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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

Biophysical Evidence for the Amyloid Formation of a Recombinant Rab2 Isoform of Leishmania donovani

Author(s): Roshanara, Shivani A. Muthu, Gulafsha, Rati Tandon, Angamuthu Selvapandiyan and Basir Ahmad*

Volume 31, Issue 4, 2024

Published on: 24 April, 2024

Page: [312 - 322] Pages: 11

DOI: 10.2174/0109298665299157240327084614

Abstract

Background: The most fatal form of Visceral leishmaniasis or kala-azar is caused by the intracellular protozoan parasite Leishmania donovani. The life cycle and the infection pathway of the parasite are regulated by the small GTPase family of Rab proteins. The involvement of Rab proteins in neurodegenerative amyloidosis is implicated in protein misfolding, secretion abnormalities and dysregulation. The inter and intra-cellular shuttlings of Rab proteins are proposed to be aggregation-prone. However, the biophysical unfolding and aggregation of protozoan Rab proteins is limited. Understanding the aggregation mechanisms of Rab protein will determine their physical impact on the disease pathogenesis and individual health.

Objective: This work investigates the acidic pH-induced unfolding and aggregation of a recombinant Rab2 protein from L. donovani (rLdRab2) using multi-spectroscopic probes.

Methods: The acidic unfolding of rLdRab2 is characterised by intrinsic fluorescence and ANS assay, while aggregation is determined by Thioflavin-T and 90⁰ light scattering assay. Circular dichroism determined the secondary structure of monomers and aggregates. The aggregate morphology was imaged by transmission electron microscopy.

Results: rLdRab2 was modelled to be a Rab2 isoform with loose globular packing. The acidinduced unfolding of the protein is a plausible non-two-state process. At pH 2.0, a partially folded intermediate (PFI) state characterised by ~ 30% structural loss and exposed hydrophobic core was found to accumulate. The PFI state slowly converted into well-developed protofibrils at high protein concentrations demonstrating its amyloidogenic nature. The native state of the protein was also observed to be aggregation-prone at high protein concentrations. However, it formed amorphous aggregation instead of fibrils.

Conclusion: To our knowledge, this is the first study to report in vitro amyloid-like behaviour of Rab proteins in L donovani. This study provides a novel opportunity to understand the complete biophysical characteristics of Rab2 protein of the lower eukaryote, L. donovani.

