Cellular Traffic Jam and Disease Due to Mutations in SRP54

In this issue of Structure, Juaire et al. use X-ray crystallography, biophysical tools, and cell-based assays to investigate disease-associated variants of the SRP54 GTPase and to demonstrate that defects in SRP-mediated protein secretion can explain phenotypes of severe neutropenia with Shwachman-Diamond-syndrome-like symptoms. In this issue of Structure, Juaire et al. use X-ray crystallography, biophysical tools, and cell-based assays to investigate disease-associated variants of the SRP54 GTPase and to demonstrate that defects in SRP-mediated protein secretion can explain phenotypes of severe neutropenia with Shwachman-Diamond-syndrome-like symptoms. The advent of modern DNA sequencing technology has revolutionized modern biology and medicine, and it has become feasible, affordable, and commonplace to correlate biological phenotypes with diagnostic alterations of the genomic DNA in genome-wide association studies (GWASs). But correlation does not imply causation. To gain mechanistic insight and establish causal links, GWAS-type experiments need to be followed up by quantitative structure-activity relationship (QSAR) analyses of the molecules in question, which frequently are still painstakingly difficult and laborious to perform. In this issue of Structure, Juaire and coworkers present a beautiful example of such QSAR-type analysis. They investigate the effect of mutations in the SRP54 GTPase, which had previously been correlated to a rare type of human genetic disease, an autosomal dominant neutropenia with Shwachman-Diamond syndrome (SDS)-like symptoms (Carapito et al., 2017Carapito R. Konantz M. Paillard C. Miao Z. Pichot A. Leduc M.S. Yang Y. Bergstrom K.L. Mahoney D.H. Shardy D.L. et al.Mutations in signal recognition particle SRP54 cause syndromic neutropenia with Shwachman-Diamond-like features.J. Clin. Invest. 2017; 127: 4090-4103Crossref PubMed Scopus (66) Google Scholar; Bellanné-Chantelot et al., 2018Bellanné-Chantelot C. Schmaltz-Panneau B. Marty C. Fenneteau O. Callebaut I. Clauin S. Docet A. Damaj G.L. Leblanc T. Pellier I. et al.Mutations in the SRP54 gene cause severe congenital neutropenia as well as Shwachman-Diamond-like syndrome.Blood. 2018; 132: 1318-1331Crossref PubMed Scopus (44) Google Scholar). A neutropenia is characterized by a severely reduced number of neutrophil granulozytes (or neutrophils), a very frequent type of white blood cell charged to phagocytose pathogens as part of the innate immune response. SDS-like symptoms additionally include insufficient development of and secretion from the pancreas, as well as neurodevelopmental and skeletal disorders. Importantly, many of the symptoms could be explained by deficiencies in co-translational protein targeting to the endoplasmic reticulum, affecting both differentiation and maturation of the affected cell types from their precursors as well as final function (Carapito et al., 2017Carapito R. Konantz M. Paillard C. Miao Z. Pichot A. Leduc M.S. Yang Y. Bergstrom K.L. Mahoney D.H. Shardy D.L. et al.Mutations in signal recognition particle SRP54 cause syndromic neutropenia with Shwachman-Diamond-like features.J. Clin. Invest. 2017; 127: 4090-4103Crossref PubMed Scopus (66) Google Scholar; Bellanné-Chantelot et al., 2018Bellanné-Chantelot C. Schmaltz-Panneau B. Marty C. Fenneteau O. Callebaut I. Clauin S. Docet A. Damaj G.L. Leblanc T. Pellier I. et al.Mutations in the SRP54 gene cause severe congenital neutropenia as well as Shwachman-Diamond-like syndrome.Blood. 2018; 132: 1318-1331Crossref PubMed Scopus (44) Google Scholar). SRP54 is a highly conserved protein and the catalytic GTPase of the signal recognition particle (SRP). The SRP is a ribonucleoprotein particle composed of a highly structured RNA (7SL RNA) and six proteins (SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72). It is responsible for targeting mRNA-ribosome-nascent chain complexes to Sec61 membrane translocation pores at the endoplasmic reticulum, where SRP54 interacts with another, structurally very similar GTPase, SRα. This complex of SRP54 and SRα is called the targeting complex (TC). Upon GTP hydrolysis, the nascent chain of the secretory protein then enters the translocation pore for co-translational translocation, whereas the SRP dissociates and becomes available for another round of targeting (Grudnik et al., 2009Grudnik P. Bange G. Sinning I. Protein targeting by the signal recognition particle.Biol. Chem. 2009; 390: 775-782Crossref PubMed Scopus (114) Google Scholar; Akopian et al., 2013Akopian D. Shen K. Zhang X. Shan S.O. Signal recognition particle: an essential protein-targeting machine.Annu. Rev. Biochem. 