110 results on '"Gambin, Y"'
Search Results
2. Lateral Mobility of Proteins in Liquid Membranes Revisited
- Author
-
Gambin, Y., Lopez-Esparza, R., Reffay, M., Sierecki, E., Gov, N. S., Genest, M., Hodges, R. S., and Urbach, W.
- Published
- 2006
- Full Text
- View/download PDF
3. A robust method for particulate detection of a genetic tag for 3D electron microscopy
- Author
-
Rae, J, Ferguson, C, Ariotti, N, Webb, RI, Cheng, HH, Mead, JL, Riches, JD, Hunter, DJB, Martel, N, Baltos, J, Christopoulos, A, Bryce, NS, Cagigas, ML, Fonseka, S, Sayre, ME, Hardeman, EC, Gunning, PW, Gambin, Y, Hall, TE, Parton, RG, Rae, J, Ferguson, C, Ariotti, N, Webb, RI, Cheng, HH, Mead, JL, Riches, JD, Hunter, DJB, Martel, N, Baltos, J, Christopoulos, A, Bryce, NS, Cagigas, ML, Fonseka, S, Sayre, ME, Hardeman, EC, Gunning, PW, Gambin, Y, Hall, TE, and Parton, RG
- Abstract
Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/ gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.
- Published
- 2021
4. Herpes simplex virus encoded ICP6 protein forms functional amyloid assemblies with necroptosis-associated host proteins
- Author
-
Shanmugam, N, Baker, MODG, Sanz-Hernandez, M, Sierecki, E, Gambin, Y, Steain, M, Pham, CLL, Sunde, M, Shanmugam, N, Baker, MODG, Sanz-Hernandez, M, Sierecki, E, Gambin, Y, Steain, M, Pham, CLL, and Sunde, M
- Abstract
The viral protein ICP6, encoded by herpes simplex virus 1 (HSV-1), harbours a RIP-homotypic interaction motif (RHIM), that plays a role in viral inhibition of host cell death pathways. Other members of the Herpesviridae family also encode RHIM-containing proteins that interfere with host-cell death pathways, including the M45 protein from murine cytomegalovirus, and ORF20 protein from varicella zoster virus. We have used amyloid assembly assays, electron microscopy and single molecule fluorescence spectroscopy to show that the ICP6 RHIM is amyloidogenic and can interact with host RHIM-containing proteins to form heteromeric amyloid complexes, in a manner similar to that of M45 and ORF20 RHIMs. The core tetrad sequence of the ICP6 RHIM is important for both amyloid formation and interaction with host RHIM-containing proteins. Notably, we show that the amyloid forming capacity of the ICP6 RHIM is affected by the redox environment. We propose that the formation of an intramolecular disulfide bond within ICP6 triggers the formation of amyloid assemblies that are distinct from previously characterised viral amyloids M45 and ORF20. Formation of viral-host heteromeric amyloid assemblies may underlie a general mechanism of viral adaptation against host immune machineries.
- Published
- 2021
5. Rapid HIV-1 capsid interaction screening using fluorescence fluctuation spectroscopy
- Author
-
Böcking, T, Jacques, DA, Lau, D, Walsh, JC, Dickson, CF, Tuckwell, A, Stear, JH, Hunter, DJB, Bhumkar, A, Shah, V, Turville, SG, Sierecki, E, Gambin, Y, Böcking, T, Jacques, DA, Lau, D, Walsh, JC, Dickson, CF, Tuckwell, A, Stear, JH, Hunter, DJB, Bhumkar, A, Shah, V, Turville, SG, Sierecki, E, and Gambin, Y
- Abstract
The HIV capsid is a multifunctional protein capsule that mediates the delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here, we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labeled capsid-binding analytes (“prey” molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using the fluorescent capsid as the bait further allows the quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for the discovery and characterization of molecules used by the HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays, utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms.
- Published
- 2021
6. Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response
- Author
-
Mcmahon, K-A, Stroud, DA, Gambin, Y, Tillu, V, Bastiani, M, Sierecki, E, Polinkovsky, ME, Hall, TE, Gomez, GA, Wu, Y, Parat, M-O, Martel, N, Lo, HP, Khanna, KK, Alexandrov, K, Daly, R, Yap, A, Ryan, MT, Parton, RG, Mcmahon, K-A, Stroud, DA, Gambin, Y, Tillu, V, Bastiani, M, Sierecki, E, Polinkovsky, ME, Hall, TE, Gomez, GA, Wu, Y, Parat, M-O, Martel, N, Lo, HP, Khanna, KK, Alexandrov, K, Daly, R, Yap, A, Ryan, MT, and Parton, RG
- Abstract
Caveolae-associated protein 3 (cavin3) is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair, with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays revealed a direct interaction between BRCA1 and cavin3 that occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. Supporting a role in DNA repair, cavin3-deficient cells were sensitive to PARP inhibition, where concomitant depletion of 53BP1 restored BRCA1-dependent sensitivity to PARP inhibition. We conclude that cavin3 functions together with BRCA1 in multiple cancer-related pathways. The loss of cavin3 function may provide tumor cell survival by attenuating apoptotic sensitivity and hindering DNA repair under chronic stress conditions.
- Published
- 2021
7. A dominant-negative SOX18 mutant disrupts multiple regulatory layers essential to transcription factor activity
- Author
-
McCann, AJ, Lou, J, Moustaqil, M, Graus, MS, Blum, A, Fontaine, F, Liu, H, Luu, W, Rudolffi-Soto, P, Koopman, P, Sierecki, E, Gambin, Y, Meunier, FA, Liu, Z, Hinde, E, Francois, M, McCann, AJ, Lou, J, Moustaqil, M, Graus, MS, Blum, A, Fontaine, F, Liu, H, Luu, W, Rudolffi-Soto, P, Koopman, P, Sierecki, E, Gambin, Y, Meunier, FA, Liu, Z, Hinde, E, and Francois, M
- Abstract
Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function.
- Published
- 2021
8. Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death
- Author
-
Steain, M, Baker, MODG, Pham, CLL, Shanmugam, N, Gambin, Y, Sierecki, E, McSharry, BP, Avdic, S, Slobedman, B, Sunde, M, Abendroth, A, Steain, M, Baker, MODG, Pham, CLL, Shanmugam, N, Gambin, Y, Sierecki, E, McSharry, BP, Avdic, S, Slobedman, B, Sunde, M, and Abendroth, A
- Abstract
Herpesviruses are known to encode a number of inhibitors of host cell death, including RIP Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein, open reading frame (ORF) 20, that acts as a host cell death inhibitor. Like the human cellular RHIMs in RIPK1 and RIPK3 that stabilise the necrosome in TNF-induced necroptosis, and the viral RHIM in M45 from murine cytomegalovirus that inhibits cell death, the ORF20 RHIM is capable of forming fibrillar functional amyloid complexes. Notably, the ORF20 RHIM forms hybrid amyloid complexes with human ZBP1, a cytoplasmic sensor of viral nucleic acid. Although VZV can inhibit TNF-induced necroptosis, the ORF20 RHIM does not appear to be responsible for this inhibition. In contrast, the ZBP1 pathway is identified as important for VZV infection. Mutation of the ORF20 RHIM renders the virus incapable of efficient spread in ZBP1- expressing HT-29 cells, an effect which can be reversed by the inhibition of caspases. Therefore we conclude that the VZV ORF20 RHIM is important for preventing ZBP1-driven apoptosis during VZV infection, and propose that it mediates this effect by sequestering ZBP1 into decoy amyloid assemblies.
