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ISSN 0582-9879 Acta Biochim et Biophysica Sinica 2004, 36(1):42-46 CN 31-1300/Q
Interaction of Plasminogen Activator Inhibitor-2
and Proteasome Subunit, Beta Type 1
Jing FAN, Yu-Qing ZHANG, Ping LI, Min HOU, Li TAN, Xia WANG, and Yun-Song ZHU*
( Department of Molecular Genetics, Shanghai Medical School of Fudan
University, Key Laboratory of Molecular Medicine, Ministry of Education,
Shanghai 200032, China )
Abstract The apoptosis protection by plasminogen activator
inhibitor -2(PAI-2) is dependent on a 33 amino acids fragment between helix
C and D of PAI-2 and this may be due to the interaction of PAI-2 with some
intracellular proteins. However ,that proteins interacting with PAI-2 remain
unknown.In this study we used the fragment between helix C and D of PAI-2
as bait to screen a HeLa cells cDNA library constructed during apoptosis in
a yeast two-hybrid system and retrieved a clone that encodes 241 amino acids
of proteasome (prosome, macropain) subunit, beta type 1(PSMB1). GST-pulldown
experiments confirmed the interaction between PAI-2 and PSMB1 in vitro.
Proteasome take part in the NF-kB activation process induced
by TNF-a. These data suggest that
the antiapopotosis activity of PAI-2 might related to its effect on proteasome
subunit, such as PSMB1.
Key words plasminogen activator inhibitor type-2; proteasome
(prosome, macropain) subunit, beta type 1 (PSMB1); yeast two-hybrid system;
ubiquitin
In 1993, Remokd-ODonell reported on a subset of serine protease
inhibitor (serpin) superfamily with high degree of sequence identity to chicken
ovalbumin called ov-serpin. The ov-serpin subfamily contains a variable length
of loop between helices C and D that may confer functional motifs involved
in, for example, nuclear localization [1] or transglutamination [2]. Plasminogen
activator inhibitor-2 (PAI-2), as an important member of ov-serpin subfamily,
is a multifunctional protein that has been involved in the regulation of fibrinolysis,
invasion and metastasis of cancer cells, and in regulation of apoptosis [3].
HeLa cells transfected with PAI-2 cDNA were protected from TNF-a-induced apoptosis. It
is known that the antiapoptotic activity of PAI-2 depending on a 33 amino
acids fragment between helix C and D of PAI-2 which may be due to the interaction
of PAI-2 with some unknown intracellular proteins [4, 5].
To explore the proteins which could interact with PAI-2,
the fragment between helix C and D of PAI-2 is used as bait to screen a HeLa
cells cDNA library constructed during apoptosis in a yeast two-hybrid system
and retrieved a clone that encodes 241 amino acids of proteasome (prosome,
macropain) subunit, beta type 1 (PSMB1). Using RT-PCR, we got the full-length
cDNA of PSMB1. GSTpulldown experiment confirmed the interaction between PAI-2
and PSMB1 in vitro.
The proteasome is a large multi-subunit proteinase
complex found in the cytoplasm and nucleus of all eukaryotic cells examined so
far. As part of the ubiquitin-mediated protein degradation machinery, it is
responsible for not only the elimination of misfolded proteins, including those
derived from the lumen of the endoplasmic reticulum [6], but also the controls
on a multitude of regulatory processes by removing unnecessary or even harmful
metabolic enzymes and by balancing the levels of many regulatory proteins
[7,8]. The pathway helps control many aspects of cell homeostasis,growth,and
development [9–11]. Examples include cell cycle progression,maintenance of
chromatin structure, DNA repair, enzymatic regulation, transcription, signal
transduction, and apopotosis, etc, as a subunit of proteasome, PSMB1 may play a
role in apopotosis regulation.
Material and Methods
DNA and Plasmid Constructs
The BD vector pAS2-1NE used for the yeast two-hybrid system
was a gift from Dr. Tian Yu (Harvard University). The interhelical region
of C and D of PAI-2 was obtained by PCR. The primers were A1(NheI):5′– AAAGCTAGCATGGCCAAGGTGCTTCAG-3′and A2 (EagI), 5′–AAACGGCCGGGATGAATGGATTTTATC-3′and the PCR condition was as follow: 80 s at 94 ℃, 60 s at 58 ℃, 40 s at 72
℃, 25 cycles. After digested by NheI and EagI,
the fragment was inserted in frame into pAS2- 1NE excised by the same two
enzymes. The plasmid pGEX-4T-1 which encodes a GST protein at the N-terminus
(Pharmacia). Inserting the full-length human PSMB1 cDNA in frame into pGEX-4T-1
generated the mammol/ Lalian recombinant vector pGEX-4T-1/PSMB1. The deletion
mutant of PAI-2, which encodes PAI-2 protein without the interhelical region
of C and D, has been constructed before [12].
