Please wait a minute...
Journal of Molecular and Clinical Medicine   2019, Vol. 2 Issue (2): 29-34    DOI: 10.31083/j.jmcm.2019.02.7161
Original Research Previous articles | Next articles
ALIX protein analysis: storage temperature may impair results
Lopes-Rodrigues Vanessa1, 2, 3, P. R. Xavier Cristina1, 2, Sousa Diana1, 2, 4, Osório Hugo1, 5, 6, G. Assaraf Yehuda7, T. Lima Raquel1, 2, 5, Helena Vasconcelos M.1, 2, 4, *()
1 i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
2 Cancer Drug Resistance Group, IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, 4200-465 Porto, Portugal
3 Institute of Biomedical Sciences Abel Salazar, ICBAS-UP–Institute of Biomedical Sciences Abel Salazar of the University of Porto, 4099-003 Porto, Portugal
4 Department of Biological Sciences, FFUP-Faculty of Pharmacy of the University of Porto, 4050-313 Porto, Portugal
5 Department of Oncology, FMUP–Faculty of Medicine of the University of Porto, 4200-319 Porto, Portugal
6 IPATIMUP-Institute of Molecular Pathology and Immunology of the University of Porto, 4200-465 Porto, Portugal
7 The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, 3200000 Haifa, Israel
Download:  PDF(4560KB)  ( 56 ) Full text   ( 13 )
Export:  BibTeX | EndNote (RIS)      

ALIX [ALG-2 (apoptosis-linked gene 2)-interacting protein X] is one of the most well-known molecular biomarkers of extracellular vesicles. Extracellular vesicles are very small vesicles released by most cells and carry in their cargo components from the donor cells, thus being potent vehicles of intercellular (horizontal) communication, influencing various physiological and pathological functions of both recipient and donor cells. The increasing interest in extracellular vesicles highlights the key importance of a reliable analysis of this protein. However, several recent studies in the extracellular vesicles field have shown discrepancies in terms of the expression pattern of apoptosis-linked gene 2-interacting protein X upon Western blot analysis, differing from the theoretical expression pattern of apoptosis-linked gene 2-interacting protein X and its predicted molecular mass. Therefore, to address and clarify this point, we analyzed total protein cell lysates from a chronic myeloid leukemia cell line (K562) for the expression of apoptosis-linked gene 2-interacting protein X by Western blot and mass spectrometry analyses, using protein samples stored at different conditions regarding freezing temperature and storage time. We found that, when stored at -20 oC, a C-terminal specific proteolytic cleavage of apoptosis-linked gene 2-interacting protein X may occur, which depends on the length of storage time. We conclude that analysis of apoptosis-linked gene 2-interacting protein X protein expression should be only carried out when using a wide range of protease inhibitors during isolation of protein cell extract, while preferentially using fresh protein cell extracts or samples that were snap frozen in liquid nitrogen and stored at -80 oC. The current study highlights the importance of proper handling and storage of protein cell lysates for downstream applications in pre-clinical or clinical studies.

Key words:  ALIX      extracellular vesicles      protein degradation      reliable protein identification      storage temperature     
Published:  20 April 2019     
*Corresponding Author(s):  Helena Vasconcelos M.     E-mail:

Cite this article: 

Lopes-Rodrigues Vanessa,P. R. Xavier Cristina,Sousa Diana,Osório Hugo,G. Assaraf Yehuda,T. Lima Raquel,Helena Vasconcelos M.. ALIX protein analysis: storage temperature may impair results. Journal of Molecular and Clinical Medicine , 2019, 2(2): 29-34.

URL:     OR

Figure 1.  Western blot analysis of ALIX protein levels in K562 cells following various storage conditions. Fresh total cell protein lysates were immediately stored at: -20 oC , -20 oC with a phosphatase inhibitor cocktail (+ PI) or at -80 oC . Samples were kept frozen for 1, 2, 3 and 6 weeks. (A) Representative Western blot images; (B) Scanning densitometry analysis of the Western blot ALIX bands, represented as mean ± SEM from three in dependent experiments. β-Actin was used as a loading control. * P ≤ 0.05 when comparing protein levels in samples stored at -20 oC (-PI) vs. other storage conditions (- 80 oC or -20 oC + PI).