2013; 82: 693-721Crossref PubMed Scopus (236) Google Scholar). SRP54 consists of three domains: N (N-terminal), G (GTP binding), and M (methionine rich). The M-domain assembles with the SRP in a defined orientation and participates in signal sequence recognition, whereas the complex of the N- and G-domains (also named NG-domain) remains tethered in a flexible fashion. Importantly, all of the mutations originally identified by Carapito et al., and subsequently also by Bellanné-Chantelot et al., map to the G-domain of SRP54. Having studied SRP structure and mechanism for more than two decades, the implication of SRP54 in human genetic disease prompted the Sinning lab to join forces with members of the Bahram lab to corroborate the causative nature of their three originally identified mutations (T115A, T117del, and G226E) and to obtain mechanistic insight that could explain the observed phenotypes. To this aim, they combined X-ray crystallography with complementary biophysical, biochemical, and cell-biological analyses. In a first step, Juaire et al. obtained a crystal structure of the targeting complex formed between the NG-domains of SRP54 and SRα in the presence of the GTP analog, GMPPNP (Figure 1, PDB: 6Y32). Furthermore, they obtained crystals of the isolated SRP54 NG-domain (PDB: 6Y2Z) as well as of the SRP54 NG variants T115A (PDB: 6Y30) and T117del (PDB: 6Y31), whereas the G226E variant did not crystallize. The structure of the complex is an important addition to the catalog of available SRP GTPase structures. It demonstrates the need of GTP binding for complex formation and illustrates the relative location of the investigated mutations in the G-domain. T115 is part of the P loop, a characteristic element in GTP-binding domains that fixes the bound GTP analog. The T117del variant lacks one of the three threonines in the T115-T116-T117 sequence with consequences for P-loop backbone conformation. In contrast, G226 is far from the GTP-binding site, in a loop contacting the N-domain, and at the interface with the G-domain of SRα (Figure 1). In agreement with these observations, the T115A and T117del variants lost their ability to bind GTP in microscale thermophoresis (MST) experiments, whereas the G226E variant showed an increased affinity for GTP. MST, which monitors the diffusion of a protein in a locally induced temperature gradient, is particularly sensitive to such small molecule binding. Nevertheless, the G226E variant also failed in TC formation. For all three variants, interaction with the SRα was tested in vitro by size exclusion chromatography combined with static light scattering, and in cells by the expression of the homologous variants from yeast in the context of a yeast two-hybrid experiment. Juaire et al. also noticed that the structures of both the T115A and the T117del variant display elevated crystallographic B-factors throughout portions of the G-domain, hinting at a more widespread destabilization of the molecules. This observation was confirmed by thermal melting experiments in solution using differential scanning fluorimetry (nano-DSF), where all three variants unfolded at a lower temperature than the wild-type SRP54 NG-domain. Importantly, however, the destabilization affected different portions of the G-domain in the T115/T117del and G226E variants, respectively. This could be concluded from hydrogen-deuterium exchange experiments followed by mass-spectrometry and where D2O-solvent-exposed protons on the surface of a protein are exchanged more rapidly than those in its core. Such observations are important, as they indicate that the mutations could regionally affect different functions of the SRP54 GTPase, eventually correlating with the strength and nature of the observed disease phenotypes. Finally, Juaire et al. also tested the SRP54 variants in living cells. In a first experiment, they used a yeast SRP54 deletion strain with a severe defect in growth and then supplemented the corresponding yeast homologs of the SRP54 variants. Only wild-type SRP54 could restore normal growth in this experiment, demonstrating that the variants are functionally insufficient to rescue the phenotype. A second experiment was conducted in human HEK293 cells, where human SRP54 or the respective variants were overexpressed, monitoring the secretion of a marker protein. Here, all three SRP54 variants appear to have a dominant-negative effect over the endogenous copies of the wild-type protein, jamming up the secretion of the reporter protein