PAC1 Gene Knockout Reveals an Essential Role of Chaperone-Mediated 20S Proteasome Biogenesis and Latent 20S Proteasomes in Cellular Homeostasis

The 26S proteasome, a central enzyme for ubiquitin-dependent proteolysis, is a highly complex structure comprising 33 distinct subunits. Recent studies have revealed multiple dedicated chaperones involved in proteasome assembly both in yeast and in mammals. However, none of these chaperones is essential for yeast viability. PAC1 is a mammalian proteasome assembly chaperone that plays a role in the initial assembly of the 20S proteasome, the catalytic core of the 26S proteasome, but does not cause a complete loss of the 20S proteasome when knocked down. Thus, both chaperone-dependent and -independent assembly pathways exist in cells, but the contribution of the chaperone-dependent pathway remains unclear. To elucidate its biological significance in mammals, we generated PAC1 conditional knockout mice. PAC1-null mice exhibited early embryonic lethality, demonstrating that PAC1 is essential for mammalian development, especially for explosive cell proliferation. In quiescent adult hepatocytes, PAC1 is responsible for producing the majority of the 20S proteasome. PAC1-deficient hepatocytes contained normal amounts of the 26S proteasome, but they completely lost the free latent 20S proteasome. They also accumulated ubiquitinated proteins and exhibited premature senescence. Our results demonstrate the importance of the PAC1-dependent assembly pathway and of the latent 20S proteasomes for maintaining cellular integrity.The 26S proteasome is a eukaryotic ATP-dependent protease responsible for the degradation of proteins tagged with polyubiquitin chains (21). The ubiquitin-dependent proteolysis by the proteasome plays a pivotal role in various cellular processes by catalyzing the selective degradation of short-lived regulatory proteins as well as damaged proteins. Thus, the proteasome is essential for the viability of all eukaryotic cells.The 26S proteasome is a large protein complex consisting of two portions; one is the catalytic 20S proteasome of approximately 700 kDa (also called the 20S core particle), and the other is the 19S regulatory particle (RP; also called PA700) of approximately 900 kDa, both of which are composed of a set of multiple distinct subunits (70). The 20S proteasome is a cylindrically shaped stack of four heptameric rings, where the outer and inner rings each are composed of seven homologous α subunits (α1 to α7) and seven homologous β subunits (β1 to β7), respectively (5). The proteolytic active sites reside within the central chamber enclosed by the two inner β-rings, while a small channel formed by the outer α-ring, which is primarily closed, restricts the access of native proteins to the catalytic chamber. Thus, the 20S proteasome is a latent enzyme. Appending 19S RP, which consists of 19 different subunits, to the α-ring enables the 20S proteasome to degrade native proteins; 19S RP accepts ubiquitin chains of substrate proteins, removes ubiquitin chains while unfolding the substrates, and feeds the substrates into the interior proteolytic chamber of the 20S proteasome through the α-ring that is opened when the C-terminal tails of the ATPase subunits of 19S RP are inserted into the intersubunit spaces of the α-ring (24, 62, 74). However, it also has been reported that some denatured or unstructured proteins can be degraded directly by the 20S proteasome even in the absence of 19S RP and ubiquitination (37, 39).Much attention has been focused on how such a highly elaborate structure is achieved. Recent studies have identified various proteasome-dedicated chaperones that assist in the assembly of the proteasome in eukaryotic cells (23, 40, 56, 57, 65, 66). In yeast, while most of the proteasome subunits are essential for viability, the deletion of any of these chaperones does not cause lethality. In fact, many, if not all, of the deletions exhibit subtle phenotypes. In mammalian cells, although the knockdown of the assembly chaperones reduced proteasome assembly and thus proteasome activity, leading to slow cell growth, the degree of reduction was much lower than that which occurred following the knockdown of the proteasome subunit itself (33, 35, 40). These results indicate that the assembly chaperones play an auxiliary role in proteasome biogenesis.Proteasome assembly chaperone 1 (PAC1) is one of the assembly chaperones originally identified in mammalian cells (34). PAC1 plays a role in α-ring formation that occurs during the initial assembly of the 20S proteasome; it also prevents the aberrant dimerization of the α-ring. As is the case for most assembly chaperones, the knockdown of PAC1 in mammalian cells decreases proteasome activity but to a lesser extent than that in, for example, β2 knockdown (34, 35). Therefore, both PAC1-dependent and -independent assembly pathways exist in cells, but the importance of the PAC1-dependent pathway remains elusive. To further elucidate the biological significance of PAC1 and PAC1-dependent proteasome biogenesis, we generated conditional mouse mutants carrying an inactivating mutation in Psmg1, the gene coding for PAC1 protein, in the whole body, the nervous system, and in the liver. Our results demonstrate that PAC1 is essential for the development of a mouse, and that it plays important roles in maintaining cellular integrity in quiescent tissue. Our study revealed for the first time the importance of chaperone-mediated proteasome biogenesis in a whole-body mammalian system and may provide valuable knowledge in medical drug development targeting proteasomes.     