- Published
- 2020
9. Competing Interactions between Viral RHIM Amyloid-forming Proteins and Host Functional Amyloid Structures Modulate the Cellular Response to Infection
- Author
-
Sunder, M, Pham, CLL, Shanmugam, N, Baker, MODG, Steain, M, O'Carroll, A, Brown, JW, Sierecki, E, Gambin, Y, Sunder, M, Pham, CLL, Shanmugam, N, Baker, MODG, Steain, M, O'Carroll, A, Brown, JW, Sierecki, E, and Gambin, Y
- Published
- 2020
10. An inverted CAV1 (caveolin 1) topology defines novel autophagy-dependent exosome secretion from prostate cancer cells
- Author
-
Ariotti, N, Wu, Y, Okano, S, Gambin, Y, Follett, J, Rae, J, Ferguson, C, Teasdale, RD, Alexandrov, K, Meunier, FA, Hill, MM, Parton, RG, Ariotti, N, Wu, Y, Okano, S, Gambin, Y, Follett, J, Rae, J, Ferguson, C, Teasdale, RD, Alexandrov, K, Meunier, FA, Hill, MM, and Parton, RG
- Abstract
CAV1 (caveolin 1) expression and secretion is associated with prostate cancer (PCa) disease progression, but the mechanisms underpinning CAV1 release remain poorly understood. Numerous studies have shown CAV1 can be secreted within exosome-like vesicles, but antibody-mediated neutralization can mitigate PCa progression; this is suggestive of an inverted (non-exosomal) CAV1 topology. Here we show that CAV1 can be secreted from specific PCa types in an inverted vesicle-associated form consistent with the features of bioactive CAV1 secretion. Characterization of the isolated vesicles by electron microscopy, single-molecule fluorescence microscopy and proteomics reveals they represent a novel class of exosomes ~40 nm in diameter containing ~50-60 copies of CAV1 and, strikingly, are released via a non-canonical secretory macroautophagy/autophagy pathway. This study provides novel insights into a mechanism whereby CAV1 translocates from a normal plasma membrane distribution to an inverted secreted form implicated in PCa disease progression. Abbreviations: 3-MA: 3-methyladenine; APEX: a modified soybean ascorbate peroxidase; ATG5: autophagy related 5; ATG9A: autophagy related 9A; ATG12: autophagy related 12; BHK: baby hamster kidney; C-exosomes: caveolin-exosomes; CAMKK2/CAMKKβ: calckum/calmodulin dependent protein kinase kinase 2; CAV1: caveolin 1; DAB: 3,3′-diaminobenzidine; DAPK: death associated protein kinase; EEA1: early endosome antigen 1; EM: electron microscopy; FCS: fluorescence correlation spectroscopy; GBP: GFP/YFP-binding peptide; GFP: green fluorescent protein; GOLGA2: golgin A2; ILVs: intralumenal vesicles; LC3: microtubule-associated protein 1 light chain 3; MBP: maltose binding protein; MTORC1: mechanistic target of rapamycin kinase complex 1; MVBs: multivesicular bodies; PBS: phosphate-buffered saline; PCa: prostate cancer; PI3K: phosphoinositide 3-kinase; PM: plasma membrane; SFM: serum-free medium; TSG101: tumor susceptibility 101; WCL: whole cell lysat
- Published
- 2020
11. SFX structure of the MyD88 TIR domain higher-order assembly
- Author
-
Clabbers, M.T.B., primary, Holmes, S., additional, Muusse, T.W., additional, Vajjhala, P., additional, Thygesen, S.J., additional, Malde, A.K., additional, Hunter, D.J.B., additional, Croll, T.I., additional, Flueckiger, L., additional, Nanson, J.D., additional, Rahaman, M.H., additional, Aquila, A., additional, Hunter, M.S., additional, Liang, M., additional, Yoon, C.H., additional, Zhao, J., additional, Zatsepin, N.A., additional, Abbey, B., additional, Sierecki, E., additional, Gambin, Y., additional, Stacey, K.J., additional, Darmanin, C., additional, Kobe, B., additional, Xu, H., additional, and Ve, T., additional
- Published
- 2021
- Full Text
- View/download PDF
12. MicroED structure of the MyD88 TIR domain higher-order assembly
- Author
-
Clabbers, M.T.B., primary, Holmes, S., additional, Muusse, T.W., additional, Vajjhala, P., additional, Thygesen, S.J., additional, Malde, A.K., additional, Hunter, D.J.B., additional, Croll, T.I., additional, Nanson, J.D., additional, Rahaman, M.H., additional, Aquila, A., additional, Hunter, M.S., additional, Liang, M., additional, Yoon, C.H., additional, Zhao, J., additional, Zatsepin, N.A., additional, Abbey, B., additional, Sierecki, E., additional, Gambin, Y., additional, Darmanin, C., additional, Kobe, B., additional, Xu, H., additional, and Ve, T., additional
- Published
- 2021
- Full Text
- View/download PDF
13. SFX structure of the MyD88 TIR domain higher-order assembly (solved, rebuilt and refined using an identical protocol to the MicroED structure of the MyD88 TIR domain higher-order assembly)
- Author
-
Clabbers, M.T.B., primary, Holmes, S., additional, Muusse, T.W., additional, Vajjhala, P., additional, Thygesen, S.J., additional, Malde, A.K., additional, Hunter, D.J.B., additional, Croll, T.I., additional, Nanson, J.D., additional, Rahaman, M.H., additional, Aquila, A., additional, Hunter, M.S., additional, Liang, M., additional, Yoon, C.H., additional, Zhao, J., additional, Zatsepin, N.A., additional, Abbey, B., additional, Sierecki, E., additional, Gambin, Y., additional, Darmanin, C., additional, Kobe, B., additional, Xu, H., additional, and Ve, T., additional
- Published
- 2021
- Full Text
- View/download PDF
14. Interaction between poly(ethylene glycol) and two surfactants investigated by diffusion coefficient measurements
- Author
-
López-Esparza, R., Guedeau-Boudeville, M.-A., Gambin, Y., Rodríguez-Beas, C., Maldonado, A., and Urbach, W.
- Published
- 2006
- Full Text
- View/download PDF
15. Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies
- Author
-
Pham, CLL, Shanmugam, N, Strange, M, O'Carroll, A, Brown, JWP, Sierecki, E, Gambin, Y, Steain, M, Sunde, M, Pham, CLL, Shanmugam, N, Strange, M, O'Carroll, A, Brown, JWP, Sierecki, E, Gambin, Y, Steain, M, and Sunde, M
- Abstract
The murine cytomegalovirus protein M45 protects infected mouse cells from necroptotic death and, when heterologously expressed, can protect human cells from necroptosis induced by tumour necrosis factor receptor (TNFR) activation. Here, we show that the N-terminal 90 residues of the M45 protein, which contain a RIP homotypic interaction motif (RHIM), are sufficient to confer protection against TNFR-induced necroptosis. This N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils and interacts with the RHIMs of the human kinases RIPK1 and RIPK3, and the Z-DNA binding protein 1 (ZBP1), to form heteromeric amyloid fibrils in vitro. Mutation of the tetrad residues in the M45 RHIM attenuates homo- and hetero-amyloid assembly by M45, suggesting that the amyloidogenic nature of the M45 RHIM underlies its biological activity. The M45 RHIM preferentially interacts with RIPK3 and ZBP1 over RIPK1 and alters the properties of the host RHIM protein assemblies. Our results indicate that M45 mimics the interactions made by RIPK1 or ZBP1 with RIPK3, thereby forming heteromeric amyloid structures, which may explain its ability to inhibit necroptosis.
- Published
- 2019
16. Unveiling a selective mechanism for the inhibition of α-synuclein aggregation by β-synuclein
- Author
-
Leitao, A, Bhumkar, A, Hunter, DJB, Gambin, Y, Sierecki, E, Leitao, A, Bhumkar, A, Hunter, DJB, Gambin, Y, and Sierecki, E
- Abstract
α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson’s disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and β-synuclein (βS) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assessing the effect of βS on the aggregation of five αS pathological mutants linked to early-onset PD (A30P, E46K, H50Q, G51D and A53T). By coupling single molecule fluorescence spectroscopy to a cell-free protein expression system, we validated the ability of βS to act as a chaperone of αS, effectively inhibiting its aggregation. Interestingly, we found that βS does so in a selective manner, i.e., is a more effective inhibitor for certain αS pathological mutants—A30P and G51D—as compared to E46K, H50Q and A53T. Moreover, two-color coincidence experiments proved that this discrepancy is due to a preferential incorporation of βS into smaller oligomers of αS. This was validated by showing that the chaperoning effect was lost when proteins were mixed after being expressed individually. This study highlights the potential of fluorescence spectroscopy to deconstruct αS aggregation cascade and its interplay with βS.