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[1]
Selvapandiyan, A.; Croft, S.L.; Rijal, S.; Nakhasi, H.L.; Ganguly, N.K. Innovations for the elimination and control of visceral leishmaniasis. PLoS Negl. Trop. Dis., 2019, 13(9), e0007616.
[http://dx.doi.org/10.1371/journal.pntd.0007616] [PMID: 31536490]
[2]
Burza, S.; Croft, S.L.; Boelaert, M. Leishmaniasis. Lancet, 2018, 392(10151), 951-970.
[http://dx.doi.org/10.1016/S0140-6736(18)31204-2] [PMID: 30126638]
[3]
Selvapandiyan, A.; Puri, N.; Kumar, P.; Alam, A.; Ehtesham, N.Z.; Griffin, G.; Hasnain, S.E. Zooming in on common immune evasion mechanisms of pathogens in phagolysosomes: Potential broad-spectrum therapeutic targets against infectious diseases. FEMS Microbiol. Rev., 2023, 47(1), fuac041.
[http://dx.doi.org/10.1093/femsre/fuac041] [PMID: 36309472]
[4]
Shanmugam, S.; Kumar, K.; Singh, P.K.; Rastogi, R.; Mukhopadhyay, A. Single GDP-dissociation Inhibitor Protein regulates endocytic and secretory pathways in Leishmania. Sci. Rep., 2016, 6(1), 37058.
[http://dx.doi.org/10.1038/srep37058] [PMID: 27841328]
[5]
Pylypenko, O.; Hammich, H.; Yu, I.M.; Houdusse, A. Rab GTPases and their interacting protein partners: Structural insights into Rab functional diversity. Small GTPases, 2018, 9(1-2), 22-48.
[http://dx.doi.org/10.1080/21541248.2017.1336191] [PMID: 28632484]
[6]
Shi, M.; Shi, C.; Xu, Y. Rab GTPases: The key players in the molecular pathway of parkinson’s disease. Front. Cell. Neurosci., 2017, 11, 1-8.
[http://dx.doi.org/10.3389/fncel.2017.00081]
[7]
Hutagalung, A.H.; Novick, P.J. Role of Rab GTPases in membrane traffic and cell physiology. Physiol. Rev., 2011, 91(1), 119-149.
[http://dx.doi.org/10.1152/physrev.00059.2009] [PMID: 21248164]
[8]
Bahl, S; Parashar, S; Malhotra, H Functional characterization of monomeric gtpase rab1 in the secretory pathway of leishmania. J. Biol. Chem., 2015, 290(5), 29993-30005.
[http://dx.doi.org/10.1074/jbc.M115.670018]
[9]
Rastogi, R.; Verma, J.K.; Kapoor, A.; Langsley, G.; Mukhopadhyay, A. Rab5 isoforms specifically regulate different modes of endocytosis in Leishmania. J. Biol. Chem., 2016, 291(28), 14732-14746.
[http://dx.doi.org/10.1074/jbc.M116.716514] [PMID: 27226564]
[10]
Veleri, S.; Punnakkal, P.; Dunbar, G.L.; Maiti, P. Molecular insights into the roles of rab proteins in intracellular dynamics and neurodegenerative diseases. Neuromolecular Med., 2018, 20(1), 18-36.
[http://dx.doi.org/10.1007/s12017-018-8479-9] [PMID: 29423895]
[11]
Jordan, K.L.; Koss, D.J.; Outeiro, T.F.; Giorgini, F. Therapeutic targeting of rab gtpases: Relevance for alzheimer’s disease. Biomedicines, 2022, 10(5), 1141.
[http://dx.doi.org/10.3390/biomedicines10051141] [PMID: 35625878]
[12]
Martínez-Menárguez, J.Á.; Martínez-Alonso, E.; Cara-Esteban, M.; Tomás, M. Focus on the small GTPase Rab1: A key player in the pathogenesis of Parkinson’s disease. Int. J. Mol. Sci., 2021, 22(21), 12087.
[http://dx.doi.org/10.3390/ijms222112087] [PMID: 34769517]
[13]
Guadagno, NA.; Progida, C. Rab GTPases: Switching to human diseases. Cells, 2019, 8(8), 909.
[14]
Maheshwari, D; Yadav, R; Rastogi, R Structural and biophysical characterization of rab5a from leishmania donovani. Biophys. J., 2018, 115(7), 1217-1230.
