The Orf9b protein of SARS-CoV-2 modulates mitochondrial protein biogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed an Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. To exclude that the observed depletion was caused by the antiviral response, we generated a yeast system in which the function of human Tom70 could be recapitulated. Upon expression of Orf9b in these cells, we again observed a specific decline of a subset of mitochondrial proteins and a general reduction of mitochondrial volume. Thus, the SARS-CoV-2 virus is able to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.

A two-step mitochondrial import pathway couples the disulfide relay with matrix complex I biogenesis.

Mitochondria critically rely on protein import and its tight regulation. Here, we found that the complex I assembly factor NDUFAF8 follows a two-step import pathway linking IMS and matrix import systems. A weak targeting sequence drives TIM23-dependent NDUFAF8 matrix import, and en route, allows exposure to the IMS disulfide relay, which oxidizes NDUFAF8. Import is closely surveyed by proteases: YME1L prevents accumulation of excess NDUFAF8 in the IMS, while CLPP degrades reduced NDUFAF8 in the matrix. Therefore, NDUFAF8 can only fulfil its function in complex I biogenesis if both oxidation in the IMS and subsequent matrix import work efficiently. We propose that the two-step import pathway for NDUFAF8 allows integration of the activity of matrix complex I biogenesis pathways with the activity of the mitochondrial disulfide relay system in the IMS. Such coordination might not be limited to NDUFAF8 as we identified further proteins that can follow such a two-step import pathway.

Effect of Human Papillomavirus Subtype on the Rate of Positive Surgical Margin After Cervical Conization.

Objective. Human papillomavirus (HPV) infection is a risk factor for cervical carcinoma. Over 100 types of HPV have been identified. The excisional procedures are recommended for women with high-grade cervical intraepithelial neoplasia. Surgical margin status is an important predictor of the risk of relapse. The aim of the current study was to evaluate whether HPV genotype is a predictive factor of positive surgical margin after cervical cone excision. Materials and Methods. The records of 448 HPV-infected patients who underwent loop electrosurgical excision or cold knife conization at a tertiary gynecological cancer center were retrospectively reviewed. The patients were divided into 6 groups according to HPV positivity: HPV 16 only, HPV 18 only, HPV 16/18, other high-risk HPV (hrHPV), HPV 16/hrHPV, and HPV 18/hrHPV. Results. There was no significant difference between the HPV groups in terms of age, parity, menopausal status, endocervical canal involvement, conization method, and the rates of positive margin (P = .15, P = .49, P = .07, P = .20, P = .24, P = .39, respectively). Conclusion. The results show that HPV subtypes might not be associated with endocervical canal involvement and the rates of positive margin. In addition, margin status was not related to the conization method and the number of excised cervical tissue.

AIFM1 is a component of the mitochondrial disulfide relay that drives complex I assembly through efficient import of NDUFS5.

The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long-lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.

Mitochondria shed their outer membrane in response to infection-induced stress.

The outer mitochondrial membrane (OMM) is essential for cellular homeostasis. Yet little is known of the mechanisms that remodel it during natural stresses. We found that large "SPOTs" (structures positive for OMM) emerge during Toxoplasma gondii infection in mammalian cells. SPOTs mediated the depletion of the OMM proteins mitofusin 1 and 2, which restrict parasite growth. The formation of SPOTs depended on the parasite effector TgMAF1 and the host mitochondrial import receptor TOM70, which is required for optimal parasite proliferation. TOM70 enabled TgMAF1 to interact with the host OMM translocase SAM50. The ablation of SAM50 or the overexpression of an OMM-targeted protein promoted OMM remodeling independently of infection. Thus, Toxoplasma hijacks the formation of SPOTs, a cellular response to OMM stress, to promote its growth.

Protein Import Assay into Mitochondria Isolated from Human Cells.

Mitochondria are essential organelles containing approximately 1,500 proteins. Only approximately 1% of these proteins are synthesized inside mitochondria, whereas the remaining 99% are synthesized as precursors on cytosolic ribosomes and imported into the organelle. Various tools and techniques to analyze the import process have been developed. Among them, in vitro reconstituted import systems are of importance to study these processes in detail. These experiments monitor the import reaction of mitochondrial precursors that were previously radiolabeled in a cell-free environment. However, the methods described have been mostly performed in mitochondria isolated from S. cerevisiae. Here, we describe the adaptation of this powerful assay to import proteins into crude mitochondria isolated from human tissue culture cells. Graphic abstract: Overview of the assay to monitor protein import into mitochondria isolated from human cells.