- Published
- 2018
17. Ultrastructural localisation of protein interactions using conditionally stable nanobodies
- Author
-
Ariotti, N, Rae, J, Giles, N, Martel, N, Sierecki, E, Gambin, Y, Hall, TE, Parton, RG, Ariotti, N, Rae, J, Giles, N, Martel, N, Sierecki, E, Gambin, Y, Hall, TE, and Parton, RG
- Abstract
We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level.
- Published
- 2018
18. Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice
- Author
-
Overman, J, Fontaine, F, Moustaqil, M, Mittal, D, Sierecki, E, Sacilotto, N, Zuegg, J, Robertson, AAB, Holmes, K, Salim, AA, Mamidyala, S, Butler, MS, Robinson, AS, Lesieur, E, Johnston, W, Alexandrov, K, Black, BL, Hogan, BM, De Val, S, Capon, RJ, Carroll, JS, Bailey, TL, Koopman, P, Jauch, R, Smyth, MJ, Cooper, MA, Gambin, Y, Francois, M, Overman, J, Fontaine, F, Moustaqil, M, Mittal, D, Sierecki, E, Sacilotto, N, Zuegg, J, Robertson, AAB, Holmes, K, Salim, AA, Mamidyala, S, Butler, MS, Robinson, AS, Lesieur, E, Johnston, W, Alexandrov, K, Black, BL, Hogan, BM, De Val, S, Capon, RJ, Carroll, JS, Bailey, TL, Koopman, P, Jauch, R, Smyth, MJ, Cooper, MA, Gambin, Y, and Francois, M
- Abstract
© Overman et al. Pharmacological targeting of transcription factors holds great promise for the development of new therapeutics, but strategies based on blockade of DNA binding, nuclear shuttling, or individual protein partner recruitment have yielded limited success to date. Transcription factors typically engage in complex interaction networks, likely masking the effects of specifically inhibiting single protein-protein interactions. Here, we used a combination of genomic, proteomic and biophysical methods to discover a suite of protein-protein interactions involving the SOX18 transcription factor, a known regulator of vascular development and disease. We describe a small-molecule that is able to disrupt a discrete subset of SOX18-dependent interactions. This compound selectively suppressed SOX18 transcriptional outputs in vitro and interfered with vascular development in zebrafish larvae. In a mouse pre-clinical model of breast cancer, treatment with this inhibitor significantly improved survival by reducing tumour vascular density and metastatic spread. Our studies validate an interactome-based molecular strategy to interfere with transcription factor activity, for the development of novel disease therapeutics.
- Published
- 2017
19. Cryo-EM structure of the MAL TIR domain filament
- Author
-
Ve, T., primary, Vajjhala, P.R., additional, Hedger, A., additional, Croll, T., additional, DiMaio, F., additional, Horsefield, S., additional, Yu, X., additional, Lavrencic, P., additional, Hassan, Z., additional, Morgan, G.P., additional, Mansell, A., additional, Mobli, M., additional, O'Carrol, A., additional, Chauvin, B., additional, Gambin, Y., additional, Sierecki, E., additional, Landsberg, M.J., additional, Stacey, K.J., additional, Egelman, E.H., additional, and Kobe, B., additional
- Published
- 2017
- Full Text
- View/download PDF
20. Nanomolar oligomerization and selective co-aggregation of α-synuclein pathogenic mutants revealed by single-molecule fluorescence
- Author
-
Sierecki, E, Giles, N, Bowden, Q, Polinkovsky, ME, Steinbeck, J, Arrioti, N, Rahman, D, Bhumkar, A, Nicovich, PR, Ross, I, Parton, RG, Böcking, T, Gambin, Y, Sierecki, E, Giles, N, Bowden, Q, Polinkovsky, ME, Steinbeck, J, Arrioti, N, Rahman, D, Bhumkar, A, Nicovich, PR, Ross, I, Parton, RG, Böcking, T, and Gambin, Y
- Abstract
Protein aggregation is a hallmark of many neurodegenerative diseases, notably Alzheimer's and Parkinson's disease. Parkinson's disease is characterized by the presence of Lewy bodies, abnormal aggregates mainly composed of α-synuclein. Moreover, cases of familial Parkinson's disease have been linked to mutations in α-synuclein. In this study, we compared the behavior of wild-type (WT) α-synuclein and five of its pathological mutants (A30P, E46K, H50Q, G51D and A53T). To this end, single-molecule fluorescence detection was coupled to cell-free protein expression to measure precisely the oligomerization of proteins without purification, denaturation or labelling steps. In these conditions, we could detect the formation of oligomeric and pre-fibrillar species at very short time scale and low micromolar concentrations. The pathogenic mutants surprisingly segregated into two classes: one group forming large aggregates and fibrils while the other tending to form mostly oligomers. Strikingly, co-expression experiments reveal that members from the different groups do not generally interact with each other, both at the fibril and monomer levels. Together, this data paints a completely different picture of α-synuclein aggregation, with two possible pathways leading to the development of fibrils.
- Published
- 2016
21. Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae
- Author
-
Gambin, Y, Ariotti, N, McMahon, KA, Bastiani, M, Sierecki, E, Kovtun, O, Polinkovsky, ME, Magenau, A, Jung, WR, Okano, S, Zhou, Y, Leneva, N, Mureev, S, Johnston, W, Gaus, K, Hancock, JF, Collins, BM, Alexandrov, K, Parton, RG, Gambin, Y, Ariotti, N, McMahon, KA, Bastiani, M, Sierecki, E, Kovtun, O, Polinkovsky, ME, Magenau, A, Jung, WR, Okano, S, Zhou, Y, Leneva, N, Mureev, S, Johnston, W, Gaus, K, Hancock, JF, Collins, BM, Alexandrov, K, and Parton, RG
- Abstract
In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae. © Gambin et al.
- Published
- 2014
22. A cell-free approach to accelerate the study of protein–protein interactions in vitro
- Author
-
Sierecki, E., primary, Giles, N., additional, Polinkovsky, M., additional, Moustaqil, M., additional, Alexandrov, K., additional, and Gambin, Y., additional
- Published
- 2013
- Full Text
- View/download PDF
23. Single-Molecule Fingerprinting Reveals Different Growth Mechanisms in Seed Amplification Assays for Different Polymorphs of α-Synuclein Fibrils.
- Author
-
Lau D, Tang Y, Kenche V, Copie T, Kempe D, Jary E, Graves NJ, Biro M, Masters CL, Dzamko N, Gambin Y, and Sierecki E
- Subjects
- Humans, Single Molecule Imaging methods, Protein Aggregates physiology, Protein Aggregation, Pathological metabolism, alpha-Synuclein metabolism, alpha-Synuclein chemistry, Parkinson Disease metabolism
- Abstract
α-Synuclein (αSyn) aggregates, detected in the biofluids of patients with Parkinson's disease (PD), have the ability to catalyze their own aggregation, leading to an increase in the number and size of aggregates. This self-templated amplification is used by newly developed assays to diagnose Parkinson's disease and turns the presence of αSyn aggregates into a biomarker of the disease. It has become evident that αSyn can form fibrils with slightly different structures, called "strains" or polymorphs, but little is known about their differential reactivity in diagnostic assays. Here, we compared the properties of two well-described αSyn polymorphs. Using single-molecule techniques, we observed that one of the polymorphs had an increased tendency to undergo secondary nucleation and we showed that this could explain the differences in reactivity observed in in vitro seed amplification assay and cellular assays. Simulations and high-resolution microscopy suggest that a 100-fold difference in the apparent rate of growth can be generated by a surprisingly low number of secondary nucleation "points" (1 every 2000 monomers added by elongation). When both strains are present in the same seeded reaction, secondary nucleation displaces proportions dramatically and causes a single strain to dominate the reaction as the major end product.