1,3-Benzoxazole-4-carbonitrile as a novel antifungal scaffold of β-1,6-glucan synthesis inhibitors

Synthesis and in vitro antifungal evaluations of 1,3-benzoxazole-7-carbonitrile 3, 1,3-benzoxazole-4-carbonitrile 4, benzofuran 5, benzoxazine 7, and benzimidazole 8 were reported. Among them, 1,3-benzoxazole-4-carbonitrile was found to be a superior scaffold structure with moderate growth inhibition against Candida species. 1,3-Benzoxazole-4-carbonitrile 6 showed potent activity against Candida species compared to 5-desmethyl compound 4 and triazolopyridine 2. Compound 6 was efficiently prepared from versatile intermediate 24, which possessed six different substituents on the benzene ring. Conversion of benzene 24 into various 1,3-benzoxazole derivatives such as 2-aliphatic 34, 2-amino 35, and lactone 38 was demonstrated.     

Regulation of IL-8 by Irsogladine maleate is involved in abolishment of Actinobacillus actinomycetemcomitans-induced reduction of gap-junctional intercellular communication

Our previous report has shown that Irsogladine maleate (IM) counters and obviates the reduction in gap junction intercellular communication (GJIC) and the increase in IL-8 levels, respectively, induced by outer membrane protein 29 from Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) in cultured human gingival epithelial cells (HGEC). In addition, IM suppresses the increase in the secretion of IL-8 caused by whole live A. actinomycetemcomitans. These findings implicate the modulation of IL-8 levels by IM in abolishment of the reduction of GJIC in HGEC. Tight junctions are also responsible for cell-cell communication. Zonula occludens protein-1 (ZO-1) is a major tight junction protein. To investigate the regulatory mechanism of intercellular communication mediated by IM, in the present study, we focused on the involvement of IL-8 in A. actinomycetemcomitans-induced change in GJIC and ZO-1 expression in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in levels of Connexin (CX) 43, suggesting that it could abolish the A. actinomycetemcomitans-induced reduction in GJIC in HGEC. CXCR-1 is a receptor of IL-8. The simultaneous addition of A. actinomycetemcomitans and anti-CXCR-1 antibody also abrogated the repression of GJIC and CX43 expression by A. actinomycetemcomitans in HGEC, although the anti-CXCR-1 antibody was less effective than IM. IM inhibited the IL-8-induced reduction in CX43 levels and GJIC in HGEC. IM countered the A. actinomycetemcomitans-induced reduction in the expression of ZO-1, although anti-CXCR-1 antibody did not influence the decrease in ZO-1 mRNA levels caused by A. actinomycetemcomitans. Furthermore, IL-8 had little effect on the mRNA levels of ZO-1. These findings suggest that IL-8 mediates the A. actinomycetemcomitans-induced reduction of GJIC and CX43 expression in HGEC. The regulation of IL-8 levels by IM in HGEC is partially involved in abrogation of the reduction of GJIC and CX43 expression by A. actinomycetemcomitans. Furthermore, the regulatory effect of IM on the expression of CX43 and ZO-1 is different.     

Probing Functional Properties of Nociceptive Axons Using a Microfluidic Culture System

Pathological changes in axonal function are integral features of many neurological disorders, yet our knowledge of the molecular basis of axonal dysfunction remains limited. Microfluidic chambers (MFCs) can provide unique insight into the axonal compartment independent of the soma. Here we demonstrate how an MFC based cell culture system can be readily adapted for the study of axonal function in vitro. We illustrate the ease and versatility to assay electrogenesis and conduction of action potentials (APs) in naïve, damaged or sensitized DRG axons using calcium imaging at the soma for pharmacological screening or patch-clamp electrophysiology for detailed biophysical characterisation. To demonstrate the adaptability of the system, we report by way of example functional changes in nociceptor axons following sensitization by neurotrophins and axotomy in vitro. We show that NGF can locally sensitize axonal responses to capsaicin, independent of the soma. Axotomizing neurons in MFC results in a significant increase in the proportion of neurons that respond to axonal stimulation, and interestingly leads to accumulation of Nav1.8 channels in regenerating axons. Axotomy also augmented AP amplitude following axotomy and altered activation thresholds in a subpopulation of regenerating axons. We further show how the system can readily be used to study modulation of axonal function by non-neuronal cells such as keratinocytes. Hence we describe a novel in vitro platform for the study of axonal function and a surrogate model for nerve injury and sensitization.     

Selection of novel structural zinc sites from a random peptide library

Zinc ion (Zn(2+)) can be coordinated with four or three amino acid residues to stabilize a protein's structure or to form a catalytic active center. We used phage display selection of a dodecamer random peptide library with Zn(2+) to identify structural zinc sites. The binding specificity for Zn(2+) of selected sequences was confirmed using enzyme-linked immunosorbent and competitive inhibition assays. Circular dichroism spectra indicated that the interaction with Zn(2+) induced a change in conformation, which means the peptide acts as a structural zinc site. Furthermore, a search of protein databases revealed that two selected sequences corresponded to parts of natural zinc sites of copper/zinc superoxide dismutase and zinc-containing ferredoxin. We demonstrated that Zn(2+)-binding sequences selected from the random combinatorial library would be candidates for artificial structural zinc sites.     

