J Cell Mol Med. 2014 Jan;18(1):59-68.

T lymphocytes export proteasomes by way of microparticles: a possible mechanism for generation of extracellular proteasomes.

Bochmann I, Ebstein F, Lehmann A, Wohlschlaeger J, Sixt SU, Kloetzel PM, Dahlmann B.

Institut für Biochemie, Charité-Universitätsmedizin Berlin, Berlin, Germany.

 

Abstract

The 20S proteasome is almost exclusively localized within cells. High levels of extracellular proteasomes are also found circulating in the blood plasma of patients suffering from a variety of inflammatory, autoimmune and neoplastic diseases. However, the origin of these proteasomes remained enigmatic. Since the proteome of microparticles, small membrane enclosed vesicles released from cells, was shown to contain proteasomal subunits, we studied whether intact proteasomes are actively released into the extracellular space. Using human primary T lymphocytes stimulated with CaCl2 and the calcium ionophore A23187 to induce membrane blebbing we demonstrate that microparticles contain proteolytically active 20S proteasomes as well as the proteasome activator PA28 and subunits of the 19S proteasome regulator. Furthermore, our experiments reveal that incubation of in vitro generated T lymphocyte-microparticles with sphingomyelinase results in the hydrolysis of the microparticle membranes and subsequent release of proteasomes from the vesicles. Thus, we here show for the first time that functional proteasomes can be exported from activated immune cells by way of microparticles, the dissolution of which may finally lead to the generation of extracellular proteasomes.

KEYWORDS: T lymphocytes; circulating; extracellular; microparticles; proteasome; sphingomyelinase

PMID: 24304442

 

Supplementary

Background

The proteasome is a multicatalytic protease involved in the degradation of most intracellular proteins and therefore crucial for maintaining intracellular proteostasis. It is composed of a multisubunit barrel-shaped 20S complex (20S proteasome) that contains peptide bond-hydrolysing activity in its interior cavity (figure 1), and one (26S proteasome) or two (30S proteasome) 19S regulators, which are responsible for substrate recognition and intake [1].

 

 

f1Figure 1. The 20S proteasome is composed of four seven-membered rings, the outer ones contain α-subunits and the two inner ones are build up by β-subunits bearing the active, proteolytic sites.

Extracellular proteasomes are 20S proteasomes present in blood plasma, broncho-alveolar and in cerebrospinal fluid [2, 3]. Their blood levels are raised under pathological conditions like autoimmune and certain cancer diseases, sometimes corresponding to the progression of the disease. Therefore circulating proteasomes are investigated as potential diagnostic biomarkers. In previous investigations, we have purified circulating proteasomes from plasma of healthy individuals as well as from rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients and have shown that these proteasomes are active proteases [4]. Additionally, we have demonstrated that their subtype pattern (composition of 20S proteasomes with different physicochemical features) did not fit to the subtype pattern of four major blood cells, namely, erythrocytes, thrombocytes, monocytes and T lymphocytes, suggesting that extracellular proteasomes originate from different cell types.

So far it is not known how extracellular proteasomes leave their parental cells. Cell death as a source for extracellular proteasomes cannot be excluded, but studies comparing markers for cell lysis with the level of circulating proteasomes in patients suffering from different diseases, point to a specific release mechanism [1, 5].

Findings

Measurements of proteasome activity in cell culture supernatants revealed that the extracellular proteasomal activity was restricted to the vesicle fraction. The scientific interest in extracellular vesicular structures increased during the last decades, especially in the fields of oncology and immunology, because they are believed to have important functions as mediators of intercellular communication by transferring molecules or inductors of signal transduction pathways in recipient cells. Therefore, we investigated whether 20S proteasomes use this pathway to enter the extracellular environment.

Induction of microvesicle shedding in human T cells lead to the formation of membrane-enclosed microparticles (called blebbing) directly from the plasma membrane carrying cytosolic components of the parental cell. In our experiments, we used a calcium-ionophore, but stimulation of vesicle shedding was also shown by endogeneous substances like cytokines, complement or C-reactive protein.

Extracellular vesicles contain proteins, nucleic acids and lipids. Depending on their size and origin they can be divided into exosomes (diameter of 0.05-0.1 µm), microparticles (0.1-1 µm) and larger vesicles like apoptotic bodies; however, there is an overlap between the different vesiclular bodies.

