An Archaeal Dim2-Like Protein, aDim2p, Forms a Ternary Complex with a/eIF2α and the 3′ End Fragment of 16S rRNA

Dim2p is a eukaryal small ribosomal subunit RNA processing factor required for the maturation of 18S rRNA. Here we show that an archaeal homolog of Dim2p, aDim2p, forms a ternary complex with the archaeal homolog of eIF2α, a/eIF2α, and the RNA fragment that possesses the 3′ end sequence of 16S rRNA both in solution and in crystal. The 2.8-Å crystal structure of the ternary complex reveals that two KH domains of aDim2p, KH-1 and -2, are involved in binding the anti-Shine-Dalgarno core sequence (CCUCC-3′) and a highly conserved adjacent sequence (5′-GGAUCA), respectively, of the target rRNA fragment. The surface plasmon resonance results show that the interaction of aDim2p with the target rRNA fragment is very strong, with a dissociation constant of 9.8 × 10^− 10 M, and that aDim2p has a strong nucleotide sequence preference for the 3′ end sequence of 16S rRNA. On the other hand, aDim2p interacts with the isolated α subunit and the intact αβγ complex of a/eIF2, irrespective of the RNA binding. These results suggest that aDim2p is a possible archaeal pre-rRNA processing factor recognizing the 3′ end sequence (5′-GAUCACCUCC-3′) of 16S rRNA and may have multiple biological roles in vivo by interacting with other proteins such as a/eIF2 and aRio2p.     

Mouse homologue of skin-specific retroviral-like aspartic protease involved in wrinkle formation

Retroviral proteases are encoded in the retroviral genome and are responsible for maturation and assembly of infectious virus particles. A number of retroviral protease sequences with retroviral elements are integrated in every eukaryotic genome as endogenous retroviruses. Recently, retroviral-like aspartic proteases that were not embedded within endogenous retroviral elements were identified throughout the eukaryotic and prokaryotic genomes. However, the physiological role of this novel protease family, especially in mammals, is not known. During the high throughput in situ hybridization screening of mouse epidermis, as a granular layer-expressing clone, we identified a mouse homologue of SASPase (Skin ASpartic Protease), a recently identified retroviral-like aspartic protease. We detected and purified the endogenous 32-kDa (mSASP32) and 15-kDa (mSASP15) forms of mSASP from mouse stratum corneum extracts and determined their amino acid sequences. Next, we bacterially produced recombinant mSASP15 via autoprocessing of GST-mSASP32. Purified recombinant mSASP15 cleaved a quenched fluorogenic peptide substrate, designed from the autoprocessing site for mSASP32 maximally at pH 5.77, which is close to the pH of the epidermal surface. Finally, we generated mSASP-deficient mice that at 5 weeks of age showed fine wrinkles that ran parallel on the lateral trunk without apparent epidermal differentiation defects. These results indicate that the retroviral-like aspartic protease, SASPase, is involved in prevention of fine wrinkle formation via activation in a weakly acidic stratum corneum environment. This study provides the first evidence that retroviral-like aspartic protease is functionally important in mammalian tissue organization.     

Not lipoteichoic acid but lipoproteins appear to be the dominant immunobiologically active compounds in Staphylococcus aureus

Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of TLR2. However, we previously demonstrated that LTA fraction prepared from bacterial cells contains lipoproteins, which activate cells via TLR2. In this study, we investigated the immunobiological activity of LTA fraction obtained from S. aureus wild-type strain, lipoprotein diacylglycerol transferase deletion (delta lgt) mutant, which lacks palmitate-labeled lipoproteins, and its complemented strain and evaluated the activity of LTA molecule. LTA fraction was prepared by butanol extraction of the bacteria followed by hydrophobic interaction chromatography. Although all LTA fractions activated cells through TLR2, the LTA from delta lgt mutant was 100-fold less potent than those of wild-type and complemented strains. However, no significant structural difference in LTA was observed in NMR spectra. Further, alanylation of LTA molecule showed no effect in immunobiological activity. These results showed that not LTA molecule but lipoproteins are dominant immunobiologically active TLR2 ligand in S. aureus.     

A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.     

Premeiotic germ cell defect in seminiferous tubules of Atm-null testis

Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16(Ink4a) activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16(Ink4a)-mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16(Ink4a)-mediated senescence.     

Establishment of a novel method for enriching osteoblast progenitors from adipose tissues using a difference in cell adhesive properties

In the clinical field, cell-based therapies are used to treat bone defects. Adipose tissues contain many osteoblast progenitors, among other cell types. We separated mouse adipose tissue-derived stromal cells (ATSCs) according to their cell adhesive properties. Cells in a fraction adherent to the culture dishes 0.5h after inoculation (AF-0.5) had a potent ability to differentiate into both osteoblasts and adipocytes in vitro. Their differentiation pathways depended on the culture conditions. In these cells, the expression of marker genes for osteoblast differentiation was induced in osteogenic medium. Moreover, the AF-0.5 cells, which were induced to differentiate into osteoblasts in vitro, formed abundant bone tissues in vivo. These results suggest that the AF-0.5 cells have been enriched with bi-potential progenitor cells destined for either osteoblasts or adipocytes. This simple and efficient method for preparing osteoblast progenitor cells from ATSCs may be utilized for bone defect treatment clinically.     

Caenorhabditis elegans kettin, a large immunoglobulin-like repeat protein, binds to filamentous actin and provides mechanical stability to the contractile apparatuses in body wall muscle

Kettin is a large actin-binding protein with immunoglobulin-like (Ig) repeats, which is associated with the thin filaments in arthropod muscles. Here, we report identification and functional characterization of kettin in the nematode Caenorhabditis elegans. We found that one of the monoclonal antibodies that were raised against C. elegans muscle proteins specifically reacts with kettin (Ce-kettin). We determined the entire cDNA sequence of Ce-kettin that encodes a protein of 472 kDa with 31 Ig repeats. Arthropod kettins are splice variants of much larger connectin/titin-related proteins. However, the gene for Ce-kettin is independent of other connectin/titin-related genes. Ce-kettin localizes to the thin filaments near the dense bodies in both striated and nonstriated muscles. The C-terminal four Ig repeats and the adjacent non-Ig region synergistically bind to actin filaments in vitro. RNA interference of Ce-kettin caused weak disorganization of the actin filaments in body wall muscle. This phenotype was suppressed by inhibiting muscle contraction by a myosin mutation, but it was enhanced by tetramisole-induced hypercontraction. Furthermore, Ce-kettin was involved in organizing the cytoplasmic portion of the dense bodies in cooperation with alpha-actinin. These results suggest that kettin is an important regulator of myofibrillar organization and provides mechanical stability to the myofibrils during contraction.