Hirohisa Shiraishi

THE ROLES OF SARCOPHAGA DEFENSE MOLECULES IN IMMUNITY AND METAMORPHOSIS

This article summarizes recent progress (1996 1998) in our studies on self-defense molecules in Sarcophaga peregrina. A new antibacterial substance was purified and its unique structure and function revealed a novel aspect of the Sarcophaga defense system. We found a novel lectin and cysteine protease in hemocytes which will assist in the understanding of immune response of hemocytes. There have been two major advances in research on the regulation of defense gene induction: (i) cDNA cloning of a new transcriptional factor binding to the kappaB-like promoter sequence of the Sarcophaga lectin gene, (ii) methylation of cytosolic factors essential for induction of immune genes in the fatbody. Metamorphosis is an interesting event from an immunological point of view: (i) a novel protease with antibacterial activity was discovered from metamorphosing gut, and (ii) a pupal hemocyte-specific surface antigen was purified and characterized in terms of its structure and possible function for larval tissue recognition and elimination.

Normal Formation of a Subset of Intestinal Granules in Caenorhabditis elegans Requires ATP-binding Cassette Transporters HAF-4 and HAF-9, Which Are Highly Homologous to Human Lysosomal Peptide Transporter TAP-Like

TAP-like (TAPL; ABCB9) is a half-type ATP-binding cassette (ABC) transporter that localizes in lysosome and putatively conveys peptides from cytosol to lysosome. However, the physiological role of this transporter remains to be elucidated. Comparison of genome databases reveals that TAPL is conserved in various species from a simple model organism, Caenorhabditis elegans, to mammals. C. elegans possesses homologous TAPL genes: haf-4 and haf-9. In this study, we examined the tissue-specific expression of these two genes and analyzed the phenotypes of the loss-of-function mutants for haf-4 and haf-9 to elucidate the in vivo function of these genes. Both HAF-4 and HAF-9 tagged with green fluorescent protein (GFP) were mainly localized on the membrane of nonacidic but lysosome-associated membrane protein homologue (LMP-1)-positive intestinal granules from larval to adult stage. The mutants for haf-4 and haf-9 exhibited granular defects in late larval and young adult intestinal cells, associated with decreased brood size, prolonged defecation cycle, and slow growth. The intestinal granular phenotype was rescued by the overexpression of the GFP-tagged wild-type protein, but not by the ATP-unbound form of HAF-4. These results demonstrate that two ABC transporters, HAF-4 and HAF-9, are related to intestinal granular formation and some other physiological aspects.

A family of membrane proteins associated with presenilin expression and γ-secretase function

ABSTRACT Presenilin 1 (PS1) forms the γ‐secretase complex with at least three components: nicastrin, APH‐1, and PEN‐2. This complex mediates intramembrane cleavage of amyloid precursor protein (APP) to generate β‐amyloid protein (Aβ) as well as other type 1 transmembrane proteins. Although PS1 mutations linked to familial Alzheimers disease influence these cleavages, their biological consequences have not been fully understood. In this study, we used mRNA differential display analysis to identify a gene, denoted adoplin‐1/ORMDL‐1, which displays significantly reduced expression in association with PS1 mutations. Adoplin‐1 and two highly homologous genes (adoplin‐2, ‐3) constitute a gene family that encodes transmembrane proteins. The mRNA and protein levels of adoplins (particularly adoplin‐1,‐2) were markedly elevated in PS‐deficient fibroblasts, compared to wild‐type cells. Moreover, knockdown of the three adoplins by RNA interference affected maturation of nicastrin and its association with PS1. Adoplin knockdown additionally resulted in elevated levels of APP C‐terminal fragments and decreased Aβ production, suggestive of reduced γ‐secretase activity. Our data collectively indicate that adoplins are unique molecules with PS‐related expression and functions that may play important role(s) in the maturation and activity of the γ‐secretase com‐plex.—Araki, W., Takahashi‐Sasaki, N., Chui, D.‐H., Saito, S., Takeda, K., Shirotani, K., Takahashi, K., Murayama, K. S., Kametani, F., Shiraishi, H., Komano, H., Tabira, T. A family of membrane proteins associated with presenilin expression and γ‐secretase function. FASEB J. 22, 819–827 (2008)

Co-operative function and mutual stabilization of the half ATP-binding cassette transporters HAF-4 and HAF-9 in Caenorhabditis elegans

