Abbreviations ANK, ankyrin repeat motif; ANKHD1, ankyrin repeat and KH domain 1; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HEK293, human embryonic kidney 293; hM
Trang 1The ankyrin repeat motif (ANK) is one of the most
common protein motifs found in the protein database
The ankyrin repeat is a 33-amino acid motif present
in repeats of 12–24 and first identified in yeast and
Drosophila [1] Ankyrin-repeat-containing proteins
regulate multiple cellular functions including
transcrip-tional regulation, cell-cycle regulation, ion channel, cell
survival, and cell signaling [2–4] In addition, ankyrin
repeat proteins also participate in protein–protein interactions via their repeat motifs [5,6] Using yeast two-hybrid system analysis, we identified a protein containing a single ankyrin repeat that interacts with HIV-1 viral protein R (Vpr) and we designated this protein as Vpr-binding ankyrin repeat protein (VBARP) This interaction was further confirmed by a mammalian hybrid system as well as in vivo interaction
Keywords
ANKHD1; ankyrin repeats; apoptosis; cell
survival; HIV-1 Vpr
Correspondence
V Ayyavoo, Department of Infectious
Diseases and Microbiology, Graduate School
of Public Health, University of Pittsburgh,
130 DeSoto Street, Pittsburgh,
PA 15261, USA
Fax: +1 412 624 5612
Tel: +1 412 624 3070
E-mail: Velpandi@pitt.edu
(Received 16 May 2005, revised 7 June
2005, accepted 14 June 2005)
doi:10.1111/j.1742-4658.2005.04821.x
Multiple ankyrin repeat motif-containing proteins play an important role
in protein–protein interactions ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1 This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif We characterized VBARP by molecular and functional ana-lysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved
in basic cellular function(s) Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm Functional ana-lysis using short interfering RNA approaches indicate that this gene prod-uct is essential for cell survival through its regulation of caspases Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s)
Abbreviations
ANK, ankyrin repeat motif; ANKHD1, ankyrin repeat and KH domain 1; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; HEK293, human embryonic kidney 293; hMASK, human multiple ankyrin repeats single KH domain; NLS, nuclear localization signal; ORF, open reading frame; PBL, peripheral blood leukocytes; PBMC, peripheral blood mononuclear cells; RPLPO, ribosomal protein large protein; RTK, receptor tyrosine kinase; siRNA, short interfering RNA; UTR, untranslated region; VBARP, Vpr-binding ankyrin repeat protein.
Trang 2studies blast searches of VBARP revealed that this
protein has homology to human ankyrin repeat and
KH domain containing 1(ANKHD1) variants, and to
an unknown protein named PP2500 [7,8] Although
ANKHD1 variants have been identified by several
human genome sequencing groups, no known function
has been identified for these proteins
ANKHD1 is a large protein containing multiple
ankyrin repeats and a single KH domain It is derived
from an 8 kb transcript, with a predicted molecular
mass of > 280 kDa The gene is present in human
chromosome 5q31.3 as a single copy To differentiate
the variants of ANKHD1, NCBI has classified three
transcript variants that code for three unique isoforms
of ANKHD1 Our clone, VBARP, codes for two open
reading frames (ORF) of 1.9 and 1.35 kb, each with
an identical poly(A) tail These two cDNAs were
desig-nated VBARP-L (1.9 kb) and VBARP-S (1.35 kb),
respectively Both VBARP-L and VBARP-S variants
have high homology to PP2500 and ANKHD1
vari-ant 2 In this study we focus on the biochemical and
functional characterization of the novel VBARP-L and
VBARP-S transcripts Bioinformatics analyses show
that VBARP-L and VBARP-S are comprised of 11
and 9 exons, respectively However, although these
two variants utilize many of the same exons, VBARP-S
lacks a portion of exon 4 and a 5¢ untranslated region
(UTR) that can be found in VBARP-L Results from
functional analyses indicate that these transcripts are
ubiquitously expressed in human tissues and cell lines
at different levels Specific loss of the VBARP
tran-scripts induced by short interfering RNA (siRNA)
caused apoptosis via caspase activation, indicating a
potentially important role for these proteins in cell
