Seminars in Cell & Developmental Biology 62 (2017) 187–193

Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

j ournal homepage: www.elsevier.com/locate/semcdb

Cell penetrating SERPINA5 ( inhibitor, PCI): More questions

than answers

∗

Hanjiang Yang, Margarethe Geiger

Medical University of Vienna, Center of Physiology and Pharmacology, Department of Vascular Biology and Thrombosis Research, Schwarzspanierstrasse

17, A-1090 Vienna, Austria

a r a

t i b s

c t

l e i n f o r a c t

Article history: SERPINA5 (protein C inhibitor, inhibitor-3) is a secreted, extracellular clade A ser-

Received 27 June 2016

pin. Its main characteristics are broad reactivity and wide tissue distribution (in man). SERPINA5

Accepted 26 October 2016

has originally been described as an inhibitor of activated protein C and independently as an inhibitor of

Available online 29 October 2016

the plasminogen activator . SERPINA5 binds glycosaminoglycans, phospholipids, and retinoic

acid. Glycosaminoglycans and certain phospholipids can modulate its inhibitory activity and specificity.

Keywords:

Studies suggest that SERPINA5 may play a role in hemostasis, in male reproduction, in host defense, and

Serpin

as a tumor suppressor. However, its biological role has not yet been defined. So far SERPINA5 deficiency

Protein C inhibitor

Phospholipid has not been described in man. Mouse models are of limited value, since in mice serpinA5 is almost exclu-

sively expressed in the reproductive tract. Consistently the only obvious phenotype of serpinA5-knockout

Cell penetrating peptide

Protease mice is infertility of homozygous males. SERPINA5 can be internalized by cells and translocated to the

Spermatogenesis nucleus. The internalization is dependent on the phospholipid phosphatidylethanolamine and on the

intact N-terminus of SERPINA5, which functions as a cell penetrating peptide. Further functional analysis

of intracellular SERPINA5 will contribute to our understanding of the biological role of this molecule.

© 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction ...... 188

2. SERPINA5 (Protein C inhibitor, PCI) is not only an inhibitor of activated protein C in plasma, but a with broad protease

reactivity and wide tissue distribution in man ...... 188

3. Non-protein ligands of SERPINA5 ...... 188

4. Biological functions of SERPINA5...... 188

4.1. Role in hemostasis ...... 188

4.2. Role in male fertility ...... 189

4.3. Protective role in cancer ...... 189

4.4. Role in host defense...... 189

5. The problem with serpinA5 mouse models ...... 189

6. Internalization of SERPINA5 by cells ...... 190

7. Nuclear translocation of SERPINA5...... 190

8. Intracellular interaction partners of SERPINA5 ...... 190

9. Conclusions ...... 190

Acknowledgements ...... 191

References ...... 191

∗

Corresponding author.

E-mail addresses: [email protected] (H. Yang), [email protected] (M. Geiger).

http://dx.doi.org/10.1016/j.semcdb.2016.10.007

1084-9521/© 2016 Elsevier Ltd. All rights reserved.

188 H. Yang, M. Geiger / Seminars in Cell & Developmental Biology 62 (2017) 187–193

1. Introduction cosaminoglycans and phospholipids involves basic amino acids

in the H-helix of serpinA5 [48,50,51]. Depending on the tar-

Members of the serpin superfamily of proteins are widely dis- get protease binding of glycosaminoglycans and phospholipids

tributed in nature. They occur not only in vertebrates, but also can stimulate or suppress the inhibitory activity of serpinA5

in arthropods [1], nematodes [2,3], plants [4], prokaryotes [5–7], [42,47,52,53]. In vivo SERPINA5 could bind to glycosaminoglycans

and viruses [8]. Most are secreted, extracellular proteins, on cell surfaces as well as to phospholipids exposed on atheroscle-

but there are also intracellular serpins [9]. Serpins are classified rotic plaques [48], on apoptotic and/or activated cells [54], and

into clades and intracellular serpins form clade B (B1-B13)[10]. on microparticles [55]. Binding of SERPINA5 to cell surface gly-

For some serpins extracellular as well as in intracellular forms cosaminoglycans could modulate its activity in a -like

have been described [11,12]; and several serpins have intra- as manner [45]. Binding of SERPINA5 to phospholipids on the sur-

well as extracellular functions [13,14]. Interestingly, a few ser- face of apoptotic cells and activated platelets interferes with the

pins that are considered as classical extracellular proteins secreted phagocytotic removal of these cells [54]. Microparticles containing

into body fluids have also been detected intracellularly in the SERPINA5 are present in normal human plasma. The origin of these

nuclear compartment [15–17]. One of these secreted serpins found microparticles are megakaryocytes and/or platelets. SERPINA5

in the nucleus of certain cells is SERPINA5 (protein C inhibitor, present on these microparticles seems to be inactive, although it

PCI)[16]. does not seem to be cleaved or complexed [55].

