Deficiency of spermatogenesis and reduced expression of spermatogenesis-related genes in prefoldin 5-mutant mice.

MM-1α is a c-Myc-binding protein and acts as a transcriptional co-repressor in the nucleus. MM-1α is also PDF5, a subunit of prefoldin that is chaperon comprised of six subunits and prevents misfolding of newly synthesized nascent polypeptides. Prefoldin also plays a role in quality control against protein aggregation. It has been reported that mice harboring the missense mutation L110R of MM-1α/PFD5 exhibit neurodegeneration in the cerebellum and also male infertility, but the phenotype of infertility has not been fully characterized. In this study, we first analyzed morphology of the testis and epididymis of L110R of MM-1α mice. During differentiation of spermatogenesis, spermatogonia, spermatocytes and round spermatids were formed, but formation of elongated spermatids was compromised in L110R MM-1α mice. Furthermore, reduced number/concentration of sperm in the epididymis was observed. MM-1α was strongly expressed in the round spermatids and sperms with round spermatids, suggesting that MM-1α affects the differentiation and maturation of germ cells. Changes in expression levels of spermatogenesis-related genes in mice testes were then examined. The fatty-acid-binding protein (fabp4) gene was up-regulated and three genes, including sperm-associated glutamate (E)-rich protein 4d (speer-4d), phospholipase A2-Group 3 (pla2g3) and phospholipase A2-Group 10 (pla2g10), were down-regulated in L110R MM-1α mice. L110R MM-1α and wild-type MM-1α bound to regions of up-regulated and down-regulated genes, respectively. Since these gene products are known to play a role in maturation and motility of sperm, a defect of at least MM-1α transcriptional activity is thought to induce expressional changes of these genes, resulting in male infertility.

Prefoldin plays a role as a clearance factor in preventing proteasome inhibitor-induced protein aggregation.

Prefoldin is a molecular chaperone composed of six subunits, PFD1-6, and prevents misfolding of newly synthesized nascent polypeptides. Although it is predicted that prefoldin, like other chaperones, modulates protein aggregation, the precise function of prefoldin against protein aggregation under physiological conditions has never been elucidated. In this study, we first established an anti-prefoldin monoclonal antibody that recognizes the prefoldin complex but not its subunits. Using this antibody, it was found that prefoldin was localized in the cytoplasm with dots in co-localization with polyubiquitinated proteins and that the number and strength of dots were increased in cells that had been treated with lactacystin, a proteasome inhibitor, and thapsigargin, an inducer of endoplasmic reticulum stress. Knockdown of prefoldin increased the level of SDS-insoluble ubiquitinated protein and reduced cell viability in lactacystin and thapsigargin-treated cells. Opposite results were obtained in prefoldin-overexpressed cells. It has been reported that mice harboring a missense mutation L110R of MM-1α/PFD5 exhibit neurodegeneration in the cerebellum. Although the prefoldin complex containing L110R MM-1α was properly formed in vitro and in cells derived from L110R MM-1α mice, the levels of ubiquitinated proteins and cytotoxicity were higher in L110R MM-1α cells than in wild-type cells under normal conditions and were increased by lactacystin and thapsigargin treatment, and growth of L110R MM-1α cells was attenuated. Furthermore, the polyubiquitinated protein aggregation level was increased in the brains of L110R MM-1α mice. These results suggest that prefoldin plays a role in quality control against protein aggregation and that dysfunction of prefoldin is one of the causes of neurodegenerative diseases.