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Abstract Peroxisomes are organelles that are central to lipid metabolism and chemical detoxification. Despite advances in our understanding of peroxisome biogenesis, the mechanisms maintaining peroxisomal membrane proteins remain to be fully elucidated. We show here that mammalian FAF2/UBXD8, a membrane-associated cofactor of p97/VCP, maintains peroxisomal homeostasis by modulating the turnover of peroxisomal membrane proteins such as PMP70. In FAF2-deficient cells, PMP70 accumulation recruits the autophagy adaptor OPTN (Optineurin) to peroxisomes and promotes their autophagic clearance (pexophagy). Pexophagy is also induced by p97/VCP inhibition. FAF2 functions together with p97/VCP to negatively regulate pexophagy rather than as a factor for peroxisome biogenesis. Our results strongly suggest that p97/VCPFAF2-mediated extraction of ubiquitylated peroxisomal membrane proteins (e.g., PMP70) prevents peroxisomes from inducing nonessential autophagy under steady state conditions. These findings provide insight into molecular mechanisms underlying the regulation of peroxisomal integrity by p97/VCP and its associated cofactors.

Abstract In many sexually reproducing organisms, oocytes are fundamentally fertilized with one sperm. In Caenorhabditis elegans, chitin layer formation after fertilization by the EGG complex is one of the mechanisms of polyspermy block, but other mechanisms remain unknown. Here, we demonstrate that MARC-3, a membrane-associated RING-CH-type ubiquitin ligase that localizes to the plasma membrane and cortical puncta in oocytes, is involved in fast polyspermy block. During polyspermy, the second sperm entry occurs within approximately 10 s after fertilization in MARC-3-deficient zygotes, whereas it occurs approximately 200 s after fertilization in egg-3 mutant zygotes defective in the chitin layer formation. MARC-3 also functions in the selective degradation of maternal plasma membrane proteins and the transient accumulation of endosomal lysine 63-linked polyubiquitin after fertilization. The RING-finger domain of MARC-3 is required for its in vitro ubiquitination activity and polyspermy block, suggesting that a ubiquitination-mediated mechanism sequentially regulates fast polyspermy block and maternal membrane protein degradation during the oocyte-to-embryo transition.

Background. Extracellular adenosine 5′-triphosphate (ATP) acts as a signaling molecule in the peripheral nerves, regulating myelination after nerve injury. The present study examined whether the cerebrospinal fluid (CSF) ATP levels in patients with Guillain–Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are related to disease severity. Methods. CSF ATP levels in 13 patients with GBS and 18 patients with CIDP were compared with those in a control group of 16 patients with other neurological diseases (ONDs). In patients with CIDP, CSF ATP levels were compared before and after treatment. The correlations between CSF ATP levels and other factors, including clinical data and CSF protein levels, were also evaluated. Results. Median CSF ATP levels were significantly higher in patients with GBS and CIDP than in those with ONDs. When patients with CIDP were classified into two groups depending on their responsiveness to immunotherapy, median CSF ATP levels were significantly higher in good responders than in ONDs. CSF ATP levels tended to decrease after treatment in patients with CIDP. In patients with CIDP, there is a negative correlation between CSF ATP and CSF protein levels. Conclusions. CSF ATP levels were increased in patients with GBS and CIDP. In particular, CSF ATP levels tended to decrease following treatment in patients with CIDP. CSF ATP levels may be useful biomarkers for the diagnosis or monitoring of therapeutic effects in patients with GBS and CIDP.

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB–HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria–lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage–induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence. [ABSTRACT FROM AUTHOR]

Damaged mitochondria are selectively eliminated in a process called mitophagy. PINK1 and Parkin amplify ubiquitin signals on damaged mitochondria, which are then recognized by autophagy adaptors to induce local autophagosome formation. NDP52 and OPTN, two essential mitophagy adaptors, facilitate de novo synthesis of pre-autophagosomal membranes near damaged mitochondria by linking ubiquitinated mitochondria and ATG8 family proteins and by recruiting core autophagy initiation components. The multifunctional serine/threonine kinase TBK1 also plays an important role in mitophagy. OPTN directly binds TBK1 to form a positive feedback loop for isolation membrane expansion. TBK1 is also thought to indirectly interact with NDP52; however, its role in NDP52-driven mitophagy remains largely unknown. Here, we focused on two TBK1 adaptors, AZI2/NAP1 and TBKBP1/SINTBAD, that are thought to mediate the TBK1-NDP52 interaction. We found that both AZI2 and TBKBP1 are recruited to damaged mitochondria during Parkin-mediated mitophagy. Further, a series of AZI2 and TBKBP1 knockout constructs combined with an OPTN knockout showed that AZI2, but not TBKBP1, impacts NDP52-driven mitophagy. In addition, we found that AZI2 at S318 is phosphorylated during mitophagy, the impairment of which slightly inhibits mitochondrial degradation. These results suggest that AZI2, in concert with TBK1, plays an important role in NDP52-driven mitophagy. [ABSTRACT FROM AUTHOR]

DJ-1, a causative gene for hereditary recessive Parkinsonism, is evolutionarily conserved across eukaryotes and prokaryotes. Structural analyses of DJ-1 and its homologs suggested the 106th Cys is a nucleophilic cysteine functioning as the catalytic center of hydratase or hydrolase activity. Indeed, DJ-1 and its homologs can convert highly electrophilic a-oxoaldehydes such as methylglyoxal into a-hydroxy acids as hydratase in vitro, and oxidation-dependent ester hydrolase (esterase) activity has also been reported for DJ-1. The mechanism underlying such plural activities, however, has not been fully characterized. To address this knowledge gap, we conducted a series of biochemical assays assessing the enzymatic activity of DJ-1 and its homologs. We found no evidence for esterase activity in any of the Escherichia coli DJ-1 homologs. Furthermore, contrary to previous reports, we found that oxidation inactivated rather than facilitated DJ-1 esterase activity. The E. coli DJ-1 homolog HchA possesses phenylglyoxalase and methylglyoxalase activities but lacks esterase activity. Since evolutionary trace analysis identified the 186th H as a candidate residue involved in functional differentiation between HchA and DJ-1, we focused on H186 of HchA and found that an esterase activity was acquired by H186A mutation. Introduction of reverse mutations into the equivalent position in DJ-1 (A107H) selectively eliminated its esterase activity without compromising a-oxoaldehyde hydratase activity. The obtained results suggest that differences in the amino acid sequences near the active site contributed to acquisition of esterase activity in vitro and provide an important clue to the origin and significance of DJ-1 esterase activity. [ABSTRACT FROM AUTHOR]