Structural basis of functional cooperation of Tim15/Zim17 with yeast mitochondrial Hsp70

Mitochondrial heat-shock protein 70 (mtHsp70) and its partner proteins drive protein import into the matrix. Tim15/Zim17/Hep1 is a mtHsp70 partner protein on the matrix side of the inner mitochondrial membrane. We determined the nuclear magnetic resonance (NMR) structure of the core domain of Tim15. On the basis of the NMR structure, we created Tim15 mutants and tested their ability to complement the functional defects of Tim15 depletion and to suppress self-aggregation of mtHsp70 in vivo. A pair of basic residues, Arg 106 and His 107, conserved Asp 111 and flexible loop 133-137, and were important (Arg 106-His 107 pair and Asp 111) or partly important (the loop 133-137) for yeast cell growth, mitochondrial protein import and the suppression of mtHsp70 aggregation. Therefore, the function of Tim15 in yeast cell growth is well correlated with its ability to suppress mtHsp70 aggregation, although it is still unknown whether inhibition of mtHsp70 aggregation is the primary function of Tim15.     

Identification of innate IL-5-producing cells and their role in lung eosinophil regulation and antitumor immunity

IL-5 is involved in a number of immune responses such as helminth infection and allergy. IL-5 also plays roles in innate immunity by maintaining B-1 B cells and mucosal IgA production. However, the identity of IL-5-producing cells has not been unambiguously characterized. In this report, we describe the generation of an IL-5 reporter mouse and identify IL-5-producing non-T lymphoid cells that reside in the intestine, peritoneal cavity, and lungs in naive mice. They share many characteristics with natural helper cells, nuocytes, and Ih2 cells, including surface Ags and responsiveness to cytokines. However, these phenotypes do not completely overlap with any particular one of these cell types. Innate non-T IL-5-producing cells localized most abundantly in the lung and proliferated and upregulated IL-5 production in response to IL-25 and IL-33. IL-33 was more effective than IL-25. These cells contribute to maintaining sufficient numbers of lung eosinophils and are important for eosinophil recruitment mediated by IL-25 and IL-33. Given that eosinophils are shown to possess antitumor activity, we studied lung tumor metastasis and showed that innate IL-5-producing cells were increased in response to tumor invasion, and their regulation of eosinophils is critical to suppress tumor metastasis. Genetic blockade or neutralization of IL-5 impaired eosinophil recruitment into the lung and resulted in increased tumor metastasis. Conversely, exogenous IL-5 treatment resulted in suppressed tumor metastasis and augmented eosinophil infiltration. These newly identified innate IL-5-producing cells thus play a role in tumor surveillance through lung eosinophils and may contribute to development of novel immunotherapies for cancer.     

Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.     

Cryptic diversity of the eel goby, genus Taenioides (Gobiidae: Amblyopinae), in Japan

The eel goby, genus Taenioides (Gobiidae: Amblyopinae), inhabits muddy bottoms of estuaries or shallow areas of seas in the Indo-Pacific Ocean. Among congeners, T. cirratus ( Blyth, 1860 ) has been thought to be distributed in Japan, but taxonomic confusions remain as to which scientific names are applicable to Japanese Taenioides species, or more fundamentally, how many Taenioides species are distributed in Japan, due in part to the rarity of this group in museum collections and the morphological similarity among species. To clarify the species diversity of the genus Taenioides in Japan, we conducted phylogenetic analysis on the basis of partial mitochondrial DNA sequences and morphological observation of more than 100 specimens. As a result, four distinct species were distinguished from each other, on the basis of both genetic divergences (2.9-5.7%, 16S rRNA gene) and morphological differences (i.e., degree of development of dermal folds on the head, numbers of barbels and vertebrae). Although the identifications of four species need additional verifications, they were identified as T. anguillaris, T. snyderi, T. gracilis and T. cf. kentalleni, and the species name T. cirratus does not seem to be appropriate to any of four detected species. Museum collections indicate that the two species, which are distributed in the main islands of Japan, were collected frequently and treated as a single species. The other two were each collected only from a single locality of Okinawa Island in this study, of which one seems to be uncommon worldwide as well.     

Bacterial collagen‐binding domain targets undertwisted regions of collagen

Clostridium histolyticum collagenase causes extensive degradation of collagen in connective tissue that results in gas gangrene. The C‐terminal collagen‐binding domain (CBD) of these enzymes is the minimal segment required to bind to a collagen fibril. CBD binds unidirectionally to the undertwisted C‐terminus of triple helical collagen. Here, we examine whether CBD could also target undertwisted regions even in the middle of the triple helix. Collageneous peptides with an additional undertwisted region were synthesized by introducing a Gly → Ala substitution [(POG)_xPOA(POG)_y]₃, where x + y = 9 and x > 3). ¹H–¹⁵N heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR) titration studies with ¹⁵N‐labeled CBD demonstrated that the minicollagen binds to a 10 Å wide 25 Å long cleft. Six collagenous peptides each labeled with a nitroxide radical were then titrated with ¹⁵N‐labeled CBD. CBD binds to either the Gly → Ala substitution site or to the C‐terminus of each minicollagen. Small‐angle X‐ray scattering measurements revealed that CBD prefers to bind the Gly → Ala site to the C‐terminus. The HSQC NMR spectra of ¹⁵N‐labeled minicollagen and minicollagen with undertwisted regions were unaffected by the titration of unlabeled CBD. The results imply that CBD binds to the undertwisted region of the minicollagen but does not actively unwind the triple helix.     

