Janus-faced Sestrin2 controls ROS and mTOR signalling through two separate functional domains.

Sestrins are stress-inducible metabolic regulators with two seemingly unrelated but physiologically important functions: reduction of reactive oxygen species (ROS) and inhibition of the mechanistic target of rapamycin complex 1 (mTORC1). How Sestrins fulfil this dual role has remained elusive so far. Here we report the crystal structure of human Sestrin2 (hSesn2), and show that hSesn2 is twofold pseudo-symmetric with two globular subdomains, which are structurally similar but functionally distinct from each other. While the N-terminal domain (Sesn-A) reduces alkylhydroperoxide radicals through its helix-turn-helix oxidoreductase motif, the C-terminal domain (Sesn-C) modified this motif to accommodate physical interaction with GATOR2 and subsequent inhibition of mTORC1. These findings clarify the molecular mechanism of how Sestrins can attenuate degenerative processes such as aging and diabetes by acting as a simultaneous inhibitor of ROS accumulation and mTORC1 activation.

Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis.

Autophagy is an essential process for eliminating ubiquitinated protein aggregates and dysfunctional organelles. Defective autophagy is associated with various degenerative diseases such as Parkinson disease. Through a genetic screening in Drosophila, we identified CG11148, whose product is orthologous to GIGYF1 (GRB10-interacting GYF protein 1) and GIGYF2 in mammals, as a new autophagy regulator; we hereafter refer to this gene as Gyf. Silencing of Gyf completely suppressed the effect of Atg1-Atg13 activation in stimulating autophagic flux and inducing autophagic eye degeneration. Although Gyf silencing did not affect Atg1-induced Atg13 phosphorylation or Atg6-Pi3K59F (class III PtdIns3K)-dependent Fyve puncta formation, it inhibited formation of Atg13 puncta, suggesting that Gyf controls autophagy through regulating subcellular localization of the Atg1-Atg13 complex. Gyf silencing also inhibited Atg1-Atg13-induced formation of Atg9 puncta, which is accumulated upon active membrane trafficking into autophagosomes. Gyf-null mutants also exhibited substantial defects in developmental or starvation-induced accumulation of autophagosomes and autolysosomes in the larval fat body. Furthermore, heads and thoraxes from Gyf-null adults exhibited strongly reduced expression of autophagosome-associated Atg8a-II compared to wild-type (WT) tissues. The decrease in Atg8a-II was directly correlated with an increased accumulation of ubiquitinated proteins and dysfunctional mitochondria in neuron and muscle, which together led to severe locomotor defects and early mortality. These results suggest that Gyf-mediated autophagy regulation is important for maintaining neuromuscular homeostasis and preventing degenerative pathologies of the tissues. Since human mutations in the GIGYF2 locus were reported to be associated with a type of familial Parkinson disease, the homeostatic role of Gyf-family proteins is likely to be evolutionarily conserved.

Clinical significance of a negative loop electrosurgical excision procedure biopsy in patients with biopsy-confirmed high-grade cervical intraepithelial neoplasia.

OBJECTIVE: We sought to determine which clinical factors can predict this phenomenon and to better understand the clinical significance of negative loop electrosurgical excision procedure (LEEP) findings through long-term follow-up. METHODS: We identified 559 patients with biopsy-confirmed cervical intraepithelial neoplasia grade 2 or 3 (CIN 2, 3) who were treated by LEEP between February 2001 and December 2010. Preconization clinical characteristics, as well as high-risk human papillomavirus (hrHPV) status, were analyzed as possible predictors of an absence of a lesion in the specimen. The clinical significance of an absence of a lesion in the specimen, as well as other factors, was evaluated by Cox hazard regression analysis in terms of recurrence. RESULTS: No lesion on the LEEP specimen was found in 102 (18.2%) of 559 patients with CIN 2,3 on punch biopsy. Punch biopsy status of CIN 2, low HPV viral load (<100 relative light units [RLU]), and negative or positive HPV infection other than type 16 were significantly related to no lesion in the LEEP specimen. Postoperative HPV persistence (≥10 RLU) and same-type HPV detection were significantly related to recurrent disease of CIN 2+ (p < .001). The recurrence of patients with no lesion in LEEP did not statistically differ from that of patients with a lesion in the LEEP specimen (p = .390). CONCLUSIONS: The absence of a lesion in the LEEP specimen is very common. A negative LEEP is associated with a persistence/recurrence rate similar to that of positive LEEP. We recommend that the follow-up for patients with no lesion in the LEEP specimen should be the same as that for patients with a lesion.

Human papillomavirus type 16 causes larger colposcopic lesions than other HPV types in patients with grade 3 cervical intraepithelial neoplasia.