In accordance with the serpin nomenclature [10,18] we will Another non-protein ligand of SERPINA5 is retinoic acid

use the term SERPINA5/SERPINA5 for the human protein/gene, [56]. Two non-inhibitory members of the serpin family, i.e.

and serpinA5/serpin for the mouse protein/gene, respectively. In corticosteroid-binding globulin (CBG) and thyroxine-binding pro-

this review we will focus on the interaction of SERPINA5 with tein (TBG), act as hormone carriers [57–59]. This prompted

non-protein ligands, on its internalization by cells, its nuclear us several years ago to study binding of different hydropho-

translocation, and on the discussion of possible biological functions. bic hormones to inhibitory serpins, i.e. to SERPINA5, SERPINC1

For all other aspects, such as its gene and protein structure, the bio- (), SERPIND1 (heparin cofactor II), and SERPINE1 (PAI-

chemistry of its interaction with , and its tissue specific 1) [56]. We have shown that serpinA5 bound retinoic acid, but

expression, we would like to refer to recent reviews [19–24]. none of the steroid hormones studies (estradiol, progesterone,

testosterone, cortisol, or aldosterone). None of the other inhibitory

serpins analyzed bound any of the hydrophobic hormones stud-

2. SERPINA5 (Protein C inhibitor, PCI) is not only an

ied. The inhibitory activity of serpinA5 was not affected by retinoid

inhibitor of activated protein C in plasma, but a serpin with

binding, and binding of retinoic acid to serpinA5 was not influ-

broad protease reactivity and wide tissue distribution in

enced by the cleavage of SERPINA5 by tissue kallikrein or pancreatic

man 3

elastase [56]. Binding of H-retinoic acid was not only observed

with purified SERPINA5, but also with SERPINA5 present in semi-

SERPINA5 (Mr = 57,000) is a member of the alpha-1-antitrypsin

nal plasma. Huntington and his group have crystalized reactive site

clade (clade A) of serpins. The gene structure, tissue specific expres-

cleaved and native SERPINA5 and analyzed its structure. They put

sion, and the characteristics of the SERPINA5 protein have been

special emphasis on its interaction with proteases and non-protein

described in a recent review [19]. Briefly: SERPINA5 has origi-

ligands such as heparin and retinoic acid [44,50]. Superposing

nally been identified as an inhibitor of the protease

the structure of the SERPINA5 with that of SERPINA1 (alpha-1-

activated protein C (aPC) and was therefore named protein C

antitrypsin) revealed a two-turn shortening of helix A and a rotation

inhibitor (PCI) [25,26]. Later it was shown that SERPINA5 has

of helix H. This gives rise to a large hydrophobic pocket, which

very broad protease reactivity. It inactivates proteases involved

they called helix A gap. They suggested that this helix A gap repre-

in blood coagulation and fibrinolysis [25–27], tissue- [28,29] and

sents the retinoic acid binding site of SERPINA5. This assumption is

plasma kallikreins [30], the sperm protease [31], hepato-

supported by data obtained by superimposing the structure of SER-

cyte growth factor activator [32], and the type II transmembrane

PINA5 with that of TBG bound to thyroxine [60]. So far no in vivo

serine protease enteropeptidase [33]. As a serpin it inactivates its

data supporting role of SERPINA5 as a retinoic acid binding protein

target proteases in a suicide substrate-like manner by forming

are available. It might also be possible that SERPINA5 accommo-

stable, enzymatically inactive 1:1 complexes [34]. In addition to

dates in vivo (an)other hydrophobic molecule(s) of similar structure

serine proteases serpinA5 also inactivates the cysteine protease

and size.

cathepsin L [35]. Details regarding proteases inhibited by serpinA5

and the respective inhibition rate constants are summarized in

[19].

4. Biological functions of SERPINA5

Human SERPINA5 is expressed in many organs and tissues, and

the protein is present in most body fluids and secretions [29,36].