and leading to cytosolic mis-targeting and/or degradation of the respective mRNPs. In summary, Juaire et al. provide compelling support for the investigated SRP54 mutations to impair co-translational protein secretion and to cause the observed neutropenias with SDS-like symptoms. It is reasonable to speculate that certain SRP54 mutations simply cause a haplo-insufficiency, such as observed also for the general mRNA translation initiation factor eIF4E in certain cancer cells (Truitt et al., 2015Truitt M.L. Conn C.S. Shi Z. Pang X. Tokuyasu T. Coady A.M. Seo Y. Barna M. Ruggero D. Differential Requirements for eIF4E Dose in Normal Development and Cancer.Cell. 2015; 162: 59-71Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). In such a case, the cells and processes affected require a high dose of the respective protein, which cannot be provided by the single remaining functional copy of the gene. Other SRP54 mutations may cause a more dominant effect that cannot be compensated by the remaining wild-type copy of the gene, such as also observed for variants in the family of the small Ras-like GTPases causing cancer (Liu et al., 2017Liu W.N. Yan M. Chan A.M. A thirty-year quest for a role of R-Ras in cancer: from an oncogene to a multitasking GTPase.Cancer Lett. 2017; 403: 59-65Crossref PubMed Scopus (22) Google Scholar). For SRP54, this could be variants that primarily affect TC formation, but otherwise assemble normally into the SRP via the M-domain, leading to a depletion of functional SRP components and to an accumulation of stalled and/or mistargeted mRNPs that need to be cleared from the cytosol (Pinarbasi et al., 2018Pinarbasi E.S. Karamyshev A.L. Tikhonova E.B. Wu I.H. Hudson H. Thomas P.J. Pathogenic Signal Sequence Mutations in Progranulin Disrupt SRP Interactions Required for mRNA Stability.Cell Rep. 2018; 23: 2844-2851Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar; Lakshminarayan et al., 2020Lakshminarayan R. Phillips B.P. Binnian I.L. Gomez-Navarro N. Escudero-Urquijo N. Warren A.J. Miller E.A. Pre-emptive Quality Control of a Misfolded Membrane Protein by Ribosome-Driven Effects.Curr. Biol. 2020; 30: 854-864.e5Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Clearly, such dominant mutations would also cause more diverse phenotypes and possibly also stronger SDS-type symptoms. The tools described by Juaire et al., together with suitable animal models (zebrafish, as in Carapito et al., or mouse, as to be developed) should facilitate the analysis and also classification of future SRP54 variants. These are expected to be identified from patients or can be generated experimentally in analogy to disease-causing mutations in other GTPases, such as in the Ras-like family (Liu et al., 2017Liu W.N. Yan M. Chan A.M. A thirty-year quest for a role of R-Ras in cancer: from an oncogene to a multitasking GTPase.Cancer Lett. 2017; 403: 59-65Crossref PubMed Scopus (22) Google Scholar). Such analyses will not only further our understanding of human genetic disease, but also lead to a more fundamental and detailed mechanistic picture of the SRP-dependent co-translational protein translocation process. Juaire et al. are very careful to point out that each single mutation in SRP54 could have distinct and multiple effects throughout development and that the effects of each mutation are modulated by the remaining genetic background. Nevertheless, the continued progress in large-scale sequencing and data analysis will lead to an ever more fine-tuned classification of disease phenotypes according to their underlying genotype and will ultimately make it possible to design more and more personalized therapies based upon particular sets of diagnostic mutations (Ashley, 2016Ashley E.A. Towards precision medicine.Nat. Rev. Genet. 2016; 17: 507-522Crossref PubMed Scopus (300) Google Scholar). I wish to acknowledge all of the individuals who have contributed to a mechanistic understanding of the co-translational protein targeting pathway, as well as those investigating its physiological relevance for human disease and who remain uncited here due to space limitations. Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital NeutropeniaJuaire et al.StructureOctober 13, 2020In BriefJuaire et al. demonstrate that mutations in SRP54, which are linked to severe congenital neutropenia, lead to critically destabilized regions in SRP54 and increase overall protein flexibility. Furthermore, the mutations negatively impact GTP binding and complex formation with the SRP receptor, thereby severely affecting protein secretion by the SRP pathway. Full-Text PDF