Cell culture and transfection
Hela cells were maintained in RPMI 1640 containing 10% bovine
calf serum, 2 mmol/L glutamine, 50 u of penicillin/ml, and 50 mg of streptomycin/ml Transfections were performed using LipofectAmine (Invitrogene)
as instructed by the manufacturer.
Yeast two-hybrid screening and colony-lift filter assay
Two-hybrid system (Clontech) was used in this study. The
interhelical region of C and D of PAI-2 was used as the bait to screen a HeLa
cells cDNA library described above. The screen was done with Saccharomyces
cerevisiae strain AH109 ( MATa, trp1–901, leu2–3, ura3–52, his3–200 ),
which expresses reporter genes conferring selective auxotrophy and b-galactosidase activity. The screen was carried out in
Ade/His/Leu/Trp-deficient medium at 30 ℃. 10 d after cotransformation, 40 clones were screened and transferred
onto a filter. It was rapidly lysed by being dipped twice into liquid nitrogen
and allowed to thaw at room temperature. Carefully place the filter, colony
side up, on another filter presoaked with Z buffer (60 mmol/L Na2HPO4・7H2O, 40 mmol/L NaH2PO4・H2O, 10 mmol/L
KCl, 0.1 mmol/L MgSO4・7H2O, pH 7.0)
containing 1 g/L X-gal and 0.27% -mercaptoethanol. Filters were incubated at 37
℃ until the blue colonies
appeared .
Isolation of the plasmids
Plasmids from positive clones were isolated as described
by Hoffam et al. [13]. In brief, a large fresh positive colony was
inoculated into 5 ml SD/-Trp and was incubated at 30 ℃ overnight with shaking at 250 r/min. After centrifugation,
the pellets were resuspended in 200 ml lysis buffer
[2% Trinton X-100, 1% SDS, 100 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 8.0), 1mmol/L
EDTA]. 0.2 mg glass beads (Sigma) and
200 ml phenol / chloroform (1∶1) were added into the lysate and the mixture was vortexed
thoroughly for 10 min. They were dipped into liquid nitrogen for 10 min and
allowed to thaw at room temperature. After vortexed again for 10 min, the
supernatant was collected by centrifugation at 12,000 r/min for 10 min. 400
ml ice-cold ethanol was added and the pellet was spinned down by centrifugation.
Analysis of homology of sequence of positive clones
The plasmids isolated from the positive clones were
introduced into E.coli strain KC8 cells by electroporation. The sequence
of the inserted DNA was analyzed by Ji Kang Inc. and subjected to Genbank to
analyze the homology using BLAST program.
Amplification of the full-length human PSMB1 cDNA by
RT-PCR
The full-length human PSMB1 cDNA was generated by RT-PCR.
Briefly, total cell ular RNA from Hela cells was prepared by Trizol. RNA was
controlled by agarose gel electrophores is and spectro-phometrically quantified.
dT15– primers and AMV- Rtase were used for first strand synthesis. Primers for
PSMB1 cDNA were: B1(EcoRI), 5′-GGC GAA TTC ATG TTG TCC TCT ACA-3′; and B2(XhoI), 5′-GGC CTC GAG TCA GTC CTT CCT TAA-3′. 1 ml total cDNA product was
mixed with Taq DNA polymerase, 50 pmol/L of each appropriate primer, 200 mmol/L each dNTP in a buffer containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L
KCl, 0.01% BSA, 2 mmol/L MgCl2 in final volume of 100 ml. The sample
was amplified for 28 cycles and the PCR condition was as follow: 40 s at 94
℃, 40 s at 60 ℃, 80 s at 72
℃.
GST-PSMB1 fusion protein expression and purification
The bacterial strain Bl21 was used for the expression of
GST or GST-PSMb1 protein. The bacterial
culture was induced with isopropyl-1-thio-D-galactopyranoside (IPTG, 0.1
mmol/L) and cultured at about 25 ℃ for 3 h. The
bacterial pellet was collected by centrifugation and lysed by sonication in 10
mmol/L phosphate buffered saline (PBS), pH 7.4 (137 mmol/L NaCl, 2.7 mmol/L
KCl, 10 mmol/L Na2HPO4, 2 mmol/L KH2PO4) and 1
mmol/L PMSF. After centrifugation at 13,000 rpm for 10 min, the supernatant was
incubated with glutathione Sepharose 4B beads (Pharmacia) for 2 h at 4 ℃ on a rotating platform. GSTPSMb1 was
expressed in an insoluble form. After denaturalization and renaturalization
process, GST-PSMb1 was purified with
glutathione Sepharose 4B .