- Published
- 2024
- Full Text
- View/download PDF
24. Expressed Protein Ligation in Flow.
- Author
-
Kambanis L, Ayoub A, Bedding MJ, Egelund PHG, Maxwell JWC, Franck C, Lambrechts L, Hawkins PME, Chisholm TS, Mackay JP, Sierecki E, Gambin Y, Kulkarni SS, and Payne RJ
- Subjects
- Proteins chemistry
- Abstract
The development of a flow chemistry platform for the generation of modified protein targets via expressed protein ligation (EPL) is described. The flow EPL platform enables efficient ligation reactions with high recoveries of target protein products and superior reaction rates compared to corresponding batch processes. The utility of the flow EPL technology was first demonstrated through the semisynthesis of the tick-derived chemokine-binding protein ACA-01 containing two tyrosine sulfate modifications. Full-length, sulfated ACA-01 could be efficiently assembled by ligating a recombinantly expressed C-terminal protein fragment and a synthetic sulfopeptide thioester in flow. Following folding, the semisynthetic sulfoprotein was shown to exhibit potent binding to a variety of pro-inflammatory chemokines. In a second modified protein target, we employed an in-line flow EPL-photodesulfurization strategy to generate both unmodified and phosphorylated forms of human β-synuclein by fusing a recombinant protein thioester, generated through cleavage of an intein fusion protein, and a synthetic (phospho)peptide. The semisynthetic proteins were assembled in 90 min in flow, a significant improvement over corresponding batch protein assembly, and enabled access to tens of milligrams of high purity material. Flow EPL has the potential to serve as a robust technology to streamline access to homogeneously modified proteins for a variety of applications in both academia, as well as in the pharmaceutical and biotechnology sector.
- Published
- 2024
- Full Text
- View/download PDF
25. Dynamic PRC1-CBX8 stabilizes a porous structure of chromatin condensates.
- Author
-
Uckelmann M, Levina V, Taveneau C, Ng XH, Pandey V, Martinez J, Mendiratta S, Houx J, Boudes M, Venugopal H, Trépout S, Zhang Q, Flanigan S, Li M, Sierecki E, Gambin Y, Das PP, Bell O, de Marco A, and Davidovich C
- Abstract
The compaction of chromatin is a prevalent paradigm in gene repression. Chromatin compaction is commonly thought to repress transcription by restricting chromatin accessibility. However, the spatial organisation and dynamics of chromatin compacted by gene-repressing factors are unknown. Using cryo-electron tomography, we solved the three-dimensional structure of chromatin condensed by the Polycomb Repressive Complex 1 (PRC1) in a complex with CBX8. PRC1-condensed chromatin is porous and stabilised through multivalent dynamic interactions of PRC1 with chromatin. Mechanistically, positively charged residues on the internally disordered regions (IDRs) of CBX8 mask negative charges on the DNA to stabilize the condensed state of chromatin. Within condensates, PRC1 remains dynamic while maintaining a static chromatin structure. In differentiated mouse embryonic stem cells, CBX8-bound chromatin remains accessible. These findings challenge the idea of rigidly compacted polycomb domains and instead provides a mechanistic framework for dynamic and accessible PRC1-chromatin condensates.
- Published
- 2024
- Full Text
- View/download PDF
26. The DDHD2-STXBP1 interaction mediates long-term memory via generation of saturated free fatty acids.
- Author
-
Akefe IO, Saber SH, Matthews B, Venkatesh BG, Gormal RS, Blackmore DG, Alexander S, Sieriecki E, Gambin Y, Bertran-Gonzalez J, Vitale N, Humeau Y, Gaudin A, Ellis SA, Michaels AA, Xue M, Cravatt B, Joensuu M, Wallis TP, and Meunier FA
- Subjects
- Animals, Mice, Brain metabolism, Memory physiology, Fatty Acids, Nonesterified metabolism, Memory, Long-Term, Munc18 Proteins genetics, Phospholipases genetics
- Abstract
The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brain's lipid landscape remain largely unexplored. The levels of saturated FFAs, particularly of myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the PLA1 isoform DDHD2 in mice dramatically reduces saturated FFA responses to memory acquisition across the brain. Furthermore, DDHD2 loss also decreases memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits that mirror human spastic paraplegia. Via pulldown-mass spectrometry analyses, we find that DDHD2 binds to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficient STXBP1
+/- mouse model of human early infantile encephalopathy associated with intellectual disability and motor dysfunction, we show that STXBP1 controls targeting of DDHD2 to the plasma membrane and generation of saturated FFAs in the brain. These findings suggest key roles for DDHD2 and STXBP1 in lipid metabolism and in the processes of synaptic plasticity, learning, and memory., (© 2024. The Author(s).)- Published
- 2024
- Full Text
- View/download PDF
27. The N-end rule pathway regulates ER stress-induced clusterin release to the cytosol where it directs misfolded proteins for degradation.
- Author
-
Satapathy S, Walker H, Brown J, Gambin Y, and Wilson MR
- Abstract
Previous work suggests that cell stress induces release of the normally secreted chaperone clusterin (CLU) into the cytosol. We analyzed the localization of CLU in healthy and stressed cells, the mechanism of its cytosolic release, and its interactions with cytosolic misfolded proteins. Key results of this study are the following: (1) full-length CLU is released to the cytosol during stress, (2) the CLU N-terminal D1 residue is recognized by the N-end rule pathway and together with the enzyme ATE1 is essential for cytosolic release, (3) CLU can form stable complexes with cytosolic misfolded proteins and direct them to the proteasome and autophagosomes, and (4) cytosolic CLU protects cells from hypoxic stress and the cytosolic overexpression of an aggregation-prone protein. Collectively, the results suggest that enhanced cytosolic release of CLU is a stress response that can inhibit the toxicity of misfolded proteins and facilitate their targeted degradation via both autophagy and the proteasome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
28. Correction: Pharmacological targeting of the transcription factor SOX18 delays breast cancer in mice.
- Author
-
Overman J, Fontaine F, Moustaqil M, Mittal D, Sierecki E, Sacilotto N, Zuegg J, Robertson AAB, Holmes K, Salim AA, Mamidyala S, Butler MS, Robinson AS, Lesieur E, Johnston W, Alexandrov K, Black BL, Hogan BM, De Val S, Capon RJ, Carroll JS, Bailey TL, Koopman P, Jauch R, Cooper MA, Gambin Y, and Francois M
- Published
- 2023
- Full Text
- View/download PDF
29. α-Synuclein Strains and Their Relevance to Parkinson's Disease, Multiple System Atrophy, and Dementia with Lewy Bodies.
- Author
-
Graves NJ, Gambin Y, and Sierecki E
- Subjects
- Humans, alpha-Synuclein metabolism, Protein Aggregates, Lewy Body Disease metabolism, Multiple System Atrophy, Parkinson Disease metabolism, Synucleinopathies
- Abstract
Like many neurodegenerative diseases, Parkinson's disease (PD) is characterized by the formation of proteinaceous aggregates in brain cells. In PD, those proteinaceous aggregates are formed by the α-synuclein (αSyn) and are considered the trademark of this neurodegenerative disease. In addition to PD, αSyn pathological aggregation is also detected in atypical Parkinsonism, including Dementia with Lewy Bodies (DLB), Multiple System Atrophy (MSA), as well as neurodegeneration with brain iron accumulation, some cases of traumatic brain injuries, and variants of Alzheimer's disease. Collectively, these (and other) disorders are referred to as synucleinopathies, highlighting the relation between disease type and protein misfolding/aggregation. Despite these pathological relationships, however, synucleinopathies cover a wide range of pathologies, present with a multiplicity of symptoms, and arise from dysfunctions in different neuroanatomical regions and cell populations. Strikingly, αSyn deposition occurs in different types of cells, with oligodendrocytes being mainly affected in MSA, while aggregates are found in neurons in PD. If multiple factors contribute to the development of a pathology, especially in the cases of slow-developing neurodegenerative disorders, the common presence of αSyn aggregation, as both a marker and potential driver of disease, is puzzling. In this review, we will focus on comparing PD, DLB, and MSA, from symptomatology to molecular description, highlighting the role and contribution of αSyn aggregates in each disorder. We will particularly present recent evidence for the involvement of conformational strains of αSyn aggregates and discuss the reciprocal relationship between αSyn strains and the cellular milieu. Moreover, we will highlight the need for effective methodologies for the strainotyping of aggregates to ameliorate diagnosing capabilities and therapeutic treatments., Competing Interests: The authors declare no conflicts of interest.
