRSF11 associates with active telomerase enzyme through an interaction with TERC and directs it to nuclear speckles specifically during S phase of the cell cycle. On the other hand, a subset of telomeres is shown to be constitutively present at nuclear speckles irrespective of cell cycle phase, suggesting that nuclear speckles could be the nuclear sites for telomerase recruitment to telomeres. SRSF11 also associates with telomeres through an interaction with TRF2, which facilitates translocation of telomerase to telomeres. Depletion of SRSF11 prevents telomerase from associating with nuclear speckles and disrupts telomerase recruitment to telomeres, thereby abrogating telomere elongation by telomerase. These findings suggest that SRSF11 acts as a nuclear speckle-targeting factor that is essential for telomerase association with telomeres through the interactions with TERC and TRF2, and provides a potential target for modulating telomerase activity in cancer.

Akt-mediated phosphorylation increases the binding affinity of hTERT for importin α to promote nuclear translocation.

Telomeres are essential for chromosome integrity and protection, and their maintenance requires the ribonucleoprotein enzyme telomerase. Previously, we have shown that human telomerase reverse transcriptase (hTERT) contains a bipartite nuclear localization signal (NLS; residues 222-240) that is responsible for nuclear import, and that Akt-mediated phosphorylation of residue S227 is important for efficient nuclear import of hTERT. Here, we show that hTERT binds to importin-α proteins through the bipartite NLS and that this heterodimer then forms a complex with importin-ß proteins to interact with the nuclear pore complex. Depletion of individual importin-α proteins results in a failure of hTERT nuclear import, and the resulting cytoplasmic hTERT is degraded by ubiquitin-dependent proteolysis. Crystallographic analysis reveals that the bipartite NLS interacts with both the major and minor sites of importin-α proteins. We also show that Akt-mediated phosphorylation of S227 increases the binding affinity for importin-α proteins and promotes nuclear import of hTERT, thereby resulting in increased telomerase activity. These data provide details of a binding mechanism that enables hTERT to interact with the nuclear import receptors and of the control of the dynamic nuclear transport of hTERT through phosphorylation.

DNA-PKcs-interacting protein KIP binding to TRF2 is required for the maintenance of functional telomeres.

Human telomeres associate with shelterin, a six-protein complex that protects chromosome ends from being recognized as sites of DNA damage. The shelterin subunit TRF2 (telomeric repeat-binding factor 2) protects telomeres by facilitating their organization into the protective capping structure. We have reported previously that the DNA-PKcs (DNA-dependent protein kinase catalytic subunit)-interacting protein KIP associates with telomerase through an interaction with hTERT (human telomerase reverse transcriptase). In the present study, we identify KIP as a novel interacting partner of TRF2. KIP is able to interact with both TRF2 and DNA-PKcs at telomeres. Because KIP is required for the association between TRF2 and DNA-PKcs, the interplay of these three proteins may provide a mechanism for the recruitment of DNA-PKcs to telomeres. We also show that KIP binding to TRF2 enhances the telomere-binding activity of TRF2, suggesting that KIP acts as a positive regulator of TRF2 function. Furthermore, depletion of KIP induces DNA-damage response foci at telomeres, thereby leading to induction of growth arrest, cellular senescence and altered cell cycle distribution. Collectively, our findings suggest that KIP, in addition to its association with catalytically active telomerase, plays important roles in the maintenance of functional telomeres and the regulation of telomere-associated DNA-damage response. Thus KIP represents a new pathway for modulating telomerase and telomere function in cancer.

The splicing factor U2AF65 stabilizes TRF1 protein by inhibiting its ubiquitin-dependent proteolysis.

The human telomeric protein TRF1 is a component of the six-subunit protein complex shelterin, which provides telomere protection by organizing the telomere into a high-order structure. TRF1 functions as a negative regulator of telomere length by controlling the access of telomerase to telomeres. Thus, the cellular abundance of TRF1 at telomeres should be maintained and tightly regulated to ensure proper telomere function. Here, we identify U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 (U2AF65), an essentialpre-mRNA splicingfactor, as a novel TRF1-interacting protein. U2AF65 interacts with TRF1 in vitro and in vivo and is capable of stabilizing TRF1 protein by inhibiting its ubiquitin-dependent proteolysis. We also found that U2AF65 interferes with the interaction between TRF1 and Fbx4, an E3 ubiquitin ligase for TRF1. Depletion of endogenous U2AF65 expression by short interfering RNA (siRNA) reduced the stability of endogenous TRF1 whereas overexpression of U2AF65 significantly extended the half-life of TRF1. These findings demonstrate that U2AF65 plays a critical role in regulating the level of TRF1 through physical interaction and ubiquitin-mediated proteolysis. Hence, U2AF65 represents a new route for modulating TRF1 function at telomeres.