Neuroprotective or neurotoxic activity of 1-methyl-1,2,3,4-tetrahydroisoquinoline and related compounds

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) 1 and various 5- or 6,7-substituted analogues were synthesized and assayed for neurotoxicity towards SH-SY5Y cells. Among mono-substituted derivatives of 1, hydroxyl substitution decreased the toxicity, while methoxyl substitution increased it. Disubstituted derivatives of 1, 5a and 5b, showed the opposite tendency. Hydroxy-1MeTIQ derivatives were tested for neuroprotective activity, and 3b and 4b exhibited greater efficacy than 1. We suggest that hydroxy-1MeTIQ derivatives, especially 4b, may have potential for the treatment of Parkinson's disease.     

Protective effects of nicaraven,a new hydroxyl radical scavenger,on the endothelial dysfunction after exposure of pig coronary artery to hydroxyl radicals

Recently, we have reported that a new synthetic compound, 1,2bis(nicotinamido)-propane (nicaraven), improved cardiac function following preservation and reperfusion. In this study, we investigated the efficacy of nicaraven as a radical scavenger by using an in vitro model of oxidative stress, to clarify mechanisms of the protective effect of this new compound on reperfusion injury in rat heart. Ring segments of epicardial right coronary arteries (RCA) of pig were suspended in organ chambers and exposed to hydroxyl radicals (·OH), generated (by two different systems ) by 0.28 mM FeSO₄/0.28 mM H₂O₂ and DHF/Fe³⁺-ADP (2.4 mM, 43 nM, and 1.56 uM, respectively) to the bathing solution for 60 min. Prior exposure of the coronary arteries to ·OH significantly produced right-ward shift of the dose-response curves of the bradykinin-induced endothelium-dependent relaxations (an increase in the ED₅₀ value for bradykinin by 4.37 and 1.98 times than control in two different ·OH generating systems, respectively), but did not affect the maximum relaxation responses. The presence of nicaraven (10^-4 and 10^-5 M) in the ·OH generating system, shifted the dose-response curves to bradykinin to the control level, suggesting a significant hydroxyl radical scavenging effect of the drug. These results indicate that nicaraven, a new hydroxyl radical scavenger, exhibits a protective effect on hydroxyl radicalinduced endothelial dysfunctions of pig coronary artery.     

On the reaction of 2-aminopropionitrile in aqueous media

The reactions of 2- and 3-aminopropionitrile (APN), and 2,2-iminodipropionitrile (IDPN) were carried out in aqueous ammoniacal media. 2-APN was found to give IDPN, N-(1-cyanoethyl)alanine amide, N-(1-cyanoethyl)alanine, N-(1-carbamoylethyl)alanine, 2,2-iminodipropionic acid, alanine amide, and alanine. Compounds of biological significance such as peptides and amino acids other than alanine were not formed. The results were well consistent with those obtained for aminoacetonitrile. IDPN which can be formed easily from 2-APN in aqueous media, also yielded the same products as with 2-APN. On the other hand, 3-APN gave 3-alanine via 3-alanine amide under similar conditions. 3-APN was found to be more stable than 2-APN in aqueous media.     

Overexpression of CDCA2 in Human Squamous Cell Carcinoma: Correlation with Prevention of G1 Phase Arrest and Apoptosis

Cell division cycle associated 2 (CDCA2) recruits protein phosphatase 1 to chromatin to antagonize activation of ataxia telangiectasia mutated (ATM)-dependent signal transduction. ATM kinase plays a critical role in the DNA damage response and its phosphorylation cascade to inhibit the p53-MDM2 interaction, which releases p53 to induce p21 and G1 cell-cycle arrest. However, the relevance of CDCA2 to human malignancy including oral squamous cell carcinoma (OSCC) is unknown. In the current study, we found that CDCA2 expression was up-regulated in OSCC cell lines. Functional studies with shRNA system showed that knockdown of CDCA2 significantly (P<0.05) inhibited cellular proliferation compared with the control cells by arresting cell-cycle progression at the G1 phase and up-regulating the cyclin-dependent kinase inhibitors (p21^Cip1, p27^Kip1, p15^INK4B, and p16^INK4A). CDCA2 knockdown also promoted apoptosis after treatment with the DNA damage reagent, cisplatin. In clinical samples, the CDCA2 protein expression level in primary OSCCs was significantly (P<0.05) greater than in matched normal oral tissues (67/85, 79%). Furthermore, CDCA2-positive cases were correlated significantly (P<0.05) with high cancer progression. Our results showed for the first time that CDCA2 frequently is overexpressed in OSCCs and might be associated closely with OSCC progression by preventing cell-cycle arrest and apoptosis.