A novel high-throughput screening strategy for targeting alpha-synuclein and other long-lived proteins.

Small-molecule high-throughput screening (HTS) campaigns have frequently been used to identify lead molecules that can alter expression of disease-relevant proteins in cell-based assays. However, most cell-based HTS assays require short compound exposure periods to avoid toxicity and ensure that compounds are stable in media for the duration of the exposure. This limits the ability of HTS assays to detect inhibitors of the synthesis of target proteins with long half-lives, which can often exceed the exposure times utilized in most HTS campaigns. One such target is alpha-synuclein (α-syn)-a protein well-known for its pathological aggregation in Parkinson's Disease (PD) and other forms of neurodegeneration known collectively as synucleinopathies. Here, we report the development of an HTS assay using a CRISPR-engineered neuroblastoma cell line expressing a destabilized luciferase reporter inserted at the end of the coding region of the SNCA locus. The resultant destabilized fusion protein exhibited a significant reduction in half-life compared to the endogenous, unmodified α-syn protein, and accurately reported reductions in α-syn levels due to known protein translation inhibitors and specific α-syn siRNAs. The robustness and utility of this approach was shown by using the resulting cell line (dsLuc-Syn) to screen a focused library of 3,192 compounds for reduction of α-syn. These data demonstrate the general utility of converting endogenous loci into destabilized reporter genes capable of identifying inhibitors of gene expression of highly stable proteins even in short-term assays.

¹H, ¹³C and ¹5N resonance assignments of the complement control protein modules of the complement component C7.

Human C7 is one of four homologous complement proteins that self-assemble on the nascent activation-specific fragment, C5b, thus forming the cytolytic membrane attack complex (MAC). In addition to the conserved modular core of the MAC/perforin protein family, C7 has four C-terminal domains comprising a pair of complement control protein modules (CCPs) preceding two Factor-I like modules (FIMs). It is proposed that the C7-CCPs might serve as a molecular arm for delivery of C7-FIMs to their binding site on C5b. Here we present the NMR chemical shift assignments for the C7-CCPs produced as a 14-kDa recombinant protein. Based upon triple-resonance experiments, 98 and 94 % of the backbone and side-chain ((1)H, (13)C and (15)N) assignments, respectively, have been completed. The chemical shifts and assignments have been deposited in the BioMagResBank database under accession number 18530.

1H, 15N and 13C resonance assignment of the pair of Factor-I like modules of the complement protein C7.

The carboxy terminus of human complement component C7 comprises two Factor I-like Modules (FIMs) which are essential for formation of the Membrane Attack Complex, the terminal pathway of the innate immune system. C7-FIMs is a 16.9 kDa, recombinant, disulphide-rich, protein encompassing this C-terminal domain. Using conventional triple resonance experiments 93% of the (1)H, (15)N and (13)C assignment has been achieved, accounting for all assignment apart from a flexible N-terminus cloning artefact and an undefined loop. The chemical shifts have been deposited in the BioMagResBank; Accession No. 15996.

Solution structure of factor I-like modules from complement C7 reveals a pair of follistatin domains in compact pseudosymmetric arrangement.

Factor I-like modules (FIMs) of complement proteins C6, C7, and factor I participate in protein-protein interactions critical to the progress of a complement-mediated immune response to infections and other trauma. For instance, the carboxyl-terminal FIM pair of C7 (C7-FIMs) binds to the C345C domain of C5 and its activated product, C5b, during self-assembly of the cytolytic membrane-attack complex. FIMs share sequence similarity with follistatin domains (FDs) of known three-dimensional structure, suggesting that FIM structures could be reliably modeled. However, conflicting disulfide maps, inconsistent orientations of subdomains within FDs, and the presence of binding partners in all FD structures led us to determine the three-dimensional structure of C7-FIMs by NMR spectroscopy. The solution structure reveals that each FIM within C7 contains a small amino-terminal FOLN subdomain connected to a larger carboxyl-terminal KAZAL domain. The open arrangement of the subdomains within FIMs resembles that of first FDs within structures of tandem FDs but differs from the more compact subdomain arrangement of second or third FDs. Unexpectedly, the two C7-FIMs pack closely together with an approximate 2-fold rotational symmetry that is rarely seen in module pairs and has not been observed in FD-containing proteins. Interfaces between subdomains and between modules include numerous hydrophobic and electrostatic contributions, suggesting that this is a physiologically relevant conformation that persists in the context of the parent protein. Similar interfaces were predicted in a homology-based model of the C6-FIM pair. The C7-FIM structures also facilitated construction of a model of the single FIM of factor I.

Recombinant C345C and factor I modules of complement components C5 and C7 inhibit C7 incorporation into the complement membrane attack complex.