4.1. Role in hemostasis

SerpinA5 expression has been shown in the liver [37], in the kidney

[38], in the skin [39], in the heart [37], and in the male and female

SERPINA5 has initially been described as an inhibitor of aPC in

reproductive tracts [31,36,40]. The highest concentrations occur

plasma [25,26] and independently as an inhibitor of the plasmino-

in seminal plasma, which contains >4 ␮M SERPINA5 [36]; human

gen activator urokinase (uPA) in urine [61]. Later it was shown that

blood plasma contains ∼100 nM [36].

the inhibitor of aPC and the inhibitor of uPA (plasminogen acti-

vator inhibitor-3) are immunologically identical proteins [43]. In

3. Non-protein ligands of SERPINA5 addition to aPC and urokinase SERPINA5 inactivates several other

proteases involved in hemostasis and fibrinolysis [27,30,41,62–65]

SERPINA5 binds heparin [41–44] and other glycosaminogly- suggesting a role in the regulation of hemostasis. In vitro SERPINA5

cans [45,46]. This has been shown not only in purified systems, can act as a procoagulant as well as an anticoagulant depend-

but also on the surface of cultivated cells [45,46]. SERPINA5 ing on the assay conditions [65]. There are indications that high

also binds certain phospholipids [47–49]. These phospholipids plasma levels of SERPINA5 represent a mild risk factor for venous

include phosphatidylserine, oxidized phosphatidylethanolamine, thrombosis [66]. Furthermore, significantly elevated plasma con-

phosphoinositides, and cardiolipin [48,49]. Binding of gly- centrations of active SERPINA5 have been described in survivors

H. Yang, M. Geiger / Seminars in Cell & Developmental Biology 62 (2017) 187–193 189

of myocardial infarction, and seem to represent a risk marker for Furthermore the reactivity of mouse serpinA5 with proteases and

acute coronary events [67]. SERPINA5 in complex with proteases glycosaminoglycans is very similar to that of the human protein

has been described in plasma from patients with activated coag- (Furtmüller et al., unpublished). However, in adult mice serpinA5 is

ulation [68–70] and patients receiving thrombolytic therapy [71]. almost exclusively expressed in the male and female reproductive

These data indicate that SERPINA5 reacts in vivo with proteases of tracts [72]. As in other rodents it is not expressed in the liver of adult

the hemostatic system. However, it is questionable to which extent mice and cannot be detected in plasma [92]. Therefore it is not sur-

this contributes to the regulation of hemostasis. prising that the only obvious phenotype of serpinA5 knockout mice

is infertility of homozygous males [72]. In these mice the struc-

4.2. Role in male fertility ture of the seminiferous tubules of the testis was disturbed and the

blood-testis barrier seemed disrupted, suggesting that the infertil-

SERPINA5 is highly expressed in the male reproductive tract ity is caused by abnormal spermatogenesis. Spermatozoa isolated

[36,40], and male serpinA5-deficient mice (see below) are infertile from the epididymis were malformed and were not able to fertil-

[72]. This suggests a prominent role of SERPINA5 in spermatogen- ize oozytes in vitro. We hypothesized that unopposed proteolytic

esis and male reproduction. Complexes of SERPINA5 and its target activity might be responsible for the observed abnormalities. We

proteases have been described in human semen [29,73–75], and a therefore generated double knockout mice, in which serpinA5 and



single-nucleotide polymorphism (SNP) located in the 3 -non cod- one of its target proteases normally expressed in the testis were

ing region of SERPINA5 has been found more frequently in men with absent. However, also these mice (double knockout serpinA5/tPA

in vitro fertilization failure as compared to controls [76]. SERPINA5 and double knockout serpinA5/uPA) were infertile [93] as were ser-

and peptides derived from SERPINA5 interfere with sperm-egg pinA5/acrosin double knockout mice (Uhrin et al., unpublished).

binding and in vitro fertilization [77,78]. Nevertheless, the role of Histological analysis of the testes of these serpinA5/tPA- and

SERPINA5 in male reproduction remains enigmatic. It seems that serpinA5/uPA- double knockout mice revealed similar abnormali-

SERPINA5 acts on several levels in the male reproductive tract. ties as seen in serpinA5 single knockout mice. These data suggest

−/−

Morphological changes seen in the testis and in epididymal sperm that the changes observed in the testis of serpinA5 are most

of serpinA5-knockout mice suggest that impaired spermatogenesis likely not or not exclusively caused by unopposed proteolytic activ-

might be sufficient for causing infertility in these mice despite pos- ity.