Figure 2.  Mass spectrometry analysis of the two ALIX bands. The sequences of both the 93 kDa (A) and 75 kDa (B) bands were analyzed and the peptides related to ALIX were detected. The C-terminal 716-745 peptide, which was present in the sequence of the intact 93 kDa protein (A, marked with a box) was absent from the sequence of the 75 kDa protein (B).

Figure S1.  Mass spectrometry analysis of the 93 kDa ALIX band. The analysis of the sequence of the 93 kDa protein is shown in detail (sequences and peaks of the peptides detected).

Figure S2.  Mass spectrometry analysis of the 75 kDa ALIX band. The analysis of the sequence of 75 kDa protein is shown in detail (sequences and peaks of the peptides detected).

[1] Holohan C, Van Schaeybroeck S, Longley DB, Johnston PG. Cancer drug resistance: an evolving paradigm. Nature Reviews Cancer, 2013; 13: 714-726.
doi: 10.1038/nrc3599
[2] Yanez-Mo M, Siljander PR, Andreu Z, Zavec AB, Borras FE, Buzas EI, et al. Biological properties of extracellular vesicles and their physiological functions. Journal of Extracellular Vesicles, 2015; 4:27066.
doi: 10.3402/jev.v4.27066 pmid: 25979354
[3] Rahbarghazi R, Jabbari N, Sani NA, Asghari R, Salimi L, Kalashani SA, et al. Tumor-derived extracellular vesicles: reliable tools for Cancer diagnosis and clinical applications. Cell Commun Signal, 2019; 17:73.
doi: 10.1186/s12964-019-0390-y pmid: 31291956
[4] Sousa D, Lima RT, Vasconcelos MH. Intercellular transfer of cancer drug resistance traits by extracellular vesicles. Trends In Molecular Medicine, 2015; 21 :595-608.
doi: 10.1016/j.molmed.2015.08.002 pmid: 26432017
[5] Jan AT, Rahman S, Khan S, Tasduq SA, Choi I. Biology, Pathophysiological Role, and Clinical Implications of Exosomes: A Critical Appraisal. Cells, 2019; 8 :pii: E99.
doi: 10.3390/cells8020199 pmid: 30823596
[6] Shah R, Patel T, Freedman JE. Circulating Extracellular Vesicles in Human Disease. New England Journal of Medicine, 2018; 379 :958-966.
doi: 10.1056/NEJMra1704286 pmid: 30184457
[7] Kelemen E, Danis J, Goblos A, Bata-Csorgo Z, Szell M. Exosomal long non-coding RNAs as biomarkers in human diseases. Electronic Journal of the IFCC, 2019; 30 :224-236.
doi: 10.1093/mutage/gez038 pmid: 31784760
[8] S ELA, Mager I, Breakefield XO, Wood MJ . Extracellular vesicles: biology and emerging therapeutic opportunities. Nature reviews Drug Discovery, 2013; 12 :347-357.
doi: 10.1038/nrd3978 pmid: 23584393
[9] Thery C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles, 2018; 7 :1535750.
doi: 10.1080/20013078.2018.1535750 pmid: 30637094
[10] Missotten M, Nichols A, Rieger K, Sadoul R. Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein. Cell Death Differ, 1999; 6 :124-129.
doi: 10.1038/sj.cdd.4400456 pmid: 10200558
[11] Mercier V, Laporte MH, Destaing O, Blot B, Blouin CM, Pernet-Gallay K, et al. ALG-2 interacting protein-X (Alix) is essential for clathrin-independent endocytosis and signaling. Scientific Reports, 2016; 6 :26986.
doi: 10.1038/srep26986 pmid: 27244115
[12] Odorizzi G. The multiple personalities of Alix. Journal of Cell Science, 2006; 119 :3025-3032.
doi: 10.1242/jcs.03072 pmid: 16868030
[13] Matsuo H, Chevallier J, Mayran N, Le Blanc I, Ferguson C, Faure J et al. Role of LBPA and Alix in multivesicular liposome formation and endosome organization. Science, 2004; 303 :531-534.