[http://dx.doi.org/10.1016/j.bpj.2018.08.032]
[15]
Chamakh-ayari, R.; Chenik, M. Chakroun, AS Leishmania major large RAB GTPase is highly immunogenic in individuals immune to cutaneous and visceral leishmaniasis. Parasit. Vectors, 2017, 10(1), 185.
[http://dx.doi.org/10.1186/s13071-017-2127-3]
[16]
Takahashi, T.; Minami, S. Tsuchiya, Y Cytoplasmic control of Rab family small GTPases through BAG6. EMBO Rep., 2019, 20(4), e46794.
[http://dx.doi.org/10.15252/embr.201846794]
[17]
Homma, Y. Rab family of small GTPases: An updated view on their regulation and functions. FEBS J., 2021, 288(1), 36-55.
[http://dx.doi.org/10.1111/febs.15453]
[18]
Downing, T.; Imamura, H.; Decuypere, S.; Clark, T.G.; Coombs, G.H.; Cotton, J.A.; Hilley, J.D.; de Doncker, S.; Maes, I.; Mottram, J.C.; Quail, M.A.; Rijal, S.; Sanders, M.; Schönian, G.; Stark, O.; Sundar, S.; Vanaerschot, M.; Hertz-Fowler, C.; Dujardin, J.C.; Berriman, M. Whole genome sequencing of multiple Leishmania donovani clinical isolates provides insights into population structure and mechanisms of drug resistance. Genome Res., 2011, 21(12), 2143-2156.
[http://dx.doi.org/10.1101/gr.123430.111] [PMID: 22038251]
[19]
Rogers, M.B.; Hilley, J.D.; Dickens, N.J.; Wilkes, J.; Bates, P.A.; Depledge, D.P.; Harris, D.; Her, Y.; Herzyk, P.; Imamura, H.; Otto, T.D.; Sanders, M.; Seeger, K.; Dujardin, J.C.; Berriman, M.; Smith, D.F.; Hertz-Fowler, C.; Mottram, J.C. Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania. Genome Res., 2011, 21(12), 2129-2142.
[http://dx.doi.org/10.1101/gr.122945.111] [PMID: 22038252]
[20]
Ahuja, K.; Beg, M.A.; Sharma, R.; Saxena, A.; Naqvi, N.; Puri, N.; Rai, P.K.; Chaudhury, A.; Duncan, R.; Salotra, P.; Nakhasi, H.; Selvapandiyan, A. A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites. Biochim. Biophys. Acta Mol. Cell Res., 2018, 1865(8), 1148-1159.
[http://dx.doi.org/10.1016/j.bbamcr.2018.05.012] [PMID: 29800602]
[21]
Vats, K.; Tandon, R. Roshanara; Beg, M.A.; Corrales, R.M.; Yagoubat, A.; Reyaz, E.; Wani, T.H.; Baig, M.S.; Chaudhury, A.; Krishnan, A.; Puri, N.; Salotra, P.; Sterkers, Y.; Selvapandiyan, A. Interaction of novel proteins, centrin4 and protein of centriole in Leishmania parasite and their effects on the parasite growth. Biochim. Biophys. Acta Mol. Cell Res., 2023, 1870(3), 119416.
[http://dx.doi.org/10.1016/j.bbamcr.2022.119416] [PMID: 36623775]
[22]
Gasteiger, E; Hoogland, C; Gattiker, A 2005.The Proteomics Protocols Handbook.,
[http://dx.doi.org/ 10.1385/1592598900]
[23]
Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. The SWISS-MODEL workspace: A web-based environment for protein structure homology modelling. Bioinformatics, 2006, 22(2), 195-201.
[http://dx.doi.org/10.1093/bioinformatics/bti770] [PMID: 16301204]
[24]
Kelley, L.A.; Mezulis, S.; Yates, C.M. Trabajo práctico No 13. Varianzas en función de variable independiente categórica. Nat. Protoc., 2016, 10, 845-858.
[http://dx.doi.org/10.1038/nprot.2015.053] [PMID: 25950237]
[25]
Rost, B.; Yachdav, G.; Liu, J. The PredictProtein server. Nucleic Acids Res. 2004, 32(Web Server), W321-W326.
[http://dx.doi.org/ 10.1093/nar/gkh377] [PMID: 15215403]
[26]
