Kunihiko Yasuda

p97/valosin-containing protein (VCP) is highly modulated by phosphorylation and acetylation.

p97/valosin‐containing protein (VCP) is a member of the AAA family proteins, which plays various important roles in cells by using its ATPase activity. But mechanism of regulating its ATPase activity is mostly unknown. We report here that VCP is highly modified throughout the protein via acetylation and phosphorylation. In addition to six previously identified phosphorylation sites, we identified at least 14 serines, 14 threonines, 6 tyrosines and 22 lysines as potential modification sites. Interestingly, these sites included Lys251 and Lys524, which are very critical for the ATP binding in Walker A motif of D1 and D2 domains, respectively. It is notable that 16 sites are in the N‐terminal region and 16 sites are clustered in D2α domain (from Pro646 to Gly765). Indeed, amino acid substitution of Lys696 and Thr761 profoundly affect VCP ATPase activities. From these results, we propose that D2α domain acts as a VCP ATPase Regulatory domain or “VAR domain”. VCP modifications including those in this VAR domain may endorse adaptive and multiple functions to VCP in different cell conditions such as in the cell cycle and with abnormal protein accumulation.

Targeted Disruption of Hsp110/105 Gene Protects Against Ischemic Stress

Background and Purpose— Hsp110/105 belongs to the HSP110 heat shock protein family, which is a subgroup of the HSP70 family. In mammals, Hsp110/105 is constitutively expressed but exhibits particularly high levels in the brain. It has recently been shown that both Hsp110/105 and Hsp70 are elevated after cerebral ischemia. To study the physiological role of this protein in vivo, we generated hsp110/105 knockout (KO) mice and investigate the effect of reduced Hsp110/105 levels on focal cerebral ischemia. Methods— hsp110/105 KO and wild-type mice were subjected to 30 minutes of transient middle cerebral artery occlusion followed by reperfusion for 24 hours. The infarct volume and neurological scores were measured and compared. The Hsp70 chaperone activity of thermally denatured firefly luciferase was measured in hsp110/105 KO embryonic fibroblasts. Results— The infarct volume and neurological deficit scores were significantly (P<0.05) reduced in hsp110/105 KO mice compared with wild-type controls. In addition, hsp110/105 KO embryonic fibroblasts exhibited a dose-dependent suppression of Hsp70 chaperone activity by the presence of Hsp110/105. Conclusions— These results demonstrate that hsp110/105 KO mice are resistant to ischemic injury and that the protective effects of hsp110/105 deficiency in cerebral ischemia may partly be mediated by an increase in the chaperone activity of Hsp70.

Characterization of stress sensitivity and chaperone activity of Hsp105 in mammalian cells.

Hsp105 is a major mammalian heat shock protein that belongs to the Hsp105/110 family, a diverged subgroup of the Hsp70 family. Hsp105 not only protects the thermal aggregation of proteins, but also regulates the Hsc70 chaperone system in vitro. Recently, it has been shown that Hsp105/110 family members act as nucleotide exchange factors for cytosolic Hsp70s. However, the biological functions of Hsp105/110 family proteins still remain to be clarified. Here, we examined the function of Hsp105 in mammalian cells, and showed that the sensitivity to various stresses was enhanced in the Hsp105-deficient cells compared with that in control cells. In addition, we found that deficiency of Hsp105 impaired the refolding of heat-denatured luciferase in mammalian cells. In contrast, overexpression of Hsp105α enhanced the ability to recover heat-inactivated luciferase in mammalian cells. Thus, Hsp105 may play an important role in the refolding of denatured proteins and protection against stress-induced cell death in mammalian cells.

Mdm20 Stimulates PolyQ Aggregation via Inhibiting Autophagy Through Akt-Ser473 Phosphorylation

Mdm20 is an auxiliary subunit of the NatB complex, which includes Nat5, the catalytic subunit for protein N-terminal acetylation. The NatB complex catalyzes N-acetylation during de novo protein synthesis initiation; however, recent evidence from yeast suggests that NatB also affects post-translational modification of tropomyosin, which is involved in intracellular sorting of aggregated proteins. We hypothesized that an acetylation complex such as NatB may contribute to protein clearance and/or proteostasis in mammalian cells. Using a poly glutamine (polyQ) aggregation system, we examined whether the NatB complex or its components affect protein aggregation in rat primary cultured hippocampal neurons and HEK293 cells. The number of polyQ aggregates increased in Mdm20 over-expressing (OE) cells, but not in Nat5-OE cells. Conversely, in Mdm20 knockdown (KD) cells, but not in Nat5-KD cells, polyQ aggregation was significantly reduced. Although Mdm20 directly associates with Nat5, the overall cellular localization of the two proteins was slightly distinct, and Mdm20 apparently co-localized with the polyQ aggregates. Furthermore, in Mdm20-KD cells, a punctate appearance of LC3 was evident, suggesting the induction of autophagy. Consistent with this notion, phosphorylation of Akt, most notably at Ser473, was greatly reduced in Mdm20-KD cells. These results demonstrate that Mdm20, the so-called auxiliary subunit of the translation-coupled protein N-acetylation complex, contributes to protein clearance and/or aggregate formation by affecting the phosphorylation level of Akt indepenently from the function of Nat5.