Complement component C5 binds to components C6 and C7 in reversible reactions that are distinct from the essentially nonreversible associations that form during assembly of the complement membrane attack complex (MAC). We previously reported that the approximately 150-aa residue C345C domain (also known as NTR) of C5 mediates these reversible reactions, and that the corresponding recombinant module (rC5-C345C) binds directly to the tandem pair of approximately 75-residue factor I modules from C7 (C7-FIMs). We suggested from these and other observations that binding of the C345C module of C5 to the FIMs of C7, but not C6, is also essential for MAC assembly itself. The present report describes a novel method for assembling a complex that appears to closely resemble the MAC on the sensor chip of a surface plasmon resonance instrument using the complement-reactive lysis mechanism. This method provides the ability to monitor individually the incorporation of C7, C8, and C9 into the complex. Using this method, we found that C7 binds to surface-bound C5b,6 with a K(d) of approximately 3 pM, and that micromolar concentrations of either rC5-C345C or rC7-FIMs inhibit this early step in MAC formation. We also found that similar concentrations of either module inhibited complement-mediated erythrocyte lysis by both the reactive lysis and classical pathway mechanisms. These results demonstrate that the interaction between the C345C domain of C5 and the FIMs of C7, which mediates reversible binding of C5 to C7 in solution, also plays an essential role in MAC formation and complement lytic activity.

Functional insights from the structure of the multifunctional C345C domain of C5 of complement.

The complement protein C5 initiates assembly of the membrane attack complex. This remarkable process results in lysis of target cells and is fundamental to mammalian defense against infection. The 150-amino acid residue domain at the C terminus of C5 (C5-C345C) is pivotal to C5 function. It interacts with enzymes that convert C5 to C5b, the first step in the assembly of the membrane attack complex; it also binds to the membrane attack complex components C6 and C7 with high affinity. Here a recombinant version of this C5-C345C domain is shown to adopt the oligosaccharide/oligonucleotide binding fold, with two helices packed against a five-stranded beta-barrel. The structure is compared with those from the netrin-like module family that have a similar fold. Residues critical to the interaction with C5-convertase cluster on a mobile, hydrophobic inter-strand loop that protrudes from the open face of the beta-barrel. The opposite, helix-dominated face of C5-C345C carries a pair of exposed hydrophobic side chains adjacent to a striking negatively charged patch, consistent with affinity for positively charged factor I modules in C6 and C7. Modeling of homologous domains from complement proteins C3 and C4, which do not participate in membrane attack complex assembly, suggests that this provisionally identified C6/C7-interacting face is indeed specific to C5.

Complement components C5 and C7: recombinant factor I modules of C7 bind to the C345C domain of C5.

Studies reported over 30 years ago revealed that latent, nonactivated C5 binds specifically and reversibly to C6 and C7. These reversible reactions are distinct from the essentially nonreversible associations with activated C5b that occur during assembly of the membrane attack complex, but they likely involve some, perhaps many, of the same molecular contacts. We recently reported that these reversible reactions are mediated by the C345C (NTR) domain at the C terminus of the C5 alpha-chain. Earlier work by others localized the complementary binding sites to a tryptic fragment of C6 composed entirely of two adjacent factor I modules (FIMs), and to a larger fragment of C7 composed of its homologous FIMs as well as two adjoining short consensus repeat modules. In this work, we expressed the tandem FIMs from C7 in bacteria. The mobility on SDS-polyacrylamide gels, lack of free sulfhydryl groups, and atypical circular dichroism spectrum of the recombinant product rC7-FIMs were all consistent with a native structure. Using surface plasmon resonance, we found that rC7-FIMs binds specifically to both C5 and the rC5-C345C domain with K(D) approximately 50 nM, and competes with C7 for binding to C5, as expected for an active domain. These results indicate that, like C6, the FIMs alone in C7 mediate reversible binding to C5. Based on available evidence, we suggest a model for an irreversible membrane attack complex assembly in which the C7 FIMs, but not those in C6, are bound to the C345C domain of C5 within the fully assembled complex.

Expression and characterization of the C345C/NTR domains of complement components C3 and C5.

Complement components C3, C4, and C5 are members of the thioester-containing alpha-macroglobulin protein superfamily. Within this superfamily, a unique feature of the complement proteins is a 150-residue-long C-terminal extension of their alpha-subunits that harbors three internal disulfide bonds. Previous reports have suggested that this is an independent structural module, homologous to modules found in other proteins, including netrins and tissue inhibitors of metalloproteinases. Because of its distribution, this putative module has been named both C345C and NTR. To assess the structures of these segments of the complement proteins, their relationships with other domains, and activities as independent structures, we expressed C345C from C3 and C5 in a bacterial strain that permits cytoplasmic disulfide bond formation. Affinity purification directly from cell lysates yielded recombinant C3- and C5-C345C with properties consistent with multiple intramolecular disulfide bonds and high beta-sheet contents. rC5-, but not rC3-C345C inhibited complement hemolytic activity, and surface plasmon resonance studies revealed that rC5-C345C binds to complement components C6 and C7 with dissociation constants of 10 and 3 nM, respectively. Our results provide strong evidence that this binding corresponds to the previously described reversible binding of C5 to C6 and C7, and taken together with earlier work, indicate that the C5-C345C module interacts directly with the factor I modules in C6 and C7. The high binding affinities suggest that complexes composed of C5 bound to C6 or C7 exist in plasma before activation and may facilitate assembly of the complement membrane attack complex.