- Published
- 2023
- Full Text
- View/download PDF
30. The blood vasculature instructs lymphatic patterning in a SOX7-dependent manner.
- Author
-
Chiang IKN, Graus MS, Kirschnick N, Davidson T, Luu W, Harwood R, Jiang K, Li B, Wong YY, Moustaqil M, Lesieur E, Skoczylas R, Kouskoff V, Kazenwadel J, Arriola-Martinez L, Sierecki E, Gambin Y, Alitalo K, Kiefer F, Harvey NL, and Francois M
- Subjects
- Humans, Mice, Animals, Gene Expression Regulation, Endothelium, Vascular, Transcription Factors metabolism, Lymphangiogenesis genetics, SOXF Transcription Factors genetics, SOXF Transcription Factors metabolism, Endothelial Cells metabolism, Lymphatic Vessels metabolism
- Abstract
Despite a growing catalog of secreted factors critical for lymphatic network assembly, little is known about the mechanisms that modulate the expression level of these molecular cues in blood vascular endothelial cells (BECs). Here, we show that a BEC-specific transcription factor, SOX7, plays a crucial role in a non-cell-autonomous manner by modulating the transcription of angiocrine signals to pattern lymphatic vessels. While SOX7 is not expressed in lymphatic endothelial cells (LECs), the conditional loss of SOX7 function in mouse embryos causes a dysmorphic dermal lymphatic phenotype. We identify novel distant regulatory regions in mice and humans that contribute to directly repressing the transcription of a major lymphangiogenic growth factor (Vegfc) in a SOX7-dependent manner. Further, we show that SOX7 directly binds HEY1, a canonical repressor of the Notch pathway, suggesting that transcriptional repression may also be modulated by the recruitment of this protein partner at Vegfc genomic regulatory regions. Our work unveils a role for SOX7 in modulating downstream signaling events crucial for lymphatic patterning, at least in part via the transcriptional repression of VEGFC levels in the blood vascular endothelium., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)
- Published
- 2023
- Full Text
- View/download PDF
31. Evolutionary Landscape of SOX Genes to Inform Genotype-to-Phenotype Relationships.
- Author
-
Underwood A, Rasicci DT, Hinds D, Mitchell JT, Zieba JK, Mills J, Arnold NE, Cook TW, Moustaqil M, Gambin Y, Sierecki E, Fontaine F, Vanderweele S, Das AS, Cvammen W, Sirpilla O, Soehnlen X, Bricker K, Alokaili M, Green M, Heeringa S, Wilstermann AM, Freeland TM, Qutob D, Milsted A, Jauch R, Triche TJ Jr, Krawczyk CM, Bupp CP, Rajasekaran S, Francois M, and Prokop JW
- Subjects
- Humans, Amino Acid Sequence, Dimerization, Genotype, SOXF Transcription Factors genetics, SOXF Transcription Factors metabolism, SOXB2 Transcription Factors genetics, SOXB2 Transcription Factors metabolism, SOXE Transcription Factors genetics, High Mobility Group Proteins chemistry, High Mobility Group Proteins genetics, High Mobility Group Proteins metabolism, SOX Transcription Factors genetics
- Abstract
The SOX transcription factor family is pivotal in controlling aspects of development. To identify genotype-phenotype relationships of SOX proteins, we performed a non-biased study of SOX using 1890 open-reading frame and 6667 amino acid sequences in combination with structural dynamics to interpret 3999 gnomAD, 485 ClinVar, 1174 Geno2MP, and 4313 COSMIC human variants. We identified, within the HMG (High Mobility Group)- box, twenty-seven amino acids with changes in multiple SOX proteins annotated to clinical pathologies. These sites were screened through Geno2MP medical phenotypes, revealing novel SOX15 R104G associated with musculature abnormality and SOX8 R159G with intellectual disability. Within gnomAD, SOX18 E137K (rs201931544), found within the HMG box of ~0.8% of Latinx individuals, is associated with seizures and neurological complications, potentially through blood-brain barrier alterations. A total of 56 highly conserved variants were found at sites outside the HMG-box, including several within the SOX2 HMG-box-flanking region with neurological associations, several in the SOX9 dimerization region associated with Campomelic Dysplasia, SOX14 K88R (rs199932938) flanking the HMG box associated with cardiovascular complications within European populations, and SOX7 A379V (rs143587868) within an SOXF conserved far C-terminal domain heterozygous in 0.716% of African individuals with associated eye phenotypes. This SOX data compilation builds a robust genotype-to-phenotype association for a gene family through more robust ortholog data integration.
- Published
- 2023
- Full Text
- View/download PDF
32. The RHIM of the Immune Adaptor Protein TRIF Forms Hybrid Amyloids with Other Necroptosis-Associated Proteins.
- Author
-
Baker MODG, Shanmugam N, Pham CLL, Ball SR, Sierecki E, Gambin Y, Steain M, and Sunde M
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, Adaptor Proteins, Vesicular Transport genetics, Adaptor Proteins, Vesicular Transport metabolism, Apoptosis physiology, Amyloid metabolism, Necroptosis
- Abstract
TIR-domain-containing adapter-inducing interferon-β (TRIF) is an innate immune protein that serves as an adaptor for multiple cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. One outcome of TRIF-directed signalling is the activation of the programmed cell death pathway necroptosis, which is governed by interactions between proteins that contain a RIP Homotypic Interaction Motif (RHIM). TRIF contains a RHIM sequence and can interact with receptor interacting protein kinases 1 (RIPK1) and 3 (RIPK3) to initiate necroptosis. Here, we demonstrate that the RHIM of TRIF is amyloidogenic and supports the formation of homomeric TRIF-containing fibrils. We show that the core tetrad sequence within the RHIM governs the supramolecular organisation of TRIF amyloid assemblies, although the stable amyloid core of TRIF amyloid fibrils comprises a much larger region than the conserved RHIM only. We provide evidence that RHIMs of TRIF, RIPK1 and RIPK3 interact directly to form heteromeric structures and that these TRIF-containing hetero-assemblies display altered and emergent properties that likely underlie necroptosis signalling in response to Toll-like receptor activation.
- Published
- 2022
- Full Text
- View/download PDF
33. Single Molecule Fingerprinting Reveals Different Amplification Properties of α-Synuclein Oligomers and Preformed Fibrils in Seeding Assay.
- Author
-
Lau D, Magnan C, Hill K, Cooper A, Gambin Y, and Sierecki E
- Subjects
- Amyloid, Biological Assay, Biomarkers analysis, Humans, Parkinson Disease diagnosis, alpha-Synuclein chemistry
- Abstract
The quantification of α-synuclein aggregates has emerged as a promising biomarker for synucleinopathies. Assays that amplify and detect such aggregates have revealed the presence of seeding-competent species in biosamples of patients diagnosed with Parkinson's disease. However, multiple species, such as oligomers and amyloid fibrils, are formed during the aggregation of α-synuclein; these species are likely to coexist in biological samples, and thus it remains unclear which species(s) are contributing to the signal detected in seeding assays. To identify individual contributions to the amplification process, recombinant oligomers and preformed fibrils were produced and purified to characterize their individual biochemical and seeding potential. Here, we used single molecule spectroscopy to track the formation and purification of oligomers and fibrils at the single particle level and compare their respective seeding potential in an amplification assay. Single molecule detection validates that size-exclusion chromatography efficiently separates oligomers from fibrils. Oligomers were found to be seeding-competent, but our results reveal that their seeding behavior is very different compared to that of preformed fibrils, in our amplification assay. Overall, our data suggest that even a low number of preformed fibrils present in biosamples is likely to dominate the response in seeding assays.
