Interaction of the spectrin-like repeats of alpha-actinin-4 with humanin peptide.
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.
Kigawa
Akihiro
A
Third Department of Internal Medicine, Akita University School of Medicine, 1-1-1 Hondo, Akita 010-8543, Japan.
Wakui
Hideki
H
Maki
Nobuki
N
Okuyama
Shin
S
Masai
Rie
R
Ohtani
Hiroshi
H
Komatsuda
Atsushi
A
Suzuki
Daisuke
D
Toyoda
Masao
M
Kobayashi
Ryoji
R
Sawada
Ken-Ichi
K
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
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