The epididymal amyloid matrix: structure and putative functions

Quynh, Hoa; Hewetson, Aveline; Muthusubramanian, Archana; Myers, Caitlyn

doi:10.1111/andr.12586

Cited by 14 publications

(13 citation statements)

References 55 publications

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“…We hypothesize that cross-seeding may be conserved among other eukaryotic functional amyloid systems, as has been previously proposed for cystatin-related epididymal spermatogenic (CRES) proteins which form a functional amyloid matrix in the epididymal lumen of mice (32)(33)(34). While bacteria achieve nucleated polymerization by clustering minor and major amyloid subunits as individual genes within the same operon, we provide an example where the same mechanism is accomplished by alternative splicing.…”

Section: Discussionmentioning

confidence: 54%

Modulating functional amyloid formation via alternative splicing of the premelanosomal protein PMEL17

Dean

Lee

2020

Journal of Biological Chemistry

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The premelanosomal protein (PMEL17) forms functional amyloid fibrils involved in melanin biosynthesis. Multiple PMEL17 isoforms are produced, two of which arise from excision of a cryptic intron within the amyloid-forming repeat (RPT) domain, leading to long (lRPT) and short (sRPT) isoforms with 10 and 7 imperfect repeats, respectively. Both lRPT and sRPT isoforms undergo similar pH-dependent mechanisms of amyloid formation and fibril dissolution. Here, using human PMEL17, we tested the hypothesis that the minor, but more aggregation-prone, sRPT facilitates amyloid formation of lRPT. We observed that cross-seeding by sRPT fibrils accelerates the rate of lRPT aggregation, resulting in propagation of an sRPT-like twisted fibril morphology, unlike the rodlike structure that lRPT normally adopts. This templating was specific, as the reversed reaction inhibited sRPT fibril formation. Despite displaying ultrastructural differences, self- and cross-seeded lRPT fibrils had a similar β-sheet structured core, revealed by Raman spectroscopy, limited-proteolysis, and fibril disaggregation experiments, suggesting the fibril twist is modulated by N-terminal residues outside the amyloid core. Interestingly, bioinformatics analysis of PMEL17 homologs from other mammals uncovered that long and short RPT isoforms are conserved among members of this phylogenetic group. Collectively, our results indicate that the short isoform of RPT serves as a “nucleator” of PMEL17 functional amyloid formation, mirroring how bacterial functional amyloids assemble during biofilm formation. Whereas bacteria regulate amyloid assembly by using individual genes within the same operon, we propose that the modulation of functional amyloid formation in higher organisms can be accomplished through alternative splicing.

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Section: Discussionmentioning

confidence: 54%

Modulating functional amyloid formation via alternative splicing of the premelanosomal protein PMEL17

Dean

Lee

2020

Journal of Biological Chemistry

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“…epididymis to one rich in fibrils in the distal epididymis (18). The epididymal amyloid matrix contains several family 2 cystatins of cysteine protease inhibitors, including cystatin C and four members of the CRES (cystatin-related epididymal spermatogenic) subgroup, a reproductive subgroup of family 2 cystatins (10).…”

mentioning

confidence: 99%

Maturation of the functional mouse CRES amyloid from globular form

Hewetson

Khan

Dominguez

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The epididymal lumen contains a complex cystatin-rich nonpathological amyloid matrix with putative roles in sperm maturation and sperm protection. Given our growing understanding for the biological function of this and other functional amyloids, the problem still remains: how functional amyloids assemble including their initial transition to early oligomeric forms. To examine this, we developed a protocol for the purification of nondenatured mouse CRES, a component of the epididymal amyloid matrix, allowing us to examine its assembly to amyloid under conditions that may mimic those in vivo. Herein we use X-ray crystallography, solution-state NMR, and solid-state NMR to follow at the atomic level the assembly of the CRES amyloidogenic precursor as it progressed from monomeric folded protein to an advanced amyloid. We show the CRES monomer has a typical cystatin fold that assembles into highly branched amyloid matrices, comparable to those in vivo, by forming β-sheet assemblies that our data suggest occur via two distinct mechanisms: a unique conformational switch of a highly flexible disulfide-anchored loop to a rigid β-strand and by traditional cystatin domain swapping. Our results provide key insight into our understanding of functional amyloid assembly by revealing the earliest structural transitions from monomer to oligomer and by showing that some functional amyloid structures may be built by multiple and distinctive assembly mechanisms.

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“…Previous studies have shown that 8 genes ( Cstl1 - Cstdc2 ) are located contiguously with Cst3 on chromosome 2 and are strongly expressed in the male reproductive tract (17–19). Although CSTs are involved in the formation of the amyloid matrix that appears to function in maturation of spermatozoa in the epididymal lumen (20, 21), Cst8 or Cst9 single mutant male mice are fertile (22, 23). In this study, we generated mice lacking the entire male reproductive tract-specific CST family and examined the phenotypes of these cluster del mice.…”

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confidence: 99%

Identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice

Fujihara

Noda

Kobayashi

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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CRISPR/Cas9-mediated genome editing technology enables researchers to efficiently generate and analyze genetically modified animals. We have taken advantage of this game-changing technology to uncover essential factors for fertility. In this study, we generated knockouts (KOs) of multiple male reproductive organ-specific genes and performed phenotypic screening of these null mutant mice to attempt to identify proteins essential for male fertility. We focused on making large deletions (dels) within 2 gene clusters encoding cystatin (CST) and prostate and testis expressed (PATE) proteins and individual gene mutations in 2 other gene families encoding glycerophosphodiester phosphodiesterase domain (GDPD) containing and lymphocyte antigen 6 (Ly6)/Plaur domain (LYPD) containing proteins. These gene families were chosen because many of the genes demonstrate male reproductive tract-specific expression. AlthoughGdpd1andGdpd4mutant mice were fertile, disruptions ofCstandPategene clusters andLypd4resulted in male sterility or severe fertility defects secondary to impaired sperm migration through the oviduct. While absence of the epididymal protein families CST and PATE affect the localization of the sperm membrane protein A disintegrin and metallopeptidase domain 3 (ADAM3), the sperm acrosomal membrane protein LYPD4 regulates sperm fertilizing ability via an ADAM3-independent pathway. Thus, use of CRISPR/Cas9 technologies has allowed us to quickly rule in and rule out proteins required for male fertility and expand our list of male-specific proteins that function in sperm migration through the oviduct.

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The epididymal amyloid matrix: structure and putative functions

Cited by 14 publications

References 55 publications

Modulating functional amyloid formation via alternative splicing of the premelanosomal protein PMEL17

Modulating functional amyloid formation via alternative splicing of the premelanosomal protein PMEL17

Maturation of the functional mouse CRES amyloid from globular form

Identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice

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