- Published
- 2022
- Full Text
- View/download PDF
34. SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species.
- Author
-
Moustaqil M, Ollivier E, Chiu HP, Van Tol S, Rudolffi-Soto P, Stevens C, Bhumkar A, Hunter DJB, Freiberg AN, Jacques D, Lee B, Sierecki E, and Gambin Y
- Subjects
- Amino Acid Sequence, Animals, COVID-19 pathology, Cell Line, Chiroptera virology, Coronavirus 3C Proteases genetics, Coronavirus Papain-Like Proteases genetics, HEK293 Cells, Humans, Mice, SARS-CoV-2 enzymology, SARS-CoV-2 genetics, Adaptor Proteins, Signal Transducing metabolism, Coronavirus 3C Proteases metabolism, Coronavirus Papain-Like Proteases metabolism, Interferon Regulatory Factor-3 metabolism, Intracellular Signaling Peptides and Proteins metabolism
- Abstract
The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.
- Published
- 2021
- Full Text
- View/download PDF
35. A dominant-negative SOX18 mutant disrupts multiple regulatory layers essential to transcription factor activity.
- Author
-
McCann AJ, Lou J, Moustaqil M, Graus MS, Blum A, Fontaine F, Liu H, Luu W, Rudolffi-Soto P, Koopman P, Sierecki E, Gambin Y, Meunier FA, Liu Z, Hinde E, and Francois M
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Disease Models, Animal, Gene Expression Regulation, Gene Regulatory Networks, Genes, Reporter, Glomerulonephritis metabolism, Glomerulonephritis pathology, HeLa Cells, Human Umbilical Vein Endothelial Cells, Humans, Hypotrichosis metabolism, Hypotrichosis pathology, Luciferases genetics, Luciferases metabolism, Lymphedema metabolism, Lymphedema pathology, MEF2 Transcription Factors genetics, MEF2 Transcription Factors metabolism, Mice, Mutation, SOXF Transcription Factors metabolism, Single Molecule Imaging, Telangiectasis metabolism, Telangiectasis pathology, Glomerulonephritis genetics, Hypotrichosis genetics, Lymphedema genetics, SOXF Transcription Factors genetics, Telangiectasis genetics, Transcription, Genetic
- Abstract
Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2021
- Full Text
- View/download PDF
36. Selectivity of Lewy body protein interactions along the aggregation pathway of α-synuclein.
- Author
-
Leitão ADG, Rudolffi-Soto P, Chappard A, Bhumkar A, Lau D, Hunter DJB, Gambin Y, and Sierecki E
- Subjects
- Amyloid metabolism, Humans, Lewy Bodies metabolism, Parkinson Disease pathology, alpha-Synuclein metabolism
- Abstract
The aggregation of alpha-synuclein (α-SYN) follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson's Disease. Although LB contain over 70 proteins, the potential for interactions along the aggregation pathway of α-SYN is unknown. Here we propose a map of interactions of 65 proteins against different species of α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead luminescence assay for detection of protein interactions. To access oligomeric species, we used the pathological mutants of α-SYN (A30P, G51D and A53T) which form oligomers with distinct properties. Finally, we generated amyloid fibrils from recombinant α-SYN. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are recruited to specific steps in the aggregation of α-SYN, uncovering future targets to modulate aggregation in synucleinopathies., (© 2021. Crown.)
- Published
- 2021
- Full Text
- View/download PDF
37. Characterising proteolysis during SARS-CoV-2 infection identifies viral cleavage sites and cellular targets with therapeutic potential.
- Author
-
Meyer B, Chiaravalli J, Gellenoncourt S, Brownridge P, Bryne DP, Daly LA, Grauslys A, Walter M, Agou F, Chakrabarti LA, Craik CS, Eyers CE, Eyers PA, Gambin Y, Jones AR, Sierecki E, Verdin E, Vignuzzi M, and Emmott E
- Subjects
- Animals, Cell Line, Dipeptides pharmacology, Humans, Mutation, Myosin-Light-Chain Kinase antagonists & inhibitors, Myosin-Light-Chain Kinase genetics, Myosin-Light-Chain Kinase metabolism, Proteolysis, Proteomics, RNA, Small Interfering pharmacology, SARS-CoV-2 genetics, Viral Proteases metabolism, Viral Proteins genetics, Viral Proteins metabolism, Virus Internalization drug effects, Virus Replication drug effects, src-Family Kinases antagonists & inhibitors, src-Family Kinases genetics, src-Family Kinases metabolism, COVID-19 Drug Treatment, Antiviral Agents pharmacology, COVID-19 metabolism, Protease Inhibitors pharmacology, SARS-CoV-2 drug effects
- Abstract
SARS-CoV-2 is the causative agent behind the COVID-19 pandemic, responsible for over 170 million infections, and over 3.7 million deaths worldwide. Efforts to test, treat and vaccinate against this pathogen all benefit from an improved understanding of the basic biology of SARS-CoV-2. Both viral and cellular proteases play a crucial role in SARS-CoV-2 replication. Here, we study proteolytic cleavage of viral and cellular proteins in two cell line models of SARS-CoV-2 replication using mass spectrometry to identify protein neo-N-termini generated through protease activity. We identify previously unknown cleavage sites in multiple viral proteins, including major antigens S and N: the main targets for vaccine and antibody testing efforts. We discover significant increases in cellular cleavage events consistent with cleavage by SARS-CoV-2 main protease, and identify 14 potential high-confidence substrates of the main and papain-like proteases. We show that siRNA depletion of these cellular proteins inhibits SARS-CoV-2 replication, and that drugs targeting two of these proteins: the tyrosine kinase SRC and Ser/Thr kinase MYLK, show a dose-dependent reduction in SARS-CoV-2 titres. Overall, our study provides a powerful resource to understand proteolysis in the context of viral infection, and to inform the development of targeted strategies to inhibit SARS-CoV-2 and treat COVID-19., (© 2021. The Author(s).)
- Published
- 2021
- Full Text
- View/download PDF
38. An inverted CAV1 (caveolin 1) topology defines novel autophagy-dependent exosome secretion from prostate cancer cells.
- Author
-
Ariotti N, Wu Y, Okano S, Gambin Y, Follett J, Rae J, Ferguson C, Teasdale RD, Alexandrov K, Meunier FA, Hill MM, and Parton RG
- Subjects
- Autophagy, Humans, Male, Caveolin 1 metabolism, Exosomes metabolism, Prostatic Neoplasms
- Abstract
CAV1 (caveolin 1) expression and secretion is associated with prostate cancer (PCa) disease progression, but the mechanisms underpinning CAV1 release remain poorly understood. Numerous studies have shown CAV1 can be secreted within exosome-like vesicles, but antibody-mediated neutralization can mitigate PCa progression; this is suggestive of an inverted (non-exosomal) CAV1 topology. Here we show that CAV1 can be secreted from specific PCa types in an inverted vesicle-associated form consistent with the features of bioactive CAV1 secretion. Characterization of the isolated vesicles by electron microscopy, single-molecule fluorescence microscopy and proteomics reveals they represent a novel class of exosomes ~40 nm in diameter containing ~50-60 copies of CAV1 and, strikingly, are released via a non-canonical secretory macroautophagy/autophagy pathway. This study provides novel insights into a mechanism whereby CAV1 translocates from a normal plasma membrane distribution to an inverted secreted form implicated in PCa disease progression. Abbreviations: 3-MA: 3-methyladenine; APEX: a modified soybean ascorbate peroxidase; ATG5: autophagy related 5; ATG9A: autophagy related 9A; ATG12: autophagy related 12; BHK: baby hamster kidney; C-exosomes: caveolin-exosomes; CAMKK2/CAMKKβ: calckum/calmodulin dependent protein kinase kinase 2; CAV1: caveolin 1; DAB: 3,3'-diaminobenzidine; DAPK: death associated protein kinase; EEA1: early endosome antigen 1; EM: electron microscopy; FCS: fluorescence correlation spectroscopy; GBP: GFP/YFP-binding peptide; GFP: green fluorescent protein; GOLGA2: golgin A2; ILVs: intralumenal vesicles; LC3: microtubule-associated protein 1 light chain 3; MBP: maltose binding protein; MTORC1: mechanistic target of rapamycin kinase complex 1; MVBs: multivesicular bodies; PBS: phosphate-buffered saline; PCa: prostate cancer; PI3K: phosphoinositide 3-kinase; PM: plasma membrane; SFM: serum-free medium; TSG101: tumor susceptibility 101; WCL: whole cell lysates; WT: wild type; YFP: yellow fluorescent protein; βoG: β-octylglucoside.
