Activity-based profiling of cullin-RING E3 networks by conformation-specific probes.

The cullin-RING ubiquitin ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We developed a synthetic antibody recognizing the active conformation of NEDD8-linked cullins. Implementing the probe to profile cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing E3s revealed the complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, and prime efficiency of targeted protein degradation. Our probe also unveiled differential rewiring of CRL networks across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate the development of degrader drugs.

Activity-based profiling of cullin-RING ligase networks by conformation-specific probes.

The cullin-RING E3 ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We report development of a synthetic antibody recognizing the active conformation of a NEDD8-linked cullin. We established a pipeline probing cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing CRLs, revealing the CRL complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, prime efficiency of targeted protein degradation, and are differentially rewired across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate development of degrader drugs.

Congenital Perineal Groove Defect in Monozygotic Twin Infants: A Literature Review.

Perineal groove is a rare benign congenital anomaly with lesion that resembles perforation of mid-perineum or perineal raphe area. Most reported cases of congenital perineal groove presented as an isolated defect in term or early-term singleton female infants. Thus far, there is no reported case of this anomaly in monozygotic twins. Embryo pathogenesis of this female predominance congenital defect remains controversial. Many clinicians are unfamiliar with this congenital anomaly. This congenital defect tends to get self-resolved at around 2 year of age. Nevertheless, the exposed nonepithelized mucous membrane can carry risk of local infection or irritation with the possibility of requiring early surgical correction. The defect can be infrequently associated with other ano-urogenital malformations that required immediate surgical intervention. Most isolated cases tend to be asymptomatic and self-healed with expectant management. Surgical correction may be considered if not healed after 2 years of age. Early diagnosis at birth is important to avoid misdiagnoses at later age for trauma, dermatitis, sexual abuse, and risk of unnecessary aggressive intervention. Early parental counseling for providing good hygiene and close follow-up is important to prevent infection or inflammation. Presentation of this anomaly in both monozygotic twins may support the hypothesis of potential disruption during embryo morphogenesis stages.

Impact of MYCN status on response of high-risk neuroblastoma to neoadjuvant chemotherapy.

BACKGROUND/PURPOSE: MYCN-amplification in neuroblastoma is associated with an aggressive clinical phenotype. We evaluated the association of MYCN amplification with tumor response to neoadjuvant chemotherapy. METHODS: Primary tumor response, assessed by percentage volume change on CT scan and degree of tumor resection, assessed by the operating surgeon, were retrospectively compared in 84 high-risk neuroblastoma patients. There were thirty-four (40%) with MYCN-amplified tumors and fifty (60%) with non-amplified tumors treated at our institution from 1999 to 2016. Metastatic disease response was assessed on MIBG scan by change in Curie score. RESULTS: MYCN-amplification was associated with a greater mean percentage reduction in primary tumor volume after neoadjuvant chemotherapy (72.27% versus 46.83% [non-amplified tumors], p = 0.001). The percentage of patients with a Curie score > 2 at diagnosis who then had a score ≤ 2 after neoadjuvant chemotherapy was not significantly different (8 [61.5%] and 8 [34.8%], respectively, p = 0.37). Twenty-eight (85.7%) patients with MYCN-amplification had ≥90% surgical resection compared to 45 (91.84%) patients with non-amplified tumors (p = 0.303). CONCLUSIONS: MYCN-amplification in high-risk neuroblastoma was associated with a better response of the primary tumor to neoadjuvant chemotherapy, but not metastatic sites, than in patients with non-amplified tumors. This did not significantly impact the ability to resect ≥90% of the primary tumor/locoregional disease. TYPE OF STUDY: Treatment Study LEVEL OF EVIDENCE: Level III.

Complications Following Nephron-Sparing Surgery for Wilms Tumor.

