Fibrillogenic Oligomers of Human Cystatin C Are Formed by Propagated Domain Swapping

Wahlbom, Maria; Wang, Xin; Lindström, Veronica; Carlemalm, Eric; Jaskólski, Mariusz; Grubb, Anders

doi:10.1074/jbc.m611368200

Cited by 118 publications

(143 citation statements)

References 46 publications

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“…Domain swapping (31,32) has been recognized as an aggregation mechanism for a number of proteins-for example, human prion protein (33) and β2-microglobulin (34). Aggregation by domain swapping in an open-ended fashion has been observed for cystatin C (35). Successive domain swapping of a single domain has been previously suggested as a mechanism for polymerization on the basis of the dimeric and/or trimeric structures of Serpin (36), RNAse A (37), and cytochrome C (38).…”

Section: Simulations Of Oligomerization Of Aggregation-prone Monomericmentioning

confidence: 95%

Aggregation of γ-crystallins associated with human cataracts via domain swapping at the C-terminal β-strands

Das

King

Zhou

2011

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

125

148

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The prevalent eye disease age-onset cataract is associated with aggregation of human γD-crystallins, one of the longest-lived proteins. Identification of the γ-crystallin precursors to aggregates is crucial for developing strategies to prevent and reverse cataract. Our microseconds of atomistic molecular dynamics simulations uncover the molecular structure of the experimentally detected aggregation-prone folding intermediate species of monomeric native γD-crystallin with a largely folded C-terminal domain and a mostly unfolded N-terminal domain. About 30 residues including a, b, and c strands from the Greek Key motif 4 of the C-terminal domain experience strong solvent exposure of hydrophobic residues as well as partial unstructuring upon N-terminal domain unfolding. Those strands comprise the domain-domain interface crucial for unusually high stability of γD-crystallin. We further simulate the intermolecular linkage of these monomeric aggregation precursors, which reveals domain-swapped dimeric structures. In the simulated dimeric structures, the N-terminal domain of one monomer is frequently found in contact with residues 135-164 encompassing the a, b, and c strands of the Greek Key motif 4 of the second molecule. The present results suggest that γD-crystallin may polymerize through successive domain swapping of those three C-terminal β-strands leading to age-onset cataract, as an evolutionary cost of its very high stability. Alanine substitutions of the hydrophobic residues in those aggregation-prone β-strands, such as L145 and M147, hinder domain swapping as a pathway toward dimerization. These findings thus provide critical molecular insights onto the initial stages of age-onset cataract, which is important for understanding protein aggregation diseases.

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Section: Simulations Of Oligomerization Of Aggregation-prone Monomericmentioning

confidence: 95%

Aggregation of γ-crystallins associated with human cataracts via domain swapping at the C-terminal β-strands

Das

King

Zhou

2011

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

125

148

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“…Interestingly, cystatin C itself also self-aggregates and forms amyloid fibrils in vitro suggesting that the normal functioning of cystatin C may be in its aggregated state (Wahlbom et al, 2007). Similar to cystatin C, CRES is aggregation-prone and will self-assemble into oligomeric and fibrillar amyloid structures (von Horsten et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Cystatin-related epididymal spermatogenic subgroup members are part of an amyloid matrix and associated with extracellular vesicles in the mouse epididymal lumen