Erv1 and Cytochrome c Mediate Rapid Electron Transfer via A Collision-Type Interaction.

Being essential for oxidative protein folding in the mitochondrial intermembrane space, the mitochondrial disulfide relay relies on the electron transfer (ET) from the sulfhydryl oxidase Erv1 to cytochrome c (Cc). Using solution NMR spectroscopy, we demonstrate that while the yeast Cc-Erv1 system is functionally active, no observable binding of the protein partners takes place. The transient interaction between Erv1 and Cc can be rationalized by molecular modeling, suggesting that a large surface area of Erv1 can sustain a fast ET to Cc via a collision-type mechanism, without the need for a canonical protein complex formation. We suggest that, by preventing the direct ET to molecular oxygen (O2), the collision-type Cc-Erv1 interaction plays a role in protecting the organism against reactive oxygen species.

The meaning of high-risk HPV other than type 16/18 in women with negative cytology: Is it really safe to wait for 1 year?

BACKGROUND: Human papillomavirus (HPV) is a primary risk factor for cervical cancer. HPV 16 and 18 are the two most carcinogenic genotypes and have been reported in the majority of cervical cancer. High-risk HPVs (hrHPVs) other than HPV 16/18 cause approximately a quarter of cervical cancers. We aimed to present the colposcopy-guided biopsy results of non-16/18 hrHPV-infected women with negative cytology. METHODS: This is a retrospective cohort study conducted on 752 patients between the ages of 30-65 years with non-16/18 hrHPV and negative cytology undergoing colposcopy-guided biopsy at a tertiary gynecological cancer center between January-2016 and January-2019. RESULTS: The mean age of the women was 42.35±9.41 years. Cervical intraepithelial neoplasia (CIN) 2+ lesion was detected in 49 (6.5%) women with negative cytology. The rate of CIN 2+ lesions in women with abnormal cytology was 12.8%. Patients with abnormal cytology had about 2.1 and 2.4 times increased the odds of CIN 2+ lesion in cervical biopsy and endocervical curettage specimens, respectively. CIN 3+ lesion was detected in 20 (2.7%) women with negative cytology. One (0.1%) of the patients with HPV 39 and negative cytology had invasive cervical cancer. The two most common HPV subtypes were HPV 31 and HPV 51. CONCLUSIONS: The risk of cervical preinvasive lesions still can be detected and cannot be completely eliminated among hrHPV other than 16/18-infected women with negative cytology. Based on the results of this study, referral of non-16/18 hrHPV-infected women with negative cytology to colposcopy is supported as a credible and feasible strategy.

Is cervical cytology testing as a part of co-test unnecessary for HPV 16/18-infected women? A retrospective cohort study of 1647 women.

BACKGROUND: We aimed to present the biopsy results of women with HPV 16/18 infection and investigate whether cytology is necessary as a part of routine cervical cancer screening in women with HPV 16/18. METHODS: This is a retrospective cohort study conducted on 1647 patients between the ages of 30 and 65 years with HPV 16/18 undergoing colposcopy-guided biopsy at a tertiary gynecological cancer center between January-2016 and January-2019. We compared the preinvasive lesion rates and the invasive cervical cancer rates of women with HPV 16/18 between the negative and the abnormal cytology group. RESULTS: Of the 1647 women, 1105 (67.1%) had negative cytology and 542 (32.9%) had abnormal cytology. Among women with initial negative cytology, cervical intraepithelial neoplasia (CIN) 2+ lesion was detected in 205 (18.6%) women. The rate of CIN 2+ lesion in women with abnormal cytology was 28%. There was a significant difference between negative and abnormal cytology group in terms of CIN 2+ lesion rates (P < .001). Among women with initial negative cytology, invasive cervical cancer was detected in 6 (0.5%) women. The rate of invasive cervical cancer in women with abnormal cytology was 8 (1.5%). There was no significant difference between negative and abnormal cytology group in terms of invasive cervical cancer rates (P = .082). CONCLUSIONS: The rate of cervical cancer among HPV 16/18-infected women with negative cytology is similar to women with abnormal cytology. Based on the results of this study, Pap testing could be unnecessary in HPV 16/18-infected women to diagnose invasive cervical cancer who will undergo colposcopy biopsy.