PURPOSE: Because of the increasing use of nephron-sparing surgery (NSS) in bilateral Wilms tumor, we sought to review the early postoperative complications associated with NSS. METHODS: A retrospective review of patients who underwent NSS at our institution from 2000 to 2017 was performed. For comparison, a cohort of patients who underwent radical nephrectomy (RN) was also reviewed. Early (30-day) postoperative complications and oncologic outcomes were assessed. RESULTS: Fifty-five patients underwent either bilateral (46) NSS or unilateral (9) NSS owing to prior resection or congenital solitary kidney. Fifty-four patients who underwent unilateral RN were also evaluated. Twenty NSS patients (36.4%) experienced 21 postoperative complications, including prolonged urine leak (9), infection (8), transient renal insufficiency (1), and intussusception (3). Seven RN patients (13.0%) experienced surgical complications, including infection (4) and intussusception (3). Average intraoperative blood loss was significantly greater in NSS as compared to RN (483.51 ± 337.92 mL and 278.15 mL ± 390.25, respectively, p < 0.001), as was the incidence of positive tumor resection margins (20 [36.4%] and 12 [22.2%], respectively, (p = 0.037). CONCLUSIONS: In our experience, prolonged urine leak, intraoperative blood loss, and positive margins were more frequent in patients undergoing NSS as compared to RN. However, the complications were successfully managed, suggesting that an aggressive approach to NSS in patients with bilateral Wilms tumor is safe and appropriate. LEVEL OF EVIDENCE: Level III TYPE OF STUDY: Treatment study.

KDM5A Regulates a Translational Program that Controls p53 Protein Expression.

The p53 tumor suppressor pathway is frequently inactivated in human cancers. However, there are some cancer types without commonly recognized alterations in p53 signaling. Here we report that histone demethylase KDM5A is involved in the regulation of p53 activity. KDM5A is significantly amplified in multiple types of cancers, an event that tends to be mutually exclusive to p53 mutation. We show that KDM5A acts as a negative regulator of p53 signaling through inhibition of p53 translation via suppression of a subgroup of eukaryotic translation initiation genes. Genetic deletion of KDM5A results in upregulation of p53 in multiple lineages of cancer cells and inhibits tumor growth in a p53-dependent manner. In addition, we have identified a regulatory loop between p53, miR-34, and KDM5A, whereby the induction of miR-34 leads to suppression of KDM5A. Thus, our findings reveal a mechanism by which KDM5A inhibits p53 translation to modulate cancer progression.

Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.

Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy.

The PTEN-induced putative kinase protein 1 (PINK1) and ubiquitin (UB) ligase PARKIN direct damaged mitochondria for mitophagy. PINK1 promotes PARKIN recruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and noncanonical UB chains. PINK1 phosphorylates both Ser65 (S65) in the UB-like domain of PARKIN and the conserved Ser in UB itself, but the temporal sequence and relative importance of these events during PARKIN activation and mitochondria quality control remain poorly understood. Using "UB(S65A)-replacement," we find that PARKIN phosphorylation and activation, and ubiquitylation of Lys residues on a cohort of MOM proteins, occur similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, polyubiquitin (poly-UB) chain synthesis, PARKIN retention on the MOM, and mitophagy are reduced in UB(S65A)-replacement cells. Analogous experiments examining roles of individual UB chain linkage types revealed the importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1 did not enhance binding to candidate mitophagy receptors optineurin (OPTN), sequestosome-1 (p62), and nuclear dot protein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKIN cannot build UB chains, and <0.16% of the monomeric UB pool underwent S65 phosphorylation upon mitochondrial damage. Combining p-S65-UB and p-S65-PARKIN in vitro showed accelerated transfer of nonphosphorylated UB to PARKIN itself, its substrate mitochondrial Rho GTPase (MIRO), and UB. Our data further define a feed-forward mitochondrial ubiquitylation pathway involving PARKIN activation upon phosphorylation, UB chain synthesis on the MOM, UB chain phosphorylation, and further PARKIN recruitment and enzymatic amplification via binding to phosphorylated UB chains.