sible additional functions of serpinA5 throughout the reproductive To study the role of SERPINA5 in blood coagulation, Wage-

tract. naar et al. generated transgenic mice, which expressed human

SERPINA5 in hepatocytes only [92]. In these mice plasma con-

4.3. Protective role in cancer centrations of human SERPINA5 were similar to those in human

plasma, and plasma of these mice inhibited human aPC. Hayashi

Several studies indicate a protective role of serpinA5 against et al. generated human SERPINA5 gene transgenic mice [37], which

tumor development, tumor invasiveness, and tumor metastasis expressed human SERPINA5 with similar tissue distribution as seen

[79–82]. SNPs of SERPINA5 have been found to be associated with in humans. SERPINA5 present in the plasma of these mice inhibited

the risk of papillary and follicular thyroid cancer [83,84]. In ovar- the anticoagulant activity of exogenously injected human aPC as

ian cancer the expression of SERPINA5 seems to promote a more well as aPC generated in mouse plasma by addition of the protein C

®

benign behavior, whereas in more aggressive tumors SERPINA5 was activator Protac . Human SERPINA5 present in the plasma of trans-

downregulated [85]. Several ex vivo and preclinical studies have genic mice furthermore inhibited the anti-inflammatory activity of

been performed to analyze the role of SERPINA5 in tumor cell pro- aPC. This pro-inflammatory effect was not observed in the mice

liferation, migration, metastasis formation, and tumor angiogenesis generated by Wagenaar et al. [92]. Transgenic mice expressing

[80,82,86,87]. Also in these studies SERPINA5 seems to induce less human SERPINA5 seem to be a useful model for the analysis of the

malignant behavior. However, the mechanism(s) for these effects role of serpinA5 in plasma, although for mice there is apparently

have not been clarified so far. Not all activities seem to depend on no need for serpinA5 in plasma.

its protease inhibitory activity [82]. Interestingly, during mouse embryogenesis serpinA5 is

expressed in several organs and tissues at clearly defined sites

4.4. Role in host defense and in a developmental stage-dependent manner [94]. These sites

include the skin, the gut, the choroid plexus of brain ventricles, the

SERPINA5 has antimicrobial activity, which has been attributed heart and skeletal muscles, the urogenital tract, and cartilage. A

to the heparin-binding helix H [88]. Also the heparin-binding site- and developmental stage-dependent expression of serpinA5

sequence of SERPINC1 (antithrombin), which is located in its D- was especially observed in the lung. Although these data suggest

helix, exhibits antimicrobial activity [89]. So far there are no in vivo a role of serpinA5 in embryonic development and morphogenesis,

data supporting the role of SERPINA5 in the defense against bac- serpinA5 knockout mice seem to develop normally and do not

teria. Interestingly, SERPINA5 seems to have a protective role show any gross morphological abnormalities [72].

against HIV-infection. High concentrations of SERPINA5 have been Since so far male infertility is the only phenotype seen in ser-

described in the cervicovaginal fluid of women who remained pinA5 knockout mice, we analyzed differences in protein expression

seronegative despite high-risk exposure to HIV as compared to between testes obtained from wild-type and serpinA5 knockout

high-risk exposed, HIV-positive women and to women with low- mice, respectively, to further explore the role of serpinA5 in male

risk HIV exposure [90]. reproduction. We used a 2-dimensional fluorescence difference

gel electrophoresis (2D-DIGE) proteomics approach [95]. The most

5. The problem with serpinA5 mouse models striking difference in testis lysates of serpinA5 knockout mice was a

10-fold upregulation of prostaglandin reductase 1 (PTGR1) as com-

To study the biological role of SERPINA5 we have developed pared to wild-type testis lysates. Besides that we observed shifts in

a mouse model. We have characterized the mouse serpinA5 gene the molecular weights of serpinA1c and serpina3k, suggesting the

[91] and have generated mice, in which the serpinA5 gene has been presence of different isoforms and/or cleaved forms of these serpins

inactivated by targeted disruption [72]. Mouse serpinA5 is highly in serpinA5-deficient and wild-type testis, respectively. Follow up

homologous to human SERPINA5, especially as far as functionally studies are needed to determine the underlying mechanisms for

important sites (e.g. reactive site, hinge region) are concerned [91]. and the consequences of these changes.

