Inhibition Mechanism of SARS-CoV-2 Infection by a Cholesterol Derivative, Nat-20(S)-yne.

The coronavirus disease 2019 (COVID-19) is caused by the etiological agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19, with the recurrent epidemics of new variants of SARS-CoV-2, remains a global public health problem, and new antivirals are still required. Some cholesterol derivatives, such as 25-hydroxycholesterol, are known to have antiviral activity against a wide range of enveloped and non-enveloped viruses, including SARS-CoV-2.

Mitochondrial phosphatidylethanolamine synthesis affects mitochondrial energy metabolism and quiescence entry through attenuation of Snf1/AMPK signaling in yeast.

The Ups2-Mdm35 complex mediates intramitochondrial phosphatidylserine (PS) transport to facilitate mitochondrial phosphatidylethanolamine (PE) synthesis. In the present study, we found that ups2∆ yeast showed increased mitochondrial ATP production and enhanced quiescence (G0) entry in the post-diauxic shift phase. Transcriptomic and biochemical analyses revealed that the depletion of Ups2 leads to overactivation of the yeast AMPK homolog Snf1.

Topology of phosphatidylserine synthase 1 in the endoplasmic reticulum membrane.

Phosphatidylserine (PS) synthase 1 (PSS1) of mammalian cells is a multiple membrane-spanning protein of the endoplasmic reticulum (ER) and regulated by inhibition with the product PS. Alanine-scanning mutagenesis of PSS1 has revealed eight amino acid residues as those crucial for its activity and six as those important for its regulation. Furthermore, three missense mutations in the human PSS1 gene, which lead to regulatory dysfunctions of PSS1 and are causative of Lenz-Majewski syndrome, have been identified.

Recent insights into peroxisome biogenesis and associated diseases.

Peroxisomes are single-membrane organelles present in eukaryotes. The functional importance of peroxisomes in humans is represented by peroxisome-deficient peroxisome biogenesis disorders (PBDs), including Zellweger syndrome. Defects in the genes that encode the 14 peroxins that are required for peroxisomal membrane assembly, matrix protein import and division have been identified in PBDs.

Porin Associates with Tom22 to Regulate the Mitochondrial Protein Gate Assembly.

Mitochondria import nearly all of their resident proteins from the cytosol, and the TOM complex functions as their entry gate. The TOM complex undergoes a dynamic conversion between the majority population of a three-channel gateway ("trimer") and the minor population that lacks Tom22 and has only two Tom40 channels ("dimer"). Here, we found that the porin Por1 acts as a sink to bind newly imported Tom22.

Cell Death or Survival Against Oxidative Stress.

Peroxisomes contain anabolic and catabolic enzymes including oxidases that produce hydrogen peroxide as a by-product. Peroxisomes also contain catalase to metabolize hydrogen peroxide. It has been recognized that catalase is localized to cytosol in addition to peroxisomes.

Porin proteins have critical functions in mitochondrial phospholipid metabolism in yeast.

Mitochondrial synthesis of cardiolipin (CL) and phosphatidylethanolamine requires the transport of their precursors, phosphatidic acid and phosphatidylserine, respectively, to the mitochondrial inner membrane. In yeast, the Ups1-Mdm35 and Ups2-Mdm35 complexes transfer phosphatidic acid and phosphatidylserine, respectively, between the mitochondrial outer and inner membranes. Moreover, a Ups1-independent CL accumulation pathway requires several mitochondrial proteins with unknown functions including Mdm31.

Cooperative function of Fmp30, Mdm31, and Mdm32 in Ups1-independent cardiolipin accumulation in the yeast Saccharomyces cerevisiae.

Cardiolipin (CL) is synthesized from phosphatidic acid (PA) through a series of enzymatic reactions occurring at the mitochondrial inner membrane (MIM). Ups1-Mdm35 mediates PA transfer from the mitochondrial outer membrane (MOM) to the MIM in the yeast Saccharomyces cerevisiae. Deletion of UPS1 leads to a ~80% decrease in the cellular CL level.

The VDAC2-BAK axis regulates peroxisomal membrane permeability.

Peroxisomal biogenesis disorders (PBDs) are fatal genetic diseases consisting of 14 complementation groups (CGs). We previously isolated a peroxisome-deficient Chinese hamster ovary cell mutant, ZP114, which belongs to none of these CGs. Using a functional screening strategy, VDAC2 was identified as rescuing the peroxisomal deficiency of ZP114 where VDAC2 expression was not detected.

Adaptation of a Genetic Screen Reveals an Inhibitor for Mitochondrial Protein Import Component Tim44.

Diverse protein import pathways into mitochondria use translocons on the outer membrane (TOM) and inner membrane (TIM). We adapted a genetic screen, based on Ura3 mistargeting from mitochondria to the cytosol, to identify small molecules that attenuated protein import. Small molecule mitochondrial import blockers of the Carla Koehler laboratory (MB)-10 inhibited import of substrates that require the TIM23 translocon.

Phosphatidylserine transport by Ups2-Mdm35 in respiration-active mitochondria.

Phosphatidylethanolamine (PE) is an essential phospholipid for mitochondrial functions and is synthesized mainly by phosphatidylserine (PS) decarboxylase at the mitochondrial inner membrane. In Saccharomyces cerevisiae, PS is synthesized in the endoplasmic reticulum (ER), such that mitochondrial PE synthesis requires PS transport from the ER to the mitochondrial inner membrane. Here, we provide evidence that Ups2-Mdm35, a protein complex localized at the mitochondrial intermembrane space, mediates PS transport for PE synthesis in respiration-active mitochondria.

Drp1-dependent mitochondrial fission via MiD49/51 is essential for apoptotic cristae remodeling.