Drozdetskiy, A.; Cole, C.; Procter, J.; Barton, G.J. JPred4: A protein secondary structure prediction server. Nucleic Acids Res., 2015, 43(W1), W389-W394.
[http://dx.doi.org/10.1093/nar/gkv332] [PMID: 25883141]
[27]
Ahmad, S.; Selvapandiyan, A.; Bhatnagar, R.K. Phylogenetic analysis of gram-positive bacteria based on grpE, encoded by the dnaK operon. Int. J. Syst. Evol. Microbiol., 2000, 50(5), 1761-1766.
[http://dx.doi.org/10.1099/00207713-50-5-1761] [PMID: 11034484]
[28]
Chaudhary, A.P.; Vispute, N.H.; Shukla, V.K.; Ahmad, B. A comparative study of fibrillation kinetics of two homologous proteins under identical solution condition. Biochimie, 2017, 132, 75-84.
[http://dx.doi.org/10.1016/j.biochi.2016.11.002] [PMID: 27825804]
[29]
Muthu, S.A.; Mothi, N.; Shiriskar, S.M.; Pissurlenkar, R.R.S.; Kumar, A.; Ahmad, B. Physical basis for the ofloxacin-induced acceleration of lysozyme aggregation and polymorphism in amyloid fibrils. Arch. Biochem. Biophys., 2016, 592, 10-19.
[http://dx.doi.org/10.1016/j.abb.2016.01.005] [PMID: 26777461]
[30]
Borana, M.S.; Mishra, P.; Pissurlenkar, R.R.S.; Hosur, R.V.; Ahmad, B. Curcumin and kaempferol prevent lysozyme fibril formation by modulating aggregation kinetic parameters. Biochim. Biophys. Acta. Proteins Proteomics, 2014, 1844(3), 670-680.
[http://dx.doi.org/10.1016/j.bbapap.2014.01.009] [PMID: 24468532]
[31]
Yang, X-Z.; Li, X-X.; Zhang, Y-J.; Rodriguez-Rodriguez, L.; Xiang, M-Q.; Wang, H-Y.; Zheng, X F S. Rab1 in cell signaling, cancer and other diseases. Oncogene, 2016, 35(44), 5699-5704.
[http://dx.doi.org/10.1038/onc.2016.81] [PMID: 27041585]
[32]
Eftink, M.R. The use of fluorescence methods to monitor unfolding transitions in proteins. Biophys. J., 1994, 66(2), 482-501.
[http://dx.doi.org/10.1016/S0006-3495(94)80799-4] [PMID: 8161701]
[33]
Ghisaidoobe, A.; Chung, S. Intrinsic tryptophan fluorescence in the detection and analysis of proteins: A focus on Förster resonance energy transfer techniques. Int. J. Mol. Sci., 2014, 15(12), 22518-22538.
[http://dx.doi.org/10.3390/ijms151222518] [PMID: 25490136]
[34]
White, A. Effect of pH on fluorescence of tyrosine, tryptophan and related compounds. Biochem. J., 1959, 71(2), 217-220.
[http://dx.doi.org/10.1042/bj0710217] [PMID: 13628557]
[35]
Uversky, V.; Finkelstein, A. Life in phases: Intra-and inter-molecular phase transitions in protein solutions. Biomolecules, 2019, 9(12), 842.
[http://dx.doi.org/10.3390/biom9120842] [PMID: 31817975]
[36]
Ivankov, D.N.; Finkelstein, A.V. Solution of levinthal’s paradox and a physical theory of protein folding times. Biomolecules, 2020, 10(2), 250.
[http://dx.doi.org/10.3390/biom10020250] [PMID: 32041303]
[37]
Arya, S.; Kumari, A.; Dalal, V.; Bhattacharya, M.; Mukhopadhyay, S. Appearance of annular ring-like intermediates during amyloid fibril formation from human serum albumin. Phys. Chem. Chem. Phys., 2015, 17(35), 22862-22871.
[http://dx.doi.org/10.1039/C5CP03782D] [PMID: 26264974]
[38]
Cardamone, M.; Puri, N.K. Spectrofluorimetric assessment of the surface hydrophobicity of proteins. Biochem. J., 1992, 282(2), 589-593.
[http://dx.doi.org/10.1042/bj2820589] [PMID: 1546973]
[39]
Gregoire, S.; Irwin, J.; Kwon, I. Techniques for monitoring protein misfolding and aggregation in vitro and in living cells. Korean J. Chem. Eng., 2012, 29(6), 693-702.