- Published
- 2021
- Full Text
- View/download PDF
39. Cavin3 released from caveolae interacts with BRCA1 to regulate the cellular stress response.
- Author
-
McMahon KA, Stroud DA, Gambin Y, Tillu V, Bastiani M, Sierecki E, Polinkovsky ME, Hall TE, Gomez GA, Wu Y, Parat MO, Martel N, Lo HP, Khanna KK, Alexandrov K, Daly R, Yap A, Ryan MT, and Parton RG
- Subjects
- Apoptosis genetics, HeLa Cells, Humans, Proteome genetics, Proteomics, BRCA1 Protein metabolism, Caveolae metabolism, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Stress, Physiological genetics
- Abstract
Caveolae-associated protein 3 (cavin3) is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair, with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays revealed a direct interaction between BRCA1 and cavin3 that occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. Supporting a role in DNA repair, cavin3-deficient cells were sensitive to PARP inhibition, where concomitant depletion of 53BP1 restored BRCA1-dependent sensitivity to PARP inhibition. We conclude that cavin3 functions together with BRCA1 in multiple cancer-related pathways. The loss of cavin3 function may provide tumor cell survival by attenuating apoptotic sensitivity and hindering DNA repair under chronic stress conditions., Competing Interests: KM, DS, YG, VT, MB, ES, MP, TH, GG, YW, MP, NM, HL, KK, KA, RD, AY, MR, RP No competing interests declared, (© 2021, McMahon et al.)
- Published
- 2021
- Full Text
- View/download PDF
40. Single-Molecule Counting Coupled to Rapid Amplification Enables Detection of α-Synuclein Aggregates in Cerebrospinal Fluid of Parkinson's Disease Patients.
- Author
-
Bhumkar A, Magnan C, Lau D, Jun ESW, Dzamko N, Gambin Y, and Sierecki E
- Subjects
- Adult, Aged, Biomarkers cerebrospinal fluid, Female, Humans, Limit of Detection, Male, Middle Aged, Parkinson Disease cerebrospinal fluid, Single Molecule Imaging methods, Parkinson Disease diagnosis, Protein Aggregates, alpha-Synuclein cerebrospinal fluid
- Abstract
α-Synuclein aggregation is a hallmark of Parkinson's disease and a promising biomarker for early detection and assessment of disease progression. The prospect of a molecular test for Parkinson's disease is materializing with the recent developments of detection methods based on amplification of synuclein seeds (e.g. RT-QuIC or PMCA). Here we adapted single-molecule counting methods for the detection of α-synuclein aggregates in cerebrospinal fluid (CSF), using a simple 3D printed microscope. Single-molecule methods enable to probe the early events in the amplification process used in RT-QuIC and a precise counting of ThT-positive aggregates. Importantly, the use of single-molecule counting also allows a refined characterization of the samples and fingerprinting of the protein aggregates present in CSF of patients. The fingerprinting of size and reactivity of individual aggregate shows a unique signature for each PD patients compared to controls and may provide new insights on synucleinopathies in the future., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)
- Published
- 2021
- Full Text
- View/download PDF
41. MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography.
- Author
-
Clabbers MTB, Holmes S, Muusse TW, Vajjhala PR, Thygesen SJ, Malde AK, Hunter DJB, Croll TI, Flueckiger L, Nanson JD, Rahaman MH, Aquila A, Hunter MS, Liang M, Yoon CH, Zhao J, Zatsepin NA, Abbey B, Sierecki E, Gambin Y, Stacey KJ, Darmanin C, Kobe B, Xu H, and Ve T
- Subjects
- Dimerization, HEK293 Cells, Humans, Membrane Glycoproteins genetics, Models, Molecular, Molecular Dynamics Simulation, Mutation, Myeloid Differentiation Factor 88 genetics, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Domains, Receptors, Interleukin-1 genetics, Recombinant Proteins, Signal Transduction genetics, Toll-Like Receptor 4 genetics, Crystallography methods, Membrane Glycoproteins chemistry, Myeloid Differentiation Factor 88 chemistry, Receptors, Interleukin-1 chemistry, Toll-Like Receptor 4 chemistry
- Abstract
MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MAL
TIR ) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR ). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR . We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR . Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques.- Published
- 2021
- Full Text
- View/download PDF
42. A robust method for particulate detection of a genetic tag for 3D electron microscopy.
- Author
-
Rae J, Ferguson C, Ariotti N, Webb RI, Cheng HH, Mead JL, Riches JD, Hunter DJ, Martel N, Baltos J, Christopoulos A, Bryce NS, Cagigas ML, Fonseka S, Sayre ME, Hardeman EC, Gunning PW, Gambin Y, Hall TE, and Parton RG
- Subjects
- Animals, Ascorbate Peroxidases, Freezing, Gold, Mice, Proteins, Electron Microscope Tomography methods, Genetic Techniques, Imaging, Three-Dimensional methods
- Abstract
Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method., Competing Interests: JR, CF, NA, RW, HC, JM, JR, DH, NM, JB, AC, NB, MC, SF, MS, EH, PG, YG, TH, RP No competing interests declared, (© 2021, Rae et al.)
- Published
- 2021
- Full Text
- View/download PDF
43. Rapid HIV-1 Capsid Interaction Screening Using Fluorescence Fluctuation Spectroscopy.
- Author
-
Lau D, Walsh JC, Dickson CF, Tuckwell A, Stear JH, Hunter DJB, Bhumkar A, Shah V, Turville SG, Sierecki E, Gambin Y, Böcking T, and Jacques DA
- Subjects
- Binding Sites, Capsid Proteins, Spectrometry, Fluorescence, Capsid, HIV-1
- Abstract
The HIV capsid is a multifunctional protein capsule that mediates the delivery of the viral genetic material into the nucleus of the target cell. Host cell proteins bind to a number of repeating binding sites on the capsid to regulate steps in the replication cycle. Here, we develop a fluorescence fluctuation spectroscopy method using self-assembled capsid particles as the bait to screen for fluorescence-labeled capsid-binding analytes ("prey" molecules) in solution. The assay capitalizes on the property of the HIV capsid as a multivalent interaction platform, facilitating high sensitivity detection of multiple prey molecules that have accumulated onto capsids as spikes in fluorescence intensity traces. By using a scanning stage, we reduced the measurement time to 10 s without compromising on sensitivity, providing a rapid binding assay for screening libraries of potential capsid interactors. The assay can also identify interfaces for host molecule binding by using capsids with defects in known interaction interfaces. Two-color coincidence detection using the fluorescent capsid as the bait further allows the quantification of binding levels and determination of binding affinities. Overall, the assay provides new tools for the discovery and characterization of molecules used by the HIV capsid to orchestrate infection. The measurement principle can be extended for the development of sensitive interaction assays, utilizing natural or synthetic multivalent scaffolds as analyte-binding platforms.
- Published
- 2021
- Full Text
- View/download PDF
44. Herpes simplex virus encoded ICP6 protein forms functional amyloid assemblies with necroptosis-associated host proteins.
- Author
-
Shanmugam N, Baker MODG, Sanz-Hernandez M, Sierecki E, Gambin Y, Steain M, Pham CLL, and Sunde M
- Subjects
- Humans, Necroptosis, Animals, Viral Proteins chemistry, Viral Proteins metabolism, Amyloid chemistry, Amyloid metabolism, Immediate-Early Proteins metabolism, Immediate-Early Proteins chemistry, Herpesvirus 1, Human
- Abstract
The viral protein ICP6, encoded by herpes simplex virus 1 (HSV-1), harbours a RIP-homotypic interaction motif (RHIM), that plays a role in viral inhibition of host cell death pathways. Other members of the Herpesviridae family also encode RHIM-containing proteins that interfere with host-cell death pathways, including the M45 protein from murine cytomegalovirus, and ORF20 protein from varicella zoster virus. We have used amyloid assembly assays, electron microscopy and single molecule fluorescence spectroscopy to show that the ICP6 RHIM is amyloidogenic and can interact with host RHIM-containing proteins to form heteromeric amyloid complexes, in a manner similar to that of M45 and ORF20 RHIMs. The core tetrad sequence of the ICP6 RHIM is important for both amyloid formation and interaction with host RHIM-containing proteins. Notably, we show that the amyloid forming capacity of the ICP6 RHIM is affected by the redox environment. We propose that the formation of an intramolecular disulfide bond within ICP6 triggers the formation of amyloid assemblies that are distinct from previously characterised viral amyloids M45 and ORF20. Formation of viral-host heteromeric amyloid assemblies may underlie a general mechanism of viral adaptation against host immune machineries., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
45. Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death.