Spatio-temporal expression pattern of the NatB complex, Nat5/Mdm20 in the developing mouse brain: Implications for co-operative versus non-co-operative actions of Mdm20 and Nat5

The NatB complex, Nat5/Mdm20 acetyltransferase mediates N-acetylation to control cell cycle progression and actin dynamics in yeast. As yet, little is known about the expression pattern of Mdm20 and Nat5 in multi-cellular organisms. Here we show that Mdm20 is highly expressed in mouse embryonic brain. At E11.5, Mdm20 was widely expressed in both neural progenitors and early differentiating neurons, whereas Nat5 was expressed in Sox1/3+/Mdm20+ neural progenitors. By E14.5, both Mdm20 and Nat5 were downregulated in most ventricular zone neural progenitors, whereas both proteins were found in differentiating neurons and co-expression was maintained at E18.5 in derivatives of these cells, such as midbrain dopaminergic (DA) neurons and septal neurons. These data suggest that Nat5/Mdm20 complex-mediated acetylation may play a role in the proliferation and differentiation of neural progenitors. Intriguingly, our data also showed that Mdm20 is not always co-expressed with Nat5 in all differentiated neurons, for example deep cerebellar neurons. Moreover, detailed examination of the subcellular localization of Mdm20 and Nat5 in cultured Nat5+/Mdm20+ midbrain DA neurons revealed that Mdm20 is also not necessarily co-localized with Nat5 within neurons. Given that Nat5 is only a known member of Nat family protein that interacts with Mdm20, our data imply that Mdm20 may function either with an unidentified Nat protein partner(s) or possibly in a Nat-independent manner.

Maintaining ATP levels via the suppression of PERK-mediated rRNA synthesis at ER stress

Currently, [(3)H]uridine is most often used to monitor rRNA synthesis in cultured cells. We show here that radiolabeled ribonucleoside triphosphates, such as [alpha-(33)P]UTP, in culture medium were also incorporated efficiently not only into cells but also into de novo RNA, particularly rRNA. Using this method, we first revealed that endoplasmic reticulum (ER) stress inducers such as tunicamycin and thapsigargin suppressed de novo rRNA synthesis, and that PERK, but not IRE1alpha or ATF6, mediated the suppression. PERK is known to mediate the suppression of de novo protein synthesis via phosphorylation of eIF2alpha. Consistently, other translational inhibitors such as PSI, proteasomal inhibitor, and cycloheximide suppressed de novo rRNA synthesis. eIF2alpha knockdown also suppressed both de novo protein and rRNA syntheses. Furthermore, ER stress reduced cellular ATP levels, and the suppression of rRNA synthesis apparently mitigated their reduction. These observations provided a close link between ATP levels and suppression of de novo rRNA synthesis at ER stress, and we proposed a novel feedback mechanism, in which ATP levels were maintained via suppression of de novo rRNA synthesis in ATP-demanding stresses, such as ER stress.

Involvement of the neuronal phosphotyrosine signal adaptor N-Shc in kainic acid-induced epileptiform activity

BDNF-TrkB signaling is implicated in experimental seizures and epilepsy. However, the downstream signaling involved in the epileptiform activity caused by TrkB receptor activation is still unknown. The aim of the present study was to determine whether TrkB-mediated N-Shc signal transduction was involved in kainic acid (KA)-induced epileptiform activity. We investigated KA-induced behavioral seizures, epileptiform activities and neuronal cell loss in hippocampus between N-Shc deficient and control mice. There was a significant reduction in seizure severity and the frequency of epileptiform discharges in N-Shc deficient mice, as compared with wild-type and C57BL/6 mice. KA-induced neuronal cell loss in the CA3 of hippocampus was also inhibited in N-Shc deficient mice. This study demonstrates that the activation of N-Shc signaling pathway contributes to an acute KA-induced epileptiform activity and neuronal cell loss in the hippocampus. We propose that the N-Shc-mediated signaling pathway could provide a potential target for the novel therapeutic approaches of epilepsy.

Mdm20 Modulates Actin Remodeling through the mTORC2 Pathway via Its Effect on Rictor Expression

NatB is an N-terminal acetyltransferase consisting of a catalytic Nat5 subunit and an auxiliary Mdm20 subunit. In yeast, NatB acetylates N-terminal methionines of proteins during de novo protein synthesis and also regulates actin remodeling through N-terminal acetylation of tropomyosin (Trpm), which stabilizes the actin cytoskeleton by interacting with actin. However, in mammalian cells, the biological functions of the Mdm20 and Nat5 subunits are not well understood. In the present study, we show for the first time that Mdm20-knockdown (KD), but not Nat5-KD, in HEK293 and HeLa cells suppresses not only cell growth, but also cellular motility. Although stress fibers were formed in Mdm20-KD cells, and not in control or Nat5-KD cells, the localization of Trpm did not coincide with the formation of stress fibers in Mdm20-KD cells. Notably, knockdown of Mdm20 reduced the expression of Rictor, an mTORC2 complex component, through post-translational regulation. Additionally, PKCαS657 phosphorylation, which regulates the organization of the actin cytoskeleton, was also reduced in Mdm20-KD cells. Our data also suggest that FoxO1 phosphorylation is regulated by the Mdm20-mTORC2-Akt pathway in response to serum starvation and insulin stimulation. Taken together, the present findings suggest that Mdm20 acts as a novel regulator of Rictor, thereby controlling mTORC2 activity, and leading to the activation of PKCαS657 and FoxO1.

VCP/Cdc48 rescues the growth defect of a GPI10 mutant in yeast.

We identified a yeast mutant with temperature‐sensitive growth defects that were rescued by VCP expression. The mutation occurred in GPI10, which encodes a mannosyl transferase for glycosylphosphatidylinositol anchor formation in the endoplasmic reticulum, and caused a Gly469Glu substitution in Gpi10. The mutant exhibited increased unfolded protein response, which was partially rescued by VCP or Cdc48, and showed sensitivity against cell‐wall stressors, which were not rescued by VCP. These results suggest a potential link between VCP/Cdc48 and Gpi10 functions in the control of cell growth.