The C-terminal region of the oxidoreductase MIA40 stabilizes its cytosolic precursor during mitochondrial import.

BACKGROUND: The mitochondrial intermembrane space (IMS) is home to proteins fulfilling numerous essential cellular processes, particularly in metabolism and mitochondrial function. All IMS proteins are nuclear encoded and synthesized in the cytosol and must therefore be correctly targeted and transported to the IMS, either through mitochondrial targeting sequences or conserved cysteines and the mitochondrial disulfide relay system. The mitochondrial oxidoreductase MIA40, which catalyzes disulfide formation in the IMS, is imported by the combined action of the protein AIFM1 and MIA40 itself. Here, we characterized the function of the conserved highly negatively charged C-terminal region of human MIA40. RESULTS: We demonstrate that the C-terminal region is critical during posttranslational mitochondrial import of MIA40, but is dispensable for MIA40 redox function in vitro and in intact cells. The C-terminal negatively charged region of MIA40 slowed import into mitochondria, which occurred with a half-time as slow as 90 min. During this time, the MIA40 precursor persisted in the cytosol in an unfolded state, and the C-terminal negatively charged region served in protecting MIA40 from proteasomal degradation. This stabilizing property of the MIA40 C-terminal region could also be conferred to a different mitochondrial precursor protein, COX19. CONCLUSIONS: Our data suggest that the MIA40 precursor contains the stabilizing information to allow for postranslational import of sufficient amounts of MIA40 for full functionality of the essential disulfide relay. We thereby provide for the first time mechanistic insights into the determinants controlling cytosolic surveillance of IMS precursor proteins.

Hyperoxidation of mitochondrial peroxiredoxin limits H2 O2 -induced cell death in yeast.

Hydrogen peroxide (H2 O2 ) plays important roles in cellular signaling, yet nonetheless is toxic at higher concentrations. Surprisingly, the mechanism(s) of cellular H2 O2 toxicity remain poorly understood. Here, we reveal an important role for mitochondrial 1-Cys peroxiredoxin from budding yeast, Prx1, in regulating H2 O2 -induced cell death. We show that Prx1 efficiently transfers oxidative equivalents from H2 O2 to the mitochondrial glutathione pool. Deletion of PRX1 abrogates glutathione oxidation and leads to a cytosolic adaptive response involving upregulation of the catalase, Ctt1. Both of these effects contribute to improved cell viability following an acute H2 O2 challenge. By replacing PRX1 with natural and engineered peroxiredoxin variants, we could predictably induce widely differing matrix glutathione responses to H2 O2 . Therefore, we demonstrated a key role for matrix glutathione oxidation in driving H2 O2 -induced cell death. Finally, we reveal that hyperoxidation of Prx1 serves as a switch-off mechanism to limit oxidation of matrix glutathione at high H2 O2 concentrations. This enables yeast cells to strike a fine balance between H2 O2 removal and limitation of matrix glutathione oxidation.

Comparison of Interactive Education Versus Fluorescent Concretization on Hand Hygiene Compliance Among Primary School Students: A Randomized Controlled Trial.

Hand hygiene for children is crucial to keep them healthy. The purpose of the study was to evaluate the effects of two educational initiatives on "handwashing effectiveness (HWE)." A randomized controlled trial was carried out during April/June 2016, and 96 primary school students were randomly assigned to Group I receiving education with fluorescent gel; Group II receiving interactive education or control group continuing its normal education. Evaluation was made by scoring the fluorescent areas on the hands with photographs. There were significant differences in handwashing scores between preprogram and postprogram for all areas in only Group II (p < .05). HWE increased from 17.9% to 18.4% in Group I, from 15.4% to 37.7% in Group II, and from 35.5% to 35.8% in control group. Only concretization with fluorescent gel is not a sufficiently strong motivator for increasing HWE. New techniques should be integrated into the training programs for children.