[http://dx.doi.org/10.1007/s11814-012-0060-x] [PMID: 23565019]
[40]
Dobson, C.M.; Knowles, T.P.J.; Vendruscolo, M. The amyloid phenomenon and its significance in biology and medicine. Cold Spring Harb. Perspect. Biol., 2020, 12(2), a033878.
[http://dx.doi.org/10.1101/cshperspect.a033878] [PMID: 30936117]
[41]
Ahmad, SP. Exposure of aggregation prone segments is the requirement for amyloid fibril formation. Curr. Protein Pept. Sci., 2018, 19, 1024-1035.
[42]
Tsolis, A.C.; Papandreou, N.C.; Iconomidou, V.A.; Hamodrakas, S.J. A consensus method for the prediction of ‘aggregation-prone’ peptides in globular proteins. PLoS One, 2013, 8(1), e54175.
[http://dx.doi.org/10.1371/journal.pone.0054175] [PMID: 23326595]
[43]
Kumar, S.; Udgaonkar, JB. Mechanisms of amyloid fibril formation by proteins. Biochemistry, 2014, 79(13), 1515-1527.
[44]
Juárez, J.; Taboada, P.; Mosquera, V. Existence of different structural intermediates on the fibrillation pathway of human serum albumin. Biophys. J., 2009, 96(6), 2353-2370.
[http://dx.doi.org/10.1016/j.bpj.2008.12.3901] [PMID: 19289061]
[45]
Frare, E.; Mossuto, M.F.; de Laureto, P.P.; Tolin, S.; Menzer, L.; Dumoulin, M.; Dobson, C.M.; Fontana, A. Characterization of oligomeric species on the aggregation pathway of human lysozyme. J. Mol. Biol., 2009, 387(1), 17-27.
[http://dx.doi.org/10.1016/j.jmb.2009.01.049] [PMID: 19361437]
[46]
Yokoyama, K.; Fisher, A.D.; Amori, A.R.; Welchons, D.R.; McKnight, R.E. Spectroscopic and calorimetric studies of congo red dye-amyloid peptide complexes. J. Biophys. Chem., 2010, 1(3), 153-163.
[http://dx.doi.org/10.4236/jbpc.2010.13018]
[47]
Patel, N; Singh, SB.; Basu, SK. Mukhopadhyay, A Leishmania requires Rab7-mediated degradation of endocytosed hemoglobin for their growth. Proc. Natl. Acad. Sci. , 2008, 105(10), 3980-3985.
[http://dx.doi.org/10.1073/pnas.0800404105]
[48]
Kovacs, G.G. Molecular pathology of neurodegenerative diseases: Principles and practice. J. Clin. Pathol., 2019, 72(11), 725-735.
[http://dx.doi.org/10.1136/jclinpath-2019-205952] [PMID: 31395625]
[49]
Castillo, V.; Ventura, S. Amyloidogenic regions and interaction surfaces overlap in globular proteins related to conformational diseases. PLOS Comput. Biol., 2009, 5(8), e1000476.
[http://dx.doi.org/10.1371/journal.pcbi.1000476] [PMID: 19696882]
[50]
Wu, J.W.; Chen, M.E.; Wen, W.S.; Chen, W.A.; Li, C.T.; Chang, C.K.; Lo, C.H.; Liu, H.S.; Wang, S.S.S. Comparative analysis of human γD-crystallin aggregation under physiological and low pH conditions. PLoS One, 2014, 9(11), e112309.
[http://dx.doi.org/10.1371/journal.pone.0112309] [PMID: 25389780]
[51]
Chaari, A.; Fahy, C.; Chevillot-Biraud, A.; Rholam, M. Insights into kinetics of agitation-induced aggregation of hen lysozyme under heat and acidic conditions from various spectroscopic methods. PLoS One, 2015, 10(11), e0142095.
[http://dx.doi.org/10.1371/journal.pone.0142095] [PMID: 26571264]
[52]
Banworth, MJ. Li, G Consequences of Rab GTPase dysfunction in genetic or acquired human diseases. Small GTPases, 2018, 9(1-2), 158-181.
[http://dx.doi.org/10.1080/21541248.2017.1397833]

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