Mitochondrial fission facilitates cytochrome c release from the intracristae space into the cytoplasm during intrinsic apoptosis, although how the mitochondrial fission factor Drp1 and its mitochondrial receptors Mff, MiD49, and MiD51 are involved in this reaction remains elusive. Here, we analyzed the functional division of these receptors with their knockout (KO) cell lines. In marked contrast to Mff-KO cells, MiD49/MiD51-KO and Drp1-KO cells completely resisted cristae remodeling and cytochrome c release during apoptosis.

VID22 is required for transcriptional activation of the PSD2 gene in the yeast Saccharomyces cerevisiae.

Phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae is synthesized through decarboxylation of phosphatidylserine (PS), catalysed by PS decarboxylase 1 (Psd1p) and 2 (Psd2p) and the cytidine 5'-diphosphate (CDP)-ethanolamine (CDP-Etn) pathway. PSD1 null (psd1Δ) and PSD2 null (psd2Δ) mutants are viable in a synthetic minimal medium, but a psd1Δ psd2Δ double mutant exhibits Etn auxotrophy, which is incorporated into PE through the CDP-Etn pathway. We have previously shown that psd1Δ is synthetic lethal with deletion of VID22 (vid22Δ) [Kuroda et al.

Pharmacologic rescue of an enzyme-trafficking defect in primary hyperoxaluria 1.

Primary hyperoxaluria 1 (PH1; Online Mendelian Inheritance in Man no. 259900), a typically lethal biochemical disorder, may be caused by the AGT(P11LG170R) allele in which the alanine:glyoxylate aminotransferase (AGT) enzyme is mistargeted from peroxisomes to mitochondria. AGT contains a C-terminal peroxisomal targeting sequence, but mutations generate an N-terminal mitochondrial targeting sequence that directs AGT from peroxisomes to mitochondria.

TMEM14C is required for erythroid mitochondrial heme metabolism.

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells.

Macrocytic anemia and mitochondriopathy resulting from a defect in sideroflexin 4.

We used exome sequencing to identify mutations in sideroflexin 4 (SFXN4) in two children with mitochondrial disease (the more severe case also presented with macrocytic anemia). SFXN4 is an uncharacterized mitochondrial protein that localizes to the mitochondrial inner membrane. sfxn4 knockdown in zebrafish recapitulated the mitochondrial respiratory defect observed in both individuals and the macrocytic anemia with megaloblastic features of the more severe case.

New insights into dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p in shuttling of PTS1-receptor Pex5p during peroxisome biogenesis.

Peroxisome is a single-membrane organelle in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient peroxisome biogenesis disorders such as Zellweger syndrome. Two AAA peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for PBDs of complementation groups 1 and 4, respectively.

AWP1/ZFAND6 functions in Pex5 export by interacting with cys-monoubiquitinated Pex5 and Pex6 AAA ATPase.

During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator.

Cysteine ubiquitination of PTS1 receptor Pex5p regulates Pex5p recycling.

Pex5p is the cytosolic receptor for peroxisome matrix proteins with peroxisome-targeting signal (PTS) type 1 and shuttles between the cytosol and peroxisomes. Here, we show that Pex5p is ubiquitinated at the conserved cysteine(11) in a manner sensitive to dithiothreitol, in a form associated with peroxisomes. Pex5p with a mutation of the cysteine(11) to alanine, termed Pex5p-C11A, abrogates peroxisomal import of PTS1 and PTS2 proteins in wild-type cells.

In vitro import of peroxisome-targeting signal type 2 (PTS2) receptor Pex7p into peroxisomes.

Pex7p, the peroxisome-targeting signal type 2 (PTS2) receptor, transports PTS2 proteins to peroxisomes from the cytosol. We here established a cell-free Pex7p translocation system. In assays using post-nuclear supernatant fractions each from wild-type CHO-K1 and pex7 ZPG207 cells, 35S-labeled Pex7p was imported into peroxisomes.

Analysis of nucleotide binding to P97 reveals the properties of a tandem AAA hexameric ATPase.

p97, an essential chaperone in endoplasmic reticulum-associated degradation and organelle biogenesis, contains two AAA domains (D1 and D2) and assembles as a stable hexamer. We present a quantitative analysis of nucleotide binding to both D1 and D2 domains of p97, the first detailed study of nucleotide binding to both AAA domains for this type of AAA+ ATPase. We report that adenosine 5'-O-(thiotriphosphate) (ATPgammaS) binds with similar affinity to D1 and D2, but ADP binds with higher affinity to D1 than D2, offering an explanation for the higher ATPase activity in D2.

Dynamic and functional assembly of the AAA peroxins, Pex1p and Pex6p, and their membrane receptor Pex26p involved in shuttling of the PTS1 receptor Pex5p in peroxisome biogenesis.

The peroxisome is a single-membrane-bound organelle found in eukaryotes. The functional importance of peroxisomes in humans is highlighted by peroxisome-deficient PBDs (peroxisome biogenesis disorders), such as Zellweger syndrome. Two AAA (ATPase associated with various cellular activities) peroxins, Pex1p and Pex6p, are encoded by PEX1 and PEX6, the causal genes for CG (complementation group) 1 and CG4 PBDs respectively.

Shuttling mechanism of peroxisome targeting signal type 1 receptor Pex5: ATP-independent import and ATP-dependent export.

Peroxisomal matrix proteins are posttranslationally imported into peroxisomes with the peroxisome-targeting signal 1 receptor, Pex5. The longer isoform of Pex5, Pex5L, also transports Pex7-PTS2 protein complexes. After unloading the cargoes, Pex5 returns to the cytosol.