- Author
-
Steain M, Baker MODG, Pham CLL, Shanmugam N, Gambin Y, Sierecki E, McSharry BP, Avdic S, Slobedman B, Sunde M, and Abendroth A
- Subjects
- Animals, Humans, Mice, Cell Death physiology, Herpesvirus 3, Human metabolism, RNA-Binding Proteins metabolism, Varicella Zoster Virus Infection metabolism, Viral Proteins metabolism
- Abstract
Herpesviruses are known to encode a number of inhibitors of host cell death, including RIP Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein, open reading frame (ORF) 20, that acts as a host cell death inhibitor. Like the human cellular RHIMs in RIPK1 and RIPK3 that stabilise the necrosome in TNF-induced necroptosis, and the viral RHIM in M45 from murine cytomegalovirus that inhibits cell death, the ORF20 RHIM is capable of forming fibrillar functional amyloid complexes. Notably, the ORF20 RHIM forms hybrid amyloid complexes with human ZBP1, a cytoplasmic sensor of viral nucleic acid. Although VZV can inhibit TNF-induced necroptosis, the ORF20 RHIM does not appear to be responsible for this inhibition. In contrast, the ZBP1 pathway is identified as important for VZV infection. Mutation of the ORF20 RHIM renders the virus incapable of efficient spread in ZBP1-expressing HT-29 cells, an effect which can be reversed by the inhibition of caspases. Therefore we conclude that the VZV ORF20 RHIM is important for preventing ZBP1-driven apoptosis during VZV infection, and propose that it mediates this effect by sequestering ZBP1 into decoy amyloid assemblies., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2020
- Full Text
- View/download PDF
46. Biophysical Techniques for Target Validation and Drug Discovery in Transcription-Targeted Therapy.
- Author
-
Moustaqil M, Gambin Y, and Sierecki E
- Subjects
- Animals, Humans, Cryoelectron Microscopy methods, Drug Discovery methods, Molecular Targeted Therapy methods, Single Molecule Imaging methods, Transcription Factors antagonists & inhibitors
- Abstract
In the post-genome era, pathologies become associated with specific gene expression profiles and defined molecular lesions can be identified. The traditional therapeutic strategy is to block the identified aberrant biochemical activity. However, an attractive alternative could aim at antagonizing key transcriptional events underlying the pathogenesis, thereby blocking the consequences of a disorder, irrespective of the original biochemical nature. This approach, called transcription therapy, is now rendered possible by major advances in biophysical technologies. In the last two decades, techniques have evolved to become key components of drug discovery platforms, within pharmaceutical companies as well as academic laboratories. This review outlines the current biophysical strategies for transcription manipulation and provides examples of successful applications. It also provides insights into the future development of biophysical methods in drug discovery and personalized medicine.
- Published
- 2020
- Full Text
- View/download PDF
47. Editorial.
- Author
-
Sierecki E and Gambin Y
- Subjects
- Humans, Protein Aggregation, Pathological genetics, Protein Aggregation, Pathological metabolism, Protein Binding, Proteins chemistry, Proteins genetics, Proteins metabolism, Gene Regulatory Networks
- Published
- 2020
- Full Text
- View/download PDF
48. Prions and Prion-like assemblies in neurodegeneration and immunity: The emergence of universal mechanisms across health and disease.
- Author
-
O'Carroll A, Coyle J, and Gambin Y
- Subjects
- Animals, Humans, Health, Immunity, Innate immunology, Neurodegenerative Diseases immunology, Neurodegenerative Diseases metabolism, Prions immunology, Prions metabolism
- Abstract
Prion-like behaviour is an abrupt process, an "all-or-nothing" transition between a monomeric species and an "infinite" fibrillated form. Once a nucleation point is formed, the process is unstoppable as fibrils self-propagate by recruiting and converting all monomers into the amyloid fold. After the "mad cow" episode, prion diseases have made the headlines, but more and more prion-like behaviours have emerged in neurodegenerative diseases, where formation of fibrils and large conglomerates of proteins deeply disrupt the cell homeostasis. More interestingly, in the last decade, examples emerged to suggest that prion-like conversion can be used as a positive gain of function, for memory storage or structural scaffolding. More recent experiments show that we are only seeing the tip of the iceberg and that, for example, prion-like amplification is found in many pathways of the immune response. In innate immunity, receptors on the cellular surface or within the cells 'sense' danger and propagate this information as signal, through protein-protein interactions (PPIs) between 'receptor', 'adaptor' and 'effector' proteins. In innate immunity, the smallest signal of a foreign element or pathogen needs to trigger a macroscopic signal output, and it was found that adaptor polymerize to create an extreme signal amplification. Interestingly, our body uses multiple structural motifs to create large signalling platform; a few innate proteins use amyloid scaffolds but most of the polymers discovered are composed by self-assembly in helical filaments. Some of these helical assemblies even have intercellular "contamination" in a "true" prion action, as demonstrated for ASC specks and MyD88 filaments. Here, we will describe the current knowledge in neurodegenerative diseases and innate immunity and show how these two very different fields can cross-seed discoveries., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2020
- Full Text
- View/download PDF
49. Single-molecule detection on a portable 3D-printed microscope.
- Author
-
Brown JWP, Bauer A, Polinkovsky ME, Bhumkar A, Hunter DJB, Gaus K, Sierecki E, and Gambin Y
- Abstract
Single-molecule assays have, by definition, the ultimate sensitivity and represent the next frontier in biological analysis and diagnostics. However, many of these powerful technologies require dedicated laboratories and trained personnel and have therefore remained research tools for specialists. Here, we present a single-molecule confocal system built from a 3D-printed scaffold, resulting in a compact, plug and play device called the AttoBright. This device performs single photon counting and fluorescence correlation spectroscopy (FCS) in a simple format and is widely applicable to the detection of single fluorophores, proteins, liposomes or bacteria. The power of single-molecule detection is demonstrated by detecting single α-synuclein amyloid fibrils, that are currently evaluated as biomarkers for Parkinson's disease, with an improved sensitivity of >100,000-fold over bulk measurements.
- Published
- 2019
- Full Text
- View/download PDF
50. The cryo-EM structure of the acid activatable pore-forming immune effector Macrophage-expressed gene 1.
- Author
-
Pang SS, Bayly-Jones C, Radjainia M, Spicer BA, Law RHP, Hodel AW, Parsons ES, Ekkel SM, Conroy PJ, Ramm G, Venugopal H, Bird PI, Hoogenboom BW, Voskoboinik I, Gambin Y, Sierecki E, Dunstone MA, and Whisstock JC
- Subjects
- Bacteria immunology, Cryoelectron Microscopy, Humans, Liposomes metabolism, Lysosomes physiology, Macrophages immunology, Microscopy, Atomic Force, Protein Domains, Protein Structure, Secondary, Cell Membrane metabolism, Membrane Proteins metabolism, Pore Forming Cytotoxic Proteins metabolism
- Abstract
Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, however, its mode of action remains unknown. We use cryo-Electron Microscopy (cryo-EM) to determine the 2.4 Å structure of a hexadecameric assembly of MPEG1 that displays the expected features of a soluble prepore complex. We further discover that MPEG1 prepore-like assemblies can be induced to perforate membranes through acidification, such as would occur within maturing phagolysosomes. We next solve the 3.6 Å cryo-EM structure of MPEG1 in complex with liposomes. These data reveal that a multi-vesicular body of 12 kDa (MVB12)-associated β-prism (MABP) domain binds membranes such that the pore-forming machinery of MPEG1 is oriented away from the bound membrane. This unexpected mechanism of membrane interaction suggests that MPEG1 remains bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.
- Published
- 2019
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.