190 H. Yang, M. Geiger / Seminars in Cell & Developmental Biology 62 (2017) 187–193

6. Internalization of SERPINA5 by cells Cleavage of serpinA5 at the N-terminus was seen not only in

purified systems with testisin. It also occurred on the surface of

Several years ago we and others have identified serpinA5 in cultured cells by a GPI-anchored protease, presumably testisin.

platelets [16,96]. Platelets also contain serpinA5 mRNA, suggesting These data suggest that one of the mechanisms regulating SER-

that they are able to synthesize serpinA5. Besides that platelets can PINA5 internalization is proteolytic removal of its N-terminus. This

internalize serpinA5 from the surrounding medium [16]. Studying N-terminal truncation does neither interfere with its inhibitory

serpinA5 synthesis by in situ hybrization and by immunohisto- activity nor with the stimulatory effect of heparin [50,98]. As

chemistry of bone marrow smears, we observed serpinA5 antigen mentioned above the cellular requirements for SERPINA5 internal-

also in leukocytes. In these leukocytes SERPINA5 was also detected ization are unclear and need to be investigated in detail.

in the nucleus [16]. Follow up experiments were performed to ana-

lyze binding and internalization of biotinylated serpinA5 by HL-60

7. Nuclear translocation of SERPINA5

cells (promyelocytic leukemia cell line). We observed staining of

intracellular compartments and especially staining of the nucleus

Internalized human and mouse serpinA5 can translocate to the

(Prendes M. et al., unpublished).

nucleus as judged from confocal microscopy [47]. A functional

Later we have shown that serpinA5 can be internalized by

nuclear localization signal in the H-helix of human SERPINA5 has

HL-60 cells and neutrophils. This internalization occurred within

been identified and seems to be sufficient for nuclear transloca-

minutes and was also observed at low temperatures suggest-

tion (Sokolikova et al., unpublished). Recently, we have confirmed

ing that internalization does not involve common endocytosis

nuclear localization of internalized serpinA5 in human Jurkat cells.

pathways [47]. After longer incubation periods serpinA5 was

We incubated the cells with purified recombinant mouse serpinA5

detected in the nucleus of HL-60 cells. So far the exact mecha-

and studied the presence of the mouse protein in different subcellu-

nism of the internalization of serpinA5 by cells and the cellular

lar fractions by Western blotting [98]. These studies revealed that

requirements are unknown. We only know that the phospholipid

internalized serpinA5 was localized to the nuclear envelop frac-

phosphatidylethanolamine is involved and that internalization is

tion. When a serpinA5 mutant lacking the N-terminal amino acids,

inhibited by heparin, but not by the receptor-associated protein

which is not able to be internalized by cells (see above) was used,

(RAP), a protein that antagonizes the internalization of serpin-

no mouse serpinA5 was detected in the nuclear fraction, indicat-

protease complexes via the LDL-receptor related protein [97].

ing that the nuclear serpinA5 is in fact the result of internalization

These data suggest involvement of cell surface glycosaminogly-

from the extracellular space. SERPINA5 is not the only secreted,

cans in SERPINA5 internalization [47]. However, heparin also

extracellular serpin, which has been detected in the nucleus. San-

inhibits the binding of serpinA5 to phosphatidylserine and oxi-

tamaria et al. have shown in human hepatocellular carcinoma cells

dized phosphatidylethanolamine [48]. Our data suggest that

that the serpin alpha-1-antichymotrypsin (serpinA3) can enter the

serpinA5 can also cross pure phospholipid membranes in a

nucleus and cause chromatin condensation [15].

phosphatidylethanolamine-dependent way [47]. It is therefore not

unlikely that it can directly cross the phospholipid bilayer of

8. Intracellular interaction partners of SERPINA5

the plasma membrane in a manner similar to cell penetrating

proteins/peptides. At present, however, we cannot exclude other

In a recent study we have shown that serpinA5 not only binds

internalization mechanisms. When we studied internalization of

phosphatidylserine and oxidized phosphatidylethanolamine, but

SERPINA5 by cultivated HL-60 cells or Jurkat T-cell leukemia cells

also inositol-containing glycerophospholipids, so called phospho-

using confocal microscopy, we observed internalization of SER-

inositides (PIs) [49]. In various in vitro assays we have shown that

PINA5 by only ∼10-15% of cells (Fig. 1). At present we don’t know

serpinA5 bound all mono- and bisphosphorylated PIs as well as

which characteristics enable these cells to internalize SERPINA5,

phosphatidylinositol-3,4,5-trisphosphate. We also observed mod-

whereas it is excluded from other cells.

erate stimulation of its protease inhibitory activity by PIs. PIs are

Recently we have shown that the intact N-terminus (A+-helix)

localized to the inner leaflet of the plasma membrane. SERPINA5

of SERPINA5 is required for its internalization into Jurkat cells [98].