sur-vival Taken together, these results suggest that HIV-1
Vpr interaction with VBARP might disrupt the cell
survival pathway thus leading to host cell apoptosis
Results
Identification of VBARP as HIV-1 Vpr-interacting
protein
The HIV-1 Vpr-interacting protein, VBARP, was
iden-tified using the yeast two-hybrid system as previously
described [9] A 915-bp fragment was initially identified
and further confirmed through both repetitions of the
yeast two-hybrid assay and using a mammalian hybrid
system (Invitrogen, Carlsbad, CA) (EDA Wheeler &
V Ayyavoo, unpublished data) A blast search
revealed that the IMAGE clone, localized in
chromo-some 5q31.3, was fully homologous to the 915-bp
fragment and revealed a possible full-length clone
containing multiple variants These variants, one mea-suring 1881 bp (designated VBARP-L) and a second
1305 bp clone (designated VBARP-S), were construc-ted and used to further confirm the Vpr and VBARP interaction using in vitro and in vivo interaction stud-ies Ten microliters of 35S-labeled in vitro translated VBARP and Vpr or Nef (as control) products were mixed and immunoprecipitated with VBARP-, Vpr- or Nef-specific antibody and analyzed by autoradiogra-phy (Fig 1A) Results indicate that both Vpr and VBARP were able to form a complex and the complex was pulled by VBARP and Vpr antibodies, respect-ively, whereas VBARP and Nef did not form a com-plex, indicating that the interaction between VBARP and Vpr is specific The input panel represents the amount of protein used in this assay indicating that an equal amount of protein was used in all our samples and that the lack of interaction between VBARP and Nef is not due to the lack of input proteins
To further confirm that a physical interaction exists between Vpr and VBARP in vivo, we tested this inter-action in HEK293 T cells expressing Vpr and VBARP
by cotransfecting Vpr and VBARP-His using calcium phosphate transfection Forty-eight hours post trans-fection, the cells were lysed and the whole cell proteins were extracted One hundred micrograms of total pro-tein were used for immunoprecipitation (IP) using anti-Vpr (Fig 1B, lanes 1–3) or anti-His (lanes 1–3) IgG The bound proteins were eluted and subjected to western blot analysis using anti-His and anti-Vpr IgG
As shown in Fig 1B, interaction of Vpr with VBARP was detected by the IP followed by immunoblotting, further confirming the specificity of this interaction Based on these results we were able to demonstrate the formation of a complex between Vpr and VBARP
Characterization of VBARP variants The two VBARP clones differed only in the 5¢-end where VBARP-L consisted of a 576-bp fragment absent from the shorter VBARP-S and contained a 114-bp UTR (Fig 2A) Intron and exon analysis of VBARP revealed that VBARP-L and VBARP-S con-tain 11 and 9 exons, respectively (Fig 2B) Interest-ingly, both clones shared the latter eight exons with corresponding splice donor and acceptor sites found in the genomic clone VBARP-L contained an additional three exons totaling 576 bp with a 114 bp 5¢-UTR, whereas VBARP-S started with a portion of exon 4 (41 bp) with no known 5¢-UTR The NCBI Geneview website predicted exons from the targeted full genomic sequence attributing to the discrepancies in numbers (i.e exon 1, 2, 4, 6 ) observed in Fig 2B, suggesting
Trang 3that alternate exons, designated as 3, 5, and 11, may
code for additional splice variants Analysis with
spi-deysoftware, another tool for intron⁄ exon
determina-tion, generated an identical exon map, confirming
the data presented in Fig 2B that VBARP-L and
VBARP-S are coded by these specific exons
VBARP-L and VBARP-S code for precursor
pro-teins of 627 and 435 amino acids and with calculated
peptide masses of 69 and 49 kDa, respectively
Addi-tional domain mapping revealed that the predicted
structure of VBARP does not contain signal peptide(s)
or transmembrane domains phd software, available
from the Predict Protein server, suggested that the
structure of VBARP-L is predominantly helical with
multiple loops (helix, 55%; coil, 3%; loop, 42%)
Sequence analysis using prosite motif scan and
netphos software, also predicted the presence of
several potential serine, threonine, and tyrosine