Specific recognition of the collagen triple helix by chaperone HSP47: minimal structural requirement and spatial molecular orientation

The unique folding of procollagens in the endoplasmic reticulum is achieved with the assistance of procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed "flying capstan model" (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319).     

Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates

Liposuction aspirates (primarily saline solution, blood, and adipose tissue fragments) separate into fatty and fluid portions. Cells isolated from the fatty portion are termed processed lipoaspirate (PLA) cells and contain adipose-derived adherent stromal cells (ASCs). Here we define cells isolated from the fluid portion of liposuction aspirates as liposuction aspirate fluid (LAF) cells. Stromal vascular fractions (SVF) were isolated separately from both portions and characterized under cultured and non-cultured conditions. A comparable number of LAF and PLA cells were freshly isolated, but fewer LAF cells were adherent. CD34+ CD45- cells from fresh LAF isolates were expanded by adherent culture, suggesting that LAF cells contain ASCs. Although freshly isolated PLA and LAF cells have distinct cell surface marker profiles, adherent PLA and LAF cells have quite similar characteristics with regard to growth kinetics, morphology, capacity for differentiation, and surface marker profiles. After plating, both PLA and LAF cells showed significant increased expression of CD29, CD44, CD49d, CD73, CD90, CD105, and CD151 and decreased expression of CD31 and CD45. Multicolor FACS analysis revealed that SVF are composed of heterogeneous cell populations including blood-derived cells (CD45+), ASCs (CD31- CD34+ CD45- CD90+ CD105- CD146-), endothelial (progenitor) cells (CD31+ CD34+ CD45- CD90+ CD105low CD146+), pericytes (CD31- CD34- CD45- CD90+ CD105- CD146+), and other cells. After plating, ASCs showed a dramatic increase in CD105 expression. Although some adherent ASCs lost CD34 expression with increasing culture time, our culture method maintained CD34 expression in ASCs for at least 10-20 weeks. These results suggest that liposuction-derived cells may be useful and valuable for cell-based therapies.     

Pituitary adenylate cyclase-activating peptide (PACAP) stimulates production of interleukin-6 in rat Müller cells

Pituitary adenylate cyclase-activating peptide (PACAP) is known to regulate not only neurons but also astrocytes. Here, we investigated, both in vitro and in vivo, the effects of PACAP38 on rat Müller cells, which are the predominant glial element in the retina. Müller cells isolated from juvenile Wistar rats were treated with PACAP38 or PACAP6-38, a PACAP selective antagonist. Cell proliferation was determined by measuring the incorporation of bromodeoxyuridine with ELISA. Interleukin-6 (IL-6) levels in the culture medium were determined by a bioassay using B9 cells, IL-6 dependent hybridoma. In adult Wistar rats, the expression of IL-6 in the retina after intravitreal injection of PACAP38 (10 pmol) was assessed by immunohistochemistry. PACAP38 stimulated IL-6 production in Müller cells at a concentration as low as 10(-12) M, which did not induce cell proliferation. This elevation of IL-6 production was inhibited by PACAP6-38. Radial IL-6 expression was observed throughout the retina at 2 and 3 days after PACAP38 injection. These data demonstrate that Müller cells are one of the target cells for PACAP. IL-6, which is released from Müller cells with stimulation by PACAP, may play a significant role in the retina.     

Interleukin-10 in the pathophysiology of inflammatory bowel disease: increased serum concentrations during the recovery phase

Using a specific enzyme-linked immunosorbent assay, IL-10 concentrations were measured in serum from 62 patients with ulcerative colitis (UC), 43 with Crohn's disease (CD), 25 with other colitides, and 44 normal control subjects. Serum IL-10 concentrations were increased in patients with active UC but not in those with active CD when compared with normal control subjects. A time course study showed that in patients with UC and CD, serum concentrations of IL-6 and C-reactive protein increased during the acute phase and returned to normal as patients go into remission. Notably, serum IL-10 concentrations increased during the phase of disease resolution and declined thereafter regardless of the treatment modality. Gel filtration analysis indicated that IL-10 circulated predominantly as a dimer. In conclusion, this study shows that serum IL-10 is increased during disease recovery in patients with inflammatory bowel disease, and may be a helpful marker in monitoring disease status.