doi: 10.1126/science.1092425 pmid: 14739459
[14] Fujii K, Hurley JH, Freed EO. Beyond Tsg101: the role of Alix in 'ESCRTing' HIV-1. Nature Reviews Microbiology, 2007; 5: 912-916.
doi: 10.1038/nrmicro1790 pmid: 17982468
[15] Sun R, Liu Y, Lu M, Ding Q, Wang P, Zhang H, et al. ALIX increases protein content and protective function of iPSC-derived exosomes. Journal of Molecular Medicine, 2019; 97 :829-844.
doi: 10.1007/s00109-019-01767-z pmid: 30944935
[16] Trioulier Y, Torch S, Blot B, Cristina N, Chatellard-Causse C, Verna JM, et al. Alix, a protein regulating endosomal trafficking, is involved in neuronal death. Journal of Biological Chemistry, 2004; 279 :2046-2052.
doi: 10.1074/jbc.M309243200 pmid: 14585841
[17] Mahul-Mellier AL, Strappazzon F, Petiot A, Chatellard-Causse C, Torch S, Blot B, et al. Alix and ALG-2 are involved in tumor necrosis factor receptor 1-induced cell death. Journal of Biological Chemistry, 2008; 283 :34954-34965.
doi: 10.1074/jbc.M803140200 pmid: 18936101
[18] Strack B, Calistri A, Craig S, Popova E, Gottlinger HG. AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding. Cell, 2003; 114 :689-699.
doi: 10.1016/s0092-8674(03)00653-6 pmid: 14505569
[19] Morita E, Colf LA, Karren MA, Sandrin V, Rodesch CK, Sundquist WI. Human ESCRT-III and VPS4 proteins are required for centrosome and spindle maintenance. Proceedings Of the National Academy of Sciences of the United States of America, 2010; 107 :12889-12894.
doi: 10.1073/pnas.1005938107 pmid: 20616062
[20] Pan S, Wang R, Zhou X, Corvera J, Kloc M, Sifers R, et al . Extracellular alix regulates integrin-mediated cell adhesions and extracellular matrix assembly. The EMBO Journal, 2008; 27 :2077-2090.
doi: 10.1038/emboj.2008.134 pmid: 18636094
[21] Jimenez AJ, Maiuri P, Lafaurie-Janvore J, Divoux S, Piel M, Perez F. ESCRT machinery is required for plasma membrane repair. Science, 2014; 343 :1247136.
doi: 10.1126/science.1247136 pmid: 24482116
[22] Scheffer LL, Sreetama SC, Sharma N, Medikayala S, Brown KJ, Defour A, et al. Mechanism of Ca2+-triggered ESCRT assembly and regulation of cell membrane repair. Nature Communications, 2014; 5 :5646.
doi: 10.1038/ncomms6646 pmid: 25534348
[23] Alrosan A, Aleidi SM, Yang A, Brown AJ, Gelissen IC. The adaptor protein alix is involved in the interaction between the ubiquitin ligase NEDD4-1 and its targets, ABCG1 and ABCG4. International Journal of Molecular Sciences, 2019; 20 :2714.
doi: 10.3390/ijms20112850 pmid: 31212704
[24] Zhao C, Ban N, Dai S, Zhang X, Zhang L, Xu P, et al. The role of Alix in the proliferation of human glioma cells. Human Pathology, 2016; 52 :110-118.
doi: 10.1016/j.humpath.2015.09.046 pmid: 26980041
[25] Christ L, Wenzel EM, Liestol K, Raiborg C, Campsteijn C, Stenmark H. ALIX and ESCRT-I/II function as parallel ESCRT-III recruiters in cytokinetic abscission. Journal of Cell Biology, 2016; 212 :499-513.
doi: 10.1083/jcb.201507009 pmid: 26929449
[26] Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A, et al. Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nature Cell Biology, 2012; 14 :677-685.
doi: 10.1038/ncb2502
[27] Mi S, Qin XW, Lin YF, He J, Chen NN, Liu C , et al. Budding of Tiger Frog Virus (an Iridovirus) from HepG2 Cells via Three Ways Recruits the ESCRT Pathway. Scientific Reports, 2016; 6 :26581.
doi: 10.1038/srep26581 pmid: 27225426