Caenorhabditis elegans HAF-4 and HAF-9 are half ABC (ATP-binding-cassette) transporters that are highly homologous to the human lysosomal peptide transporter TAPL [TAP (transporter associated with antigen processing)-like; ABCB9]. We reported previously that both HAF-4 and HAF-9 localize to the membrane of a subset of intestinal organelles, and are required for the formation of these organelles and other physiological aspects. In the present paper, we report the genetic and physical interactions between HAF-4 and HAF-9. Overexpression of HAF-4 and HAF-9 did not rescue the intestinal organelle defect of the haf-9 and haf-4 deletion mutants respectively, indicating that they cannot substitute for each other. Double haf-4 and haf-9 mutants do not exhibit more severe phenotypes than the single mutants, suggesting their co-operative function. Immunoprecipitation experiments demonstrated their physical interaction. The results of the present study suggest that HAF-4 and HAF-9 form a heterodimer. Furthermore, Western blot analysis of the deletion mutants and RNAi (RNA interference) knockdown experiments in GFP (green fluorescent protein)-tagged HAF-4 or HAF-9 transgenic worms suggest that HAF-4-HAF-9 heterodimer formation is required for their stabilization. The findings provide a clue as to how ABC transporters adopt a stable functional form.

Differential activation of the lectin and antimicrobial peptide genes in Sarcophaga peregrina (the flesh fly)

Sarcophaga lectin is an immune defense protein which is transcriptionally induced upon immune challenge in the flesh fly, Sarcophaga peregrina. So far, we have revealed that the Sarcophaga lectin gene has multiple NF-kappaB -binding motifs in its promoter. Here we showed that the nuclear extracts from Sarcophaga-derived culture cells, NIH-Sape-4, and larval fat bodies have binding activity to the multiple kappaB motifs in the lectin gene promoter, some of which were responsive to immune stimuli. We also compared the expression profiles of the lectin gene with those of the antibacterial peptide genes from the point of view of inducers, expression tissues and local induction in digestive tracts. In each case, the lectin gene was activated in different manners from other inducible defense genes. These results indicate the complex regulation of the lectin gene, possibly by NF-kappaB -related transcription factors.

Characterization of HAF-4- and HAF-9-localizing organelles as distinct organelles in Caenorhabditis elegans intestinal cells

BackgroundThe intestinal cells of Caenorhabditis elegans are filled with heterogeneous granular organelles that are associated with specific organ functions. The best studied of these organelles are lipid droplets and acidified gut granules associated with GLO-1, a homolog of the small GTPase Rab38. In this study, we characterized a subset of the intestinal granules in which HAF-4 and HAF-9 localize on the membrane. HAF-4 and HAF-9 are ATP-binding cassette (ABC) transporter proteins that are homologous to the mammalian lysosomal peptide transporter TAPL (transporter associated with antigen processing-like, ABCB9).ResultsUsing transgenic worms expressing fluorescent protein-tagged marker proteins, we demonstrated that the HAF-4- and HAF-9-localizing organelles are not lipid droplets and do not participate in yolk protein transport. They were also ruled out as GLO-1-positive acidified gut granules. Furthermore, we clarified that the late endosomal protein RAB-7 localizes to the HAF-4- and HAF-9-localizing organelles and is required for their biogenesis.ConclusionsOur results indicate that the HAF-4- and HAF-9-localizing organelles are distinct intestinal organelles associated with the endocytic pathway.

Tissue and developmental expression of SRAM, an unconventional Rel-family protein.

Previously we have reported the purification and cDNA cloning of a novel Rel/Ankyrin-family protein named SRAM from the flesh fly, Sarcophaga peregrina. Rel proteins generally translocate into the nucleus upon immune stimuli by dissociating from an inhibitory ankyrin domain, while SRAM is unique in terms of its constitutive nuclear localization with its internal ankyrin domain accompanied, at least in a Sarcophaga cell line and fat body cells. Although SRAM had been originally identified as a sole factor that binds to the κB motif of the inducible Sarcophaga lectin gene promoter, its transcriptional activity remained controversial. Moreover, homologues of SRAM have not been found in any other established model organisms including Drosophila. Here we report that the developmental expression of SRAM was up-regulated at the early stages of embryogenesis and metamorphosis. Furthermore, SRAM expression was prominent in the digestive tracts of the third instar larvae. We argue the hypothesis that SRAM has evolved as a quite unconventional Rel-family protein in Sarcophaga.