11 internalized by cells might bind to these PIs and thereby influ-

Cleavage of a peptide bond close to the N-terminus (between Arg

12 18 19 ence PI-functions. It could interfere with the binding of other

and Val in human SERPINA5 and between Ala and Val in

proteins to PIs and/or influence PI-dependent signaling. Since

mouse serpinA5) by the serine protease testisin abolished inter-

one of the SERPINA5-binding PIs (PtdIns(4,5)P2) is a substrate

nalization of the serpin. This was seen with human as well as

for PI3-kinase, we were interested if SERPINA5 might influence

with mouse serpinA5. Also a truncated serpinA5 mutant lacking

the PI3-kinase/AKT-signaling pathway. We have shown that SER-

the N-terminal amino acids was not internalized by cells. On the

PINA5 caused an increase in phosphorylated AKT, not only, when

other hand, synthetic peptides corresponding to the N-terminal

overexpressed in HEK293 cells, but also when exogenously added

11 amino acids of human serpinA5 or to the N-terminal 18 amino

to and incubated with human umbilical vein endothelial cells

acids of mouse serpinA5, respectively, were internalized by Jurkat

(HUVECs) [49]. In a purified system the presence of SERPINA5

cells. These peptides can therefore be considered as cell penetrat-

furthermore stimulated the activity of the phospholipid phos-

ing peptides [98]. The mechanism of internalization is not clear

phatase SHIP2 [49]. In preliminary, unpublished studies we also

at present. However, the internalization of the SERPINA5 peptides

identified proteins interacting with intracellular SERPINA5. These

seems to occur via a different mechanism as compared to that used

proteins are synaptotagmin-like protein1 (JFC-1) (Sokolikova et al.,

by the cell penetrating HIV-1 TAT peptide, a well-known exam-

unpublished) and cathepsin L [103]. Further studies are needed to

ple for cell penetrating peptides (for review see refs. [99,100]).

determine the biological relevance of the interactions of SERPINA5

As can be seen from Fig. 2 incubation of Jurkat cells with FITC-

with its intracellular partners.

conjugated helix A+-peptide resulted in homogenous staining of

the cells, whereas incubation of the cells with FITC-conjugated

TAT-peptide resulted in a punctate staining. Internalization of 9. Conclusions

TAT has been shown to occur via endocytosis [101,102]. Since

internalization of SERPINA5 also occurs at low temperatures, we What we know so far about serpinA5 does not justify any con-

propose a different and/or an additional internalization mecha- clusions about the physiological role of this serpin. It might be

nism. relevant as a protease inhibitor in several extracellular protease

H. Yang, M. Geiger / Seminars in Cell & Developmental Biology 62 (2017) 187–193 191

Fig 1. HL-60 cells were incubated for 15 or 60 min with biotin-labeled SERPINA5 (90 nM) and analyzed by confocal microscopy as described [47]. Internalized biotin was

visualized with Cy-2-labeled streptavidin (green), nuclei were stained with To-Pro-3 (red).

◦

Fig. 2. Jurkat cells were incubated with FITC-conjugated mouse helix A+-peptide (5 ␮M) or FITC-conjugated TAT (5 ␮M), respectively, for 1 h at 37 C. Cellular fluorescence

was analyzed by confocal microscopy as described [98]. Z-stack galleries are shown. The scale bars show a length of 5 ␮m.

systems. SerpinA5 can be internalized by cells and translocated to to the analysis of SERPINA5 internalization and nuclear translo-

the nucleus, and this internalization can be regulated by proteases cation, and of its interaction with phospholipids. This work was

present on the cell membrane and/or in the extracellular space. supported by grant P22792-B11 from the Austrian Science Fund

Therefore intracellular functions of this serpin have to be consid- (FWF) and 14064 from the “Medizinisch-wissenschaftlicher Fonds

ered as well. Further studies are needed to analyze its interaction des Bürgermeisters der Bundeshauptstadt Wien” (both to MG).

with intracellular targets in detail.

References

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