 

f2

Figure 2. Cell shedding of 20S proteasome-containing microparticles.

 

Our experiments revealed that proteolytically active 20S proteasomes of T lymphocytes were packaged into microparticles (figure 2).

Since proteasomes in blood plasma can easily be quantified by ELISA technique, they obviously are not enclosed by membrane-bilayers. Therefore, we next investigated how extracellular proteasomes are released from microvesicles.

In patients suffering from pathological conditions like rheumatoid arthritis or sepsis phospholipases like cytosolic phospholipase A2 or sphingomyelinase were reported to have elevated activity in blood plasma [6-8]. Our experiments actually showed that by the action of sphingomyelinase, an enzyme that cleaves phospholipids in biomembranes, catalytically active 20S proteasomes could be released from the microvesicles in vitro.

Outlook

Thus, our study provides evidence that 20S proteasomes are exported from T cells by microvesicles. Whether proteasomes still entrapped in vesicles might have specific functions, e.g. because they could be taken up by endocytosis of recipient cells, has to be elucidated. On the other hand, microvesicles may enzymatically be broken down in the extracellular space to release free extracellularly circulating 20S proteasomes, which were shown to have diagnostic value during several pathological conditions.

 

f3Figure 3. Proposed model for generation of circulating 20S proteasomes by shedding of extracellular vesicles.

 

References:

[1]          S.U. Sixt and B. Dahlmann, Extracellular, circulating proteasomes and ubiquitin – incidence and relevance, Biochim Biophys Acta. 1782 (2008) 817-823.

[2]          S.U. Sixt, M. Adamzik, D. Spyrka, B. Saul, J. Hakenbeck, J. Wohlschlaeger, U. Costabel, A. Kloss, J. Giesebrecht, B. Dahlmann and J. Peters, Alveolar extracellular 20S proteasome in patients with acute respiratory distress syndrome, Am J Respir Crit Care Med. 179 (2009) 1098-1106.

[3]          O. Mueller, T. Anlasik, J. Wiedemann, J. Thomassen, J. Wohlschlaeger, V. Hagel, K. Keyvani, I. Schwieger, B. Dahlmann, U. Sure and S.U. Sixt, Circulating Extracellular Proteasome in the Cerebrospinal Fluid: A Study on Concentration and Proteolytic Activity, J Mol Neurosci.  (2011).

[4]          A. Zoeger, M. Blau, K. Egerer, E. Feist and B. Dahlmann, Circulating proteasomes are functional and have a subtype pattern distinct from 20S proteasomes in major blood cells, Clin Chem. 52 (2006) 2079-2086.

[5]          K. Egerer, U. Kuckelkorn, P.E. Rudolph, J.C. Ruckert, T. Dorner, G.R. Burmester, P.M. Kloetzel and E. Feist, Circulating proteasomes are markers of cell damage and immunologic activity in autoimmune diseases, J Rheumatol. 29 (2002) 2045-2052.

[6]          R.A. Claus, A.C. Bunck, C.L. Bockmeyer, F.M. Brunkhorst, W. Losche, R. Kinscherf and H.P. Deigner, Role of increased sphingomyelinase activity in apoptosis and organ failure of patients with severe sepsis, FASEB J. 19 (2005) 1719-1721.

[7]          M.K. Lin, V. Farewell, P. Vadas, A.A. Bookman, E.C. Keystone and W. Pruzanski, Secretory phospholipase A2 as an index of disease activity in rheumatoid arthritis. Prospective double blind study of 212 patients, J Rheumatol. 23 (1996) 1162-1166.

[8]          J.A. Green, G.M. Smith, R. Buchta, R. Lee, K.Y. Ho, I.A. Rajkovic and K.F. Scott, Circulating phospholipase A2 activity associated with sepsis and septic shock is indistinguishable from that associated with rheumatoid arthritis, Inflammation. 15 (1991) 355-367.

 

Contact

Isabel Bochmann

Charité-Universitätsmedizin Berlin

Institut für Biochemie, CCO

Charitéplatz 1, 10117 Berlin

isabel.bochmann@charite.de

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