GST-pulldown experiment and Western blot analysis
HeLa cells were grown as a monolayer in 10-cmdiameter dishes
and transfected with 4 mg pcDNA3-PAI- 2 or pcDNA3-PAI-2.CD (encoding PAI-2 protein
without the interhelical region of C and D) using LipofectAmine. 48 hours
after transfection, cells were scraped from the dish, washed with ice-cold
PBS, and lysed with ice-cold lysis buffer (137 mmol/L NaCl, 20 mmol/L Tris-HCl,
pH 8.0, 0.1 mmol/L CaCl2, 1 mmol/L MgCl2, 1% NP40, 10% glycerol, 1 mmol/L PMSF, 1 g/L aprotinin)
for 15 min at 4 ℃
on a rotating
platform. Samples were centrifugated at13,000 rpm for 10 min, and incubated
the supernatant with glutathione Sepharose 4B beads prepared in the above
step “GST-PSMb1 fusion protein expression and purification” overnight
at 4 ℃ on a rotating
platform. The beads were washed three times with ice-cold lysis buffer, resuspended
in 100 ml loading buffer and boiled
for 5 min. After centrifugation, proteins of the samples were separated by
10% SDS-PAGE and transferred to nitrocellulose membranes (Amersham Pharmacia).
The membranes were blocked with 5% skimmed milk and sequentially incubated
with monoclonal PAI-2 antibody (Santa Cruz) and horseradish peroxidase-conjugated
secondary antibodies (Santa Cruz). Results were analyzed by ECL (Amersham
Pharmacia) with X-ray film (Kodak).
Results
Identification of the positive clones and analysis of
homology
Our previous study indicated that the interhelical region
of C and D of PAI-2 could interact with some proteins, and IRF3 interacted with
PAI-2 was identified [14]. In this study 10 d after cotransformation with
recombinant vector pAS2-1NE-PAI-2CD and blank vector pACT2, 40 positive clones
were screened and 36 clones showed b– galactosidase
activities (data not shown).
The 36 plasmids DNA isolated from candidate clones were
cloned with blank vector pAS2-1NE into AH109. Those with transcriptional
activities, were chosen for further analysis. 24 clones were considered to be the candidates of PSMb1 partners. The cDNAs of these clones were amplified by PCR and the
sequences were analyzed using BLAST program of GenBank at NCBI. Our result
showed that one clone was 100% homologous to the proteasome (prosome,
macropain) subunit, beta type 1 (PSMb1) (Fig.1).
Amplification of human full-length PSMb1 cDNA and construction of pGEX-4T-1-PSMb1
The full-length human PSMb1 cDNA was
obtained by RT-PCR (Fig.2). The agarose gel electrophoresis showed the fragment
was about 1.3 kb. DNA sequencing indicated that the RT-PCR product was PSMb1 gene. The plasmid pGEX-4T-1 encoding GST protein was inserted with
full-length human PSMb1 cDNA to generate the
mammol/ Lalian recombinant vector pGEX-4T-1-PSMb1.
Fig. 1 Nucleotide sequence of the positive clone cDNA
encoding the PSMb1 (nucleotide sequence of pAS2-1NE in bold)
Fig. 2 RT-PCR amplification of PSMb1 cDNA
M, marker; 1, RT-PCR product of PSMb1.
GST-PSMb1 fusion protein
expression and purification
The bacterial strain Bl21 was used for the expression of
GST or GST-PSMb1 protein. After induced
with IPTG, each bacterial culture expressed a certain amout of corresponding
protein in an insoluble form. GST-PSMb1 was denatured with 8
mol/L urea and renatured by diluting the solution to 0.5 mol/L urea and
incubated with glutathione beads (Fig. 3).
PAI-2 can interact with PSMb1 in vitro
In order to further investigate the interaction of PAI-2
and PSMb1, whole lysates of HeLa cells transfected with
pcDNA3-PAI-2 or pcDNA3-PAI-2.CD were incubated with
Sepharose 4B beads bound with GST or GST-PSMb1 fusion
protein. The Sepharose 4B beads were collected centrifugation. The samples were
analyzed by SDS-PAGE and immunoblotting with anti-PAI-2 antibody. As shown in
Fig. 4, PAI-2 can interact with PSMb1, however PAI-2 mutant
without the interhelical region of C and D (.CD) can not
interact with PSMb1, indicating that PAI-2
interacts with PSMb1 via its interhelical
regions C and D.
