The Functional Mammalian CRES (Cystatin-Related Epididymal Spermatogenic) Amyloid is Antiparallel β-Sheet Rich and Forms a Metastable Oligomer During Assembly
Abstract:An amyloid matrix composed of several family 2 cystatins, including the reproductive cystatin CRES, is an integral structure in the mouse epididymal lumen and has proposed functions in sperm maturation and protection. Understanding how CRES amyloid assembles
in vitro
may provide clues on how the epididymal amyloid matrix forms
in vivo
. We therefore purified full-length CRES under nondenaturing conditions and followed its aggregation from monomer to amyloid under c… Show more
“…The former has been shown to promote unilateral nucleation of IAPP, albeit with a lower efficiency compared to homologous assembly, whereby these proteins harbour stretches sharing sequence homology [172]. Several additional proteins have been associated to templated amyloid cross-talk via sharing of common structural architectures [161,[173][174][175], with the list expanding even to cases of functional amyloids [176][177][178][179][180][181][182]. Finally, cross-seeding and heterotypic co-aggregation has also been demonstrated in cancer-associated aggregation pathologies, where mutant p53 causes sequence-dependent co-deposition of its homologs p63 and p73 by virtue of their similar APRs [183][184][185].…”
Section: Is Sequence Specificity a Determining Factor In Amyloid Hetementioning
Amyloid aggregation results from the self‐assembly of identical aggregation‐prone sequences into cross‐beta‐sheet structures. The process is best known for its association with a wide range of human pathologies but also as a functional mechanism in all kingdoms of life. Less well elucidated is the role of heterotypic interactions between amyloids and other proteins and macromolecules and how this contributes to disease. We here review current data with a focus on neurodegenerative amyloid‐associated diseases. Evidence indicates that heterotypic interactions occur in a wide range of amyloid processes and that these interactions modify fundamental aspects of amyloid aggregation including seeding, aggregation rates and toxicity. More work is required to understand the mechanistic origin of these interactions, but current understanding suggests that both supersaturation and sequence‐specific binding can contribute to heterotypic amyloid interactions. Further unravelling these mechanisms may help to answer outstanding questions in the field including the selective vulnerability of cells types and tissues and the stereotypical spreading patterns of amyloids in disease.
“…The former has been shown to promote unilateral nucleation of IAPP, albeit with a lower efficiency compared to homologous assembly, whereby these proteins harbour stretches sharing sequence homology [172]. Several additional proteins have been associated to templated amyloid cross-talk via sharing of common structural architectures [161,[173][174][175], with the list expanding even to cases of functional amyloids [176][177][178][179][180][181][182]. Finally, cross-seeding and heterotypic co-aggregation has also been demonstrated in cancer-associated aggregation pathologies, where mutant p53 causes sequence-dependent co-deposition of its homologs p63 and p73 by virtue of their similar APRs [183][184][185].…”
Section: Is Sequence Specificity a Determining Factor In Amyloid Hetementioning
Amyloid aggregation results from the self‐assembly of identical aggregation‐prone sequences into cross‐beta‐sheet structures. The process is best known for its association with a wide range of human pathologies but also as a functional mechanism in all kingdoms of life. Less well elucidated is the role of heterotypic interactions between amyloids and other proteins and macromolecules and how this contributes to disease. We here review current data with a focus on neurodegenerative amyloid‐associated diseases. Evidence indicates that heterotypic interactions occur in a wide range of amyloid processes and that these interactions modify fundamental aspects of amyloid aggregation including seeding, aggregation rates and toxicity. More work is required to understand the mechanistic origin of these interactions, but current understanding suggests that both supersaturation and sequence‐specific binding can contribute to heterotypic amyloid interactions. Further unravelling these mechanisms may help to answer outstanding questions in the field including the selective vulnerability of cells types and tissues and the stereotypical spreading patterns of amyloids in disease.
“…1 C ). This observation, however, was not unexpected based on prior CD, FTIR, X-ray crystallization, and NMR studies of the related CRES that also revealed an antiparallel β-sheet-rich structure ( 25 , 26 ). Figure 1 CRES3 forms SDS-sensitive and SDS-resistant aggregates.…”
Section: Resultsmentioning
confidence: 75%
“…We next determined if the CRES3 seeds could template assembly of the related amyloidogenic precursor CRES. Because CRES is less aggregation-prone than CRES3 and other subgroup members, in previous studies we were able to isolate a tagless full-length mouse CRES C48A under nondenaturing conditions from the soluble fraction of bacteria and follow its assembly to amyloid ( 25 ). The free cysteine (C) at position 48 was mutated to alanine (A) to prevent improper disulfide bond formation during purification.…”
Section: Resultsmentioning
confidence: 99%
“…The free cysteine (C) at position 48 was mutated to alanine (A) to prevent improper disulfide bond formation during purification. These studies showed that under physiological conditions, CRES C48A rapidly assembled from its natively folded monomer into an early oligomer that, if kept at high concentrations, remained stable ( 25 ). Only with extended time oligomers slowly transitioned into higher-ordered amyloids ( 25 ).…”
Section: Resultsmentioning
confidence: 99%
“…These studies showed that under physiological conditions, CRES C48A rapidly assembled from its natively folded monomer into an early oligomer that, if kept at high concentrations, remained stable ( 25 ). Only with extended time oligomers slowly transitioned into higher-ordered amyloids ( 25 ). From these studies we hypothesized that in vivo the CRES oligomer may function as a stable building block for the assembly of the highly branched and elaborate amyloid matrix that is present in the mouse caput epididymal lumen.…”
Accumulating evidence shows that amyloids perform biological roles. We previously showed that an amyloid matrix composed of four members of the CRES subgroup of reproductive family 2 cystatins is a normal component of the mouse epididymal lumen. The cellular mechanisms that control the assembly of these and other functional amyloid structures, however, remain unclear. We speculated that cross-seeding between CRES members could be a mechanism to control the assembly of the endogenous functional amyloid. Herein we used thioflavin T assays and negative stain transmission electron microscopy to explore this possibility. We show that CRES3 rapidly formed large networks of beaded chains that possessed the characteristic cross-β reflections of amyloid when examined by X-ray diffraction. The beaded amyloids accelerated the amyloidogenesis of CRES, a less amyloidogenic family member, in seeding assays during which beads transitioned into films and fibrils. Similarly, CRES seeds expedited CRES3 amyloidogenesis, although less efficiently than the CRES3 seeding of CRES. These studies suggest that CRES and CRES3 hetero-oligomerize and that CRES3 beaded amyloids may function as stable preassembled seeds. The CRES3 beaded amyloids also facilitated assembly of the unrelated amyloidogenic precursor Aβ by providing a surface for polymerization though, intriguingly, CRES3 (and CRES) monomer/early oligomer profoundly inhibited Aβ assembly. The cross-seeding between the CRES subgroup members is similar to that which occurs between bacterial curli proteins suggesting that it may be an evolutionarily conserved mechanism to control the assembly of some functional amyloids. Further, interactions between unrelated amyloidogenic precursors may also be a means to regulate functional amyloid assembly.
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