OBJECTIVE: Recent studies have shown that human papillomavirus (HPV) type 16 causes more definite visual abnormalities on cervigram than other HPV types and is thus easier to evaluate colposcopically. We examined factors, including HPV-16, related to colposcopic lesions in patients with grade 3 cervical intraepithelial neoplasia (CIN 3). METHODS: A retrospective chart review included 108 women with CIN 3 who underwent the loop electrosurgical excision procedure (LEEP). Lesions were assessed according to the number of cervical quadrant(s) involved by colposcopy, dichotomized as 2 or fewer or 3 or more quadrants involved. The Hybrid Capture 2 (HC2) test and HPV DNA chip assay (MyGene Co, Seoul, Korea) were used to detect HPV before punch biopsy or loop electrosurgical excision procedure. The type of HPV was dichotomized as HPV-16 or other (including negative cases). The HC2 viral load cutoff was 300 relative light units. Cytology was dichotomized as (1) low grade, less than, or equal to low-grade squamous lesions; or (2) high-grade, with high-grade squamous lesions or worse. Age and menopausal status were also assessed. RESULTS: The mean (SD) age of the 108 women was 41.9 (10.7) years (range = 22-76 y). Seventy-one (65.7%) had lesions involving 2 quadrants or fewer and 37 (34.3%) had lesions involving 3 quadrants or more. Multiple logistic regression revealed that larger lesions (≥3 quadrants involved) were significantly associated with HPV-16 (p = .032, odds ratio [OR] = 2.552, 95% confidence interval = 1.085-6.000) but not with age, menopausal status, cytologic grade, or HPV HC2 viral load. CONCLUSIONS: Our data suggest that colposcopic lesions differ according to HPV type and that HPV-16 is associated with larger lesions, facilitating lesion detection by colposcopy.

Early human papillomavirus testing predicts residual/recurrent disease after LEEP.

OBJECTIVE: The purpose of this study was to determine the predictive factors for residual/recurrent disease and to analyze the timing for Pap smears and human papillomavirus (HPV) testing during follow-up after loop electrosurgical excision procedure (LEEP) for cervical intraepithelial neoplasia (CIN) 2 or worse. METHODS: We retrospectively analyzed 183 patients (mean age, 39.3 years) with CIN 2/3 who were treated with LEEP. Post-LEEP follow-up was performed by Pap smear and HPV hybrid capture2 (HC2) testing. The definition of persistent/recurrent disease was biopsy-proven CIN 2 or worse. RESULTS: Among 183 patients, punch biopsies were CIN 2 in 31 (16.9%) and CIN 3 in 152 (83.1%). HPV HC2 tests before LEEP were positive in 170 (95.5%) of 178 patients. During follow-up, 12 patients (6.6%) had residual/recurrent CIN 2+. LEEP margin status was a significant predictive factor for persistent/recurrent disease. Other factors such as age, HPV HC2 viral load (≥100 relative light units), and HPV typing (type 16/18 vs. other types) did not predict recurrence. Early HPV HC2 testing at 3 months after LEEP detected all cases of residual/recurrent disease. The sensitivity and negative predictive value of the HPV HC2 test for residual/recurrent disease were both 100% at 3 and 6 months. CONCLUSION: Margin involvement in conization specimens was a significant factor predicting residual/recurrent disease after LEEP. HPV test results at 3 and 6 months after treatment were comparable. Early 3-month follow-up testing after LEEP can offer timely information about residual/recurrent disease and alleviate patient anxiety early about treatment failure.

Factors associated with HPV persistence after conization in patients with negative margins.

OBJECTIVE: The clearance rate of human papillomavirus (HPV) after conization is generally high, although some HPV infections persist. We investigated the factors that affect the clearance of HPV after conization in patients with negative margins. METHODS: We retrospectively analyzed 77 patients (mean age 39.9 years, range 25 to 51 years) with CIN 2/3 who underwent loop electrosurgical excision procedure (LEEP) conization with negative margins. All patients had a Pap smear and high-risk (HR) HPV testing using Hybrid Capture II system and HPV DNA chip before conization. We used>/=1 relative light units (RLUs) as the cutoff for persistence of HPV after conization. RESULTS: High-risk HPV was detected in 73 of 77 (94.8%) patients before conization. At the 6-months follow-up, the high-risk HPV was eliminated in 60 of 73 (82.2%) patients. The HPV persistence rate after conization was 17.8% (13/73). Univariate analysis showed that persistent HPV infection after conization with negative margins was more likely to occur when the pretreatment viral load was high (RLU/positive control >100 (p=0.027) and the HPV was type 16 (p=0.021). Logistic regression analysis showed that preoperative HPV type 16 infection was the only significant independent factor (p=0.021) for HPV persistence out of age, cytology, punch biopsy histology, HPV viral load, and conization histology. CONCLUSION: Conization effectively removes HR-HPV infection. HPV type 16 infection before conization was significantly related to HR-HPV persistence after conization with negative margins. Therefore, patients with HPV 16 infection before conization need to be followed closely.