title: Interaction of the spectrin-like repeats of alpha-actinin-4 with humanin peptide.
abstract: BACKGROUND
  Podocyte alpha-actinin-4 (actinin-4) is an essential component of the glomerular filtration barrier. We recently reported that the central rod spectrin-like repeats (R1-R4) of actinin-4 have a high affinity to puromycin aminonucleoside (PAN), which can induce nephro-sis in animals. The aim of this study was to identify endogenous molecules that interact with the actinin-4 R1-R4 domain.
  
  
  METHODS
  To identify such molecules, we performed a bacterial two-hybrid screening of a human kidney cDNA library using as a bait human actinin-4 R1-R4. We further verified the identified interactions by in vitro affinity assays and immunofluorescent studies of cultured human embryonic kidney HEK293 cells. To investigate the expression of the identified molecules in podocytes, in situ hybridization, and immunohistochemical studies were performed.
  
  
  RESULTS
  One isolated cDNA from the library encoded humanin, a recently identified antiapoptotic peptide. In vitro affinity assays showed specific interactions of recombinant actinin-4 R1-R4, R1, R2, R3, and R4 proteins with humanin-Sepharose. PAN had no effect on these interactions. Green fluorescent protein-fused humanin and endogenous actinin colocalized mainly in the perinuclear cytoplasm of HEK293 cells. Altered colocalization was not observed by the addition of PAN. In situ hybridization and immunohistochemistry showed the expression of humanin in podocytes.
  
  
  CONCLUSIONS
  Our results suggest that humanin is a novel binding partner of the actinin-4 R1-R4 domain in podocytes. Humanin and PAN are unlikely to compete for the same binding surface in actinin-4.
authors.affiliation: Third Department of Internal Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kigawa
authors.forename: Hideki
authors.initials: H
authors.lastname: Wakui
authors.forename: Nobuki
authors.initials: N
authors.lastname: Maki
authors.forename: Shin
authors.initials: S
authors.lastname: Okuyama
authors.forename: Rie
authors.initials: R
authors.lastname: Masai
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ohtani
authors.forename: Atsushi
authors.initials: A
authors.lastname: Komatsuda
authors.forename: Daisuke
authors.initials: D
authors.lastname: Suzuki
authors.forename: Masao
authors.initials: M
authors.lastname: Toyoda
authors.forename: Ryoji
authors.initials: R
authors.lastname: Kobayashi
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Sawada
mesh_terms: D000185:Actinin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000378:metabolism; D002460:Cell Line; D017930:Genes, Reporter; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D007678:Kidney Glomerulus / Q000166:cytology / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011692:Puromycin Aminonucleoside / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: C111453:ACTN4 protein, human; D000903:Antibiotics, Antineoplastic; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D010455:Peptides; C427211:humanin; D000185:Actinin; D011692:Puromycin Aminonucleoside