Fig. 3 ST-PSMb1 fusion protein
expression and purification
M, marker; 1, lysed bacterial pellet transformed with pGEX-4T-1 after
induced with IPTG; 2, glutathione Sepharose 4B beads after incubated with GST
supernatant; 3, lysed bacterial pellet transformed with pGEX-4T-1/PSMb1 before induced with
IPTG; 4, lysed bacterial pellet transformd with pGEX-4T-1/PSMb1 after induced with
IPTG; 5, supernatant of lane 4 after centrifugation; 6, deposition of lane 4
after centrifugation; 7, glutathione Sepharose 4B beads after incubated with
GSTPSMb1 fusion protein after the denaturalization and
renaturalization. *GSTPSMb1 fusion protein.
Fig. 4 Interaction between PAI-2 and PSMb1 in vitro
Western blot analysis with PAI-2 antibody. 1, GST incubated with HeLa cell
which transfectected with pcDNA3-PAI-2 total protein; 2, GST-PSMb1 fusion protein
incubated with HeLa cells tracfected with pcDNA3-PAI-2 total protein; 3,
GST-TIAR fusion protein incubated with HeLa cells tracfected with pcDNA3-PAI- 2.CD total protein; 4,
GST-PSMb1 fusion protein incubated with HeLa cells total protein;
5–7, the supernatant of 2–4, respectively.
Discussion
In this study, we found that PAI-2 could interact with
PSMb1 via its interhelical region of C and D. As a part of
the ubiquitin-mediated protein degradation machinery, proteasome is responsible
for the elimination of misfolded proteins and the removal unnecessary or
harmful metabolic enzymes. In the ubiquitin-mediated protein degradation
pathway, proteins are first enzymatically tagged for breakdown by the covalent
attachment of one or more chains of ubiquitin monomers. Attachment is via an
isopeptide bond between the C-terminal glycine of ubiqutin and free lysines either
in the target or in the preceding ubiqutin in the chain [15–17]. Once
assembled, the multi-ubiquitin chain functions as a recognition signal for
substrate degradation by the 26 S proteasome. There are 27 lysines in PAI-2
including one Lys87 in the interhelical region of C and D, it is probably degraded by the biquitin/proteasome system after
ubiquitination. In our study, the protein bands of PAI-2 and PAI-2.CD only presente at the site of 46 kD and 42 kD respectively, suggesting
that PAI-2 did not attach to ubiquitin chain and the interaction between PSMb1 and PAI-2 was not involved in ubiquitin-mediated protein degradation
pathway.
Although tumor necrosis factor alpha (TNF-a) can trigger cellular apoptosis through binding to members of TNF-a receptor (TNFR) superfamily, there is evidence that TNF-a itself has little effect on the apoptosis of some kinds of cells [18]. In
addition to apoptosis, treatment of cells with TNF-a can result in
activation of transcription factors AP-1 and NF-kB which can
induce gene expression [19,20]. The activation of NF-kB requires the
degradation of its inhibitor protein IkB. In response
to TNF-a, the IkBs are phosphorylated by the IkB kinase complex, resulting
in the ubiquitination degradation by proteasome, and nuclear translocation
of free NF-kB [19]. Some genes induced by NF-kB act to suppress TNF-a-induced apoptosis. In
brief , TNF-a initiate two reverse pathways
in the cells, one is to induce apoptosis, the other is to suppress apoptosis,
and whether TNF-a can induce cellular apoptosis
or not is dependent on the balance between the two pathways. Proteasome is
an important member in the latter pathway. It can activate NF-kB indirectly by decreacing the level of IkB in cells.
In this study, we found that PAI-2 can interact with one subuit of proteasome.
Based on the discovery, we suggest that the antiapoptosis activity of PAI-2
is probably associated with proteasome system, and PAI-2 may activate proteasome
to degrade IkB and/or protect it from
the inhibition by some inhibiting factors in the cells.
In this study,we found that PAI-2 interacted with one kind
of b subunit. Is it the only kind of proteasome subunits
interacting with PAI-2? We compared the sequence identity of the 12 b subunit known so far (b1, b2, b3, b4, b5, b6, b7, variant 1 of b8, variant 2 of b8, variant 1of b9, variant 2 of b9, b10) by insightII, the sequence identity is 41.3%. Secondary structure simulation
showed that the 12 b subunits were all constructed
by several a-helices and b-sheets conjugated by some b or ã turner, and there were five conserved regions (data not shown). If PSMb1 interacts with PAI-2 through one of the conserved region, it is possible
that PAI-2 can interact with other b subunits.
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Received: September 12, 2003 Accepted: November 14, 2003
This work was supported by a grant from the National Natural Sciences
Foundation of China (No. 30070412)
*Corresponding author: Tel, 86-21-54237278; E-mail, [email protected]