et al. 2016

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STUDY QUESTION: Do the CRES (cystatin-related epididymal spermatogenic) subgroup members, including CRES2, CRES3 and cystatin E2, contribute to the formation of a nonpathological, functional amyloid matrix in the mouse epididymal lumen?SUMMARY ANSWER: CRES2, CRES3 and cystatin E2 self-assemble with different aggregation properties into amyloids in vitro, are part of a common amyloid matrix in the mouse epididymal lumen and are present in extracellular vesicles.WHAT IS KNOWN ALREADY: Although previously thought only to be pathological, accumulating evidence has established that amyloids, which are highly ordered protein aggregates, can also carry out functional roles in the absence of pathology. We previously demonstrated that nonpathological amyloids are present in the epididymis; specifically, that the reproductive cystatin CRES forms amyloid and is present in the mouse epididymal lumen in a film-like amyloid matrix that is intimately associated with spermatozoa. Because the related proteins CRES2, CRES3 and cystatin E2 are also expressed in the epididymis, the present studies were carried out to determine if these proteins are also amyloidogenic in vitro and in vivo and thus may coordinately function with CRES as an amyloid structure. STUDY DESIGN, SAMPLES/MATERIALS, METHODS:The epididymides from CD1 and Cst8 (CRES)129SvEv/B6 gene knockout (KO) and wild-type mice and antibodies that specifically recognize each CRES subgroup member were used for immunohistochemical and biochemical analyzes of CRES subgroup proteins. Methods classically used to identify amyloid, including the conformation-dependent dyes thioflavin S (ThS) and thioflavin T (ThT), conformation-dependent antibodies, protein aggregation disease ligand (which binds any amyloid independent of sequence) and negative stain electron microscopy (EM) were carried out to examine the amyloidogenic properties of CRES subgroup members. Immunofluorescence analysis and confocal microscopy were used for colocalization studies. MAIN RESULTS AND THE ROLE OF CHANCE:Immunoblot and immunofluorescence analyzes showed that CRES2, CRES3 and cystatin E2 were primarily found in the initial segment and intermediate zone of the epididymis and were profoundly downregulated in epididymides from CRES KO mice, suggesting integrated functions. Except for CRES3, which was only detected in a particulate form, proteins were present in the epididymal lumen in both soluble and particulate forms including in a film-like matrix and in extracellular vesicles. The use of amyloid-specific reagents determined that all CRES subgroup members were present as amyloids and colocalized to a common amyloid matrix present in the epididymal lumen. Negative stain EM, dot blot analysis and ThT plate assays showed that recombinant CRES2, CRES3 and cystatin E2 formed amyloid in vitro, albeit with different aggregation properties. Together, our studies demonstrate that a unique amyloid matrix composed of the CRES family of reproductive-specific cystatins and cystatin C is a normal component of the mouse ...

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“…To resolve the ambiguity of HCC fibril formation, which could proceed according to scenario number 2 or 3, a set of experiments has been conducted with control of redox conditions (Wahlbom et al, 2007). First, we note that it is possible to detect the presence of highorder oligomers using SDS-PAGE electrophoresis.…”

Section: D Domain Swapping and Amyloid Fibril Formation Of Hccmentioning

confidence: 99%

3D domain swapping – implications for conformational disorders and ways of control

Jaskólski¹

2011

bta

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Abstract3D Domain swapping is a mechanism of protein aggregation, in which a structural element of a protein fold is replaced by an identical element from another subunit. Some proteins, for instance RNase A, can assume many domain-swapped forms, thus undermining the dogma, "one sequence -one structure" in a particularly spectacular way. Completed in a mutual fashion, it is a mechanism of protein oligomerization. In an open-ended fashion, 3D domain swapping could be a mechanism of amyloid fibril formation. In another mechanism, possibly operating together with domain swapping, a specific sequence, such as a glutamine expansion, could form a β-spine of the fibril in a motif called steric zipper. The first connection between 3D domain swapping and amyloidogenicity was established in human cystatin C (HCC), the second -in the prion protein (PrP). In both cases, a disulfide bridge (natural in PrP, engineered in HCC) can be used for redox control of 3D domain swapping and to demonstrate that it is indeed involved in amyloid fibril formation. HCC has a naturally occurring L68Q mutant with drastically increased propensity for aggregation. The L68Q mutation occurs at the closed interface, or protein core. Mutations in other areas, such as the flexible hinge (especially deletions and insertions) can also be used to control 3D domain swapping and aggregation. Paradoxically, 3D domain swapping can also be used by Nature for prevention of nucleation processes that lead to high-order aggregates or crystals. Such a situation exists in the eye lens, where despite astronomical concentration of crystallins, the solution remains clear. One of the Nature's tricks to achieve polydispersity is to use a palindromic sequence for the swapped domain, thereby frustrating the growth of aggregates by constantly changing the interaction topology.

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Fibrillogenic Oligomers of Human Cystatin C Are Formed by Propagated Domain Swapping

Cited by 118 publications

References 46 publications

Aggregation of γ-crystallins associated with human cataracts via domain swapping at the C-terminal β-strands

Aggregation of γ-crystallins associated with human cataracts via domain swapping at the C-terminal β-strands

Cystatin-related epididymal spermatogenic subgroup members are part of an amyloid matrix and associated with extracellular vesicles in the mouse epididymal lumen

3D domain swapping – implications for conformational disorders and ways of control

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