[28] Ghossoub R, Lembo F, Rubio A, Gaillard CB, Bouchet J, Vitale N, et al. Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2. Nature Communications, 2014; 5 :3477.
doi: 10.1038/ncomms4477 pmid: 24637612
[29] Campos Y, Qiu X, Gomero E, Wakefield R, Horner L, Brutkowski W, et al. Alix-mediated assembly of the actomyosin-tight junction polarity complex preserves epithelial polarity and epithelial barrier. Nature Communications, 2016; 7 :11876.
doi: 10.1038/ncomms11876 pmid: 27336173
[30] Simpson RJ. Stabilization of proteins for storage. Cold Spring Harb Protoc, 2010; 2010: pdb. top79.
doi: 10.1101/pdb.top79 pmid: 20439424
[31] Verhamme IM, Leonard SE, Perkins RC. Proteases: Pivot points in functional proteomics. Molecular Biology Reports, 2019; 1871 :313-392.
doi: 10.1007/978-1-4939-8814-3_20 pmid: 30276748
[32] Goodwin RJA. Sample preparation for mass spectrometry imaging: small mistakes can lead to big consequences. Journal of Proteomics, 2012; 75 :4893-4911.
doi: 10.1016/j.jprot.2012.04.012
[33] Hancock WS, Wu SL, Shieh P. The challenges of developing a sound proteomics strategy. Proteomics, 2002; 2 :352-359.
doi: 10.1002/1615-9861(200204)2:4<352::AID-PROT352>3.0.CO;2-U pmid: 12164694
[34] Iavello A, Frech VS, Gai C, Deregibus MC, Quesenberry PJ, Camussi G . Role of Alix in miRNA packaging during extracellular vesicle biogenesis. International Journal of Molecular Medicine, 2016; 37 :958-966.
doi: 10.3892/ijmm.2016.2488 pmid: 26935291
[35] Lopes-Rodrigues V, Di Luca A, Mleczko J, Meleady P, Henry M, Pesic M , et al. Identification of the metabolic alterations associated with the multidrug resistant phenotype in cancer and their intercellular transfer mediated by extracellular vesicles. Scientific Reports, 2017; 7 :44541.
doi: 10.1038/srep44541 pmid: 28303926
[36] Lopes-Rodrigues V, Di Luca A, Sousa D, Seca H, Meleady P, Henry M, et al. Multidrug resistant tumour cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells. Biochimica et Biophysica Acta, 2016; 1860 :618-627.
doi: 10.1016/j.bbagen.2015.12.011 pmid: 26708992
[37] Gomes C, Almeida A, Ferreira JA, Silva L, Santos-Sousa H, Pinto-de-Sousa J, et al. Glycoproteomic analysis of serum from patients with gastric precancerous lesions. Journal of Proteome Research, 2013; 12 :1454-1466.
doi: 10.1021/pr301112x pmid: 23312025
[38] Osorio H, Reis CA. Mass spectrometry methods for studying glycosylation in cancer. Molecular Biology Reports, 2013; 1007 :301-316.
doi: 10.1007/978-1-62703-392-3_13 pmid: 23666732
[39] Ferreirinha P, Correia A, Teixeira-Coelho M, Osorio H, Teixeira L, Rocha A , et al. Mucosal immunization confers long-term protection against intragastrically established Neospora caninum infection. Vaccine, 2016; 34 :6250-6258.
doi: 10.1016/j.vaccine.2016.10.056 pmid: 27814932
[40] Biniossek ML, Nagler DK, Becker-Pauly C, Schilling O . Proteomic identification of protease cleavage sites characterizes prime and non-prime specificity of cysteine cathepsins B, L, and S. Journal of Proteome Research, 2011; 10 :5363-5373.
doi: 10.1021/pr200621z
[41] Impens F, Colaert N, Helsens K, Ghesquiere B, Timmerman E, De Bock PJ, et al. A quantitative proteomics design for systematic identification of protease cleavage events. Molecular & Cellular Proteomics, 2010; 9 :2327-2333.
doi: 10.1002/psc.3239 pmid: 31847053
No related articles found!
No Suggested Reading articles found!