Catalytically active telomerase holoenzyme is assembled in the dense fibrillar component of the nucleolus during S phase.

The maintenance of human telomeres requires the ribonucleoprotein enzyme telomerase, which is composed of telomerase reverse transcriptase (TERT), telomerase RNA component, and several additional proteins for assembly and activity. Telomere elongation by telomerase in human cancer cells involves multiple steps including telomerase RNA biogenesis, holoenzyme assembly, intranuclear trafficking, and telomerase recruitment to telomeres. Although telomerase has been shown to accumulate in Cajal bodies for association with telomeric chromatin, it is unclear where and how the assembly and trafficking of catalytically active telomerase is regulated in the context of nuclear architecture. Here, we show that the catalytically active holoenzyme is initially assembled in the dense fibrillar component of the nucleolus during S phase. The telomerase RNP is retained in nucleoli through the interaction of hTERT with nucleolin, a major nucleolar phosphoprotein. Upon association with TCAB1 in S phase, the telomerase RNP is transported from nucleoli to Cajal bodies, suggesting that TCAB1 acts as an S-phase-specific holoenzyme component. Furthermore, depletion of TCAB1 caused an increase in the amount of telomerase RNP associated with nucleolin. These results suggest that the TCAB1-dependent trafficking of telomerase to Cajal bodies occurs in a step separate from the holoenzyme assembly in nucleoli. Thus, we propose that the dense fibrillar component is the provider of active telomerase RNP for supporting the continued proliferation of cancer and stem cells.

Predisposition to apoptosis in keratin 8-null liver is related to inactivation of NF-κB and SAPKs but not decreased c-Flip.

Keratin 8 and 18 (K8/K18) are major intermediate filament proteins of liver hepatocytes. They provide mechanical and nonmechanical stability, thereby protecting cells from stress. Hence, K8-null mice are highly sensitive to Fas-mediated liver cell apoptosis. However, the role of c-Flip protein in K8-null related susceptibility to liver injury is controversial. Here we analyzed c-Flip protein expression in various K8 or K18 null/mutant transgenic livers and show that they are similar in all analyzed transgenic livers and that previously reported c-Flip protein changes are due to antibody cross-reaction with mouse K18. Furthermore, analysis of various apoptosis- or cell survival-related proteins demonstrated that inhibition of phosphorylation of NF-κB and various stress activated protein kinases (SAPKs), such as p38 MAPK, p44/42 MAPK and JNK1/2, is related to the higher sensitivity of K8-null hepatocytes whose nuclear NF-κB is rapidly depleted through Fas-mediated apoptosis. Notably, we found that NF-κB and the studied protein kinases are associated with the K8/K18 complex and are released upon phosphorylation. Therefore, interaction of keratins with cell survival-related protein kinases and transcription factors is another important factor for hepatocyte survival.

p300-mediated acetylation of TRF2 is required for maintaining functional telomeres.

The human telomeric protein TRF2 is required to protect chromosome ends by facilitating their organization into the protective capping structure. Post-translational modifications of TRF2 such as phosphorylation, ubiquitination, SUMOylation, methylation and poly(ADP-ribosyl)ation have been shown to play important roles in telomere function. Here we show that TRF2 specifically interacts with the histone acetyltransferase p300, and that p300 acetylates the lysine residue at position 293 of TRF2. We also report that p300-mediated acetylation stabilizes the TRF2 protein by inhibiting its ubiquitin-dependent proteolysis and is required for efficient telomere binding of TRF2. Furthermore, overexpression of the acetylation-deficient mutant, K293R, induces DNA-damage response foci at telomeres, thereby leading to induction of impaired cell growth, cellular senescence and altered cell cycle distribution. A small but significant number of metaphase chromosomes show no telomeric signals at chromatid ends, suggesting an aberrant telomere structure. These findings demonstrate that acetylation of TRF2 by p300 plays a crucial role in the maintenance of functional telomeres as well as in the regulation of the telomere-associated DNA-damage response, thus providing a new route for modulating telomere protection function.