phosphorylation sites (cAMP, PKC, and CK2), and the presence of nine potential myristoylation sites in the VBARP protein (Fig 2C) psort software predic-ted with high accuracy the subcellular localization of VBARP, and indicated that it is a cytoplasmic protein The Expert Protein Analysis System predicted that this protein belongs to the family of ankyrin repeat proteins (Fig 2C) Comparative analysis of VBARP ankyrin repeats with other known ankyrin repeat proteins, using the consensus established by Kohl et al [10] and Mosavi et al [11], which contains ankyrin repeats from over 4000 proteins, indicated that the ankyrin repeat domains in VBARP exhibit a high homology to those present in the consensus within the conserved, semiconserved and the nonconserved regions of the ankyrin motifs (data not shown) Sequence homology searches indicated that VBARP shares strong homology across many phyla, including
B
Fig 1 Interaction of Vpr and VBARP
pro-teins in vitro and in vivo (A) In vitro
transla-ted35S-labeled VBARP-His isoforms were
incubated with Vpr or Nef and
immunopre-cipitated using a-His (lanes 1–3), a-Vpr
(lanes 4–6) or a-Nef (lanes 7–9) antibodies
and resolved on SDS ⁄ PAGE Arrows
indi-cate the respective protein size (Inset)
In vitro translated products of VBARP
iso-forms, Vpr and Nef used in
coimmunopre-cipitation assay (B) Total cell lysates were
prepared from HEK293T cells transfected
with Vpr, VBARP-L, VBARP-S or control
vector plasmids One hundred micrograms
protein equivalent of cell lysates was used
in IP followed by western blot utilizing
anti-Vpr and anti-His IgG (A, B) In parallel, 50 lg
of total cell lysates from the same samples
were detected by western blot assay to
detect the input protein (Input) Arrows
depict the position of the VBARP-L,
VBARP-S and Vpr.
Trang 4proteins from mouse, rat, Drosophila and Anopheles.
Multiple sequence alignment of these orthologous
pro-teins revealed the presence of a conserved region of
human VBARP-L protein that shared 84% similarity
with mouse, 69% similarity with rat, 46% similarity
with Drosophila melanogaster and 48% similarity with
Anopheles gambiae.Interestingly, all the compared
spe-cies contained the 12 ankyrin repeat domains and
exhibited high homology between them, suggesting a
conserved function for this protein
Identification of VBARP splice variants in normal
human tissue
To precisely quantitate the amount of VBARP in
dif-ferent tissues and cell lines, real time RT-PCR was
performed Total RNA was extracted from the brain, spleen, lymph node, liver, cervix, muscle and kidney, and real-time RT-PCR was carried out in triplicate using ANKHD1⁄ VBARP isoform primer and probe sets Based on human genome sequencing, ABI has identified and constructed several primer probe sets at multiple intron–exon junctions to quantitate the vari-ants We used three sets of primers⁄ probe to distinctly identify the 8.0 kb ANKHD1, 1.9 kb VBARP-L and MASK-BP3 (splice variant of ANKHD fused with BP3) Using RNA derived from multiple tissues
we quantitated the various transcripts of VBARP using real-time PCR (Fig 3A) Human ribosomal large protein (RPLPO), a housekeeping gene, was used as an internal control and all ratios are pre-sented relative to RPLPO Results indicate that
A
B
C
Fig 2 (A) Schematic representation of VBARP-L and VBARP-S in comparison with ANKHD1 variant (B) Exon–intron analysis of VBARP isoforms Exons and introns present
in VBARP-L and VBARP-S are represented
as boxes with numbers The length in base pairs of each exon is marked on top of the boxes representing the exons Exon numbers are derived from the genomic sequence, after designating the first coding exon number 1 and counting all other exons located at this site (C) Predicted post-trans-lational modification and domain distribution
of VBARP-L translated amino acid sequences The grayshade regions in the amino acid sequence of VBARP-L protein represent the presence of ankyrin (ANK) repeats, amino acids underlined indicate predicted phosphorylation sites, and dark shaded regions represent the predicted N-myristylation sites.
Trang 5ANKHD1 isoform 1 (codes for an 8.0 kb transcript)
exhibits a high ratio in cervix tissue followed by
spleen, brain and lung Other tissues such as kidney,
lymph node, and muscle expressed relatively low levels
of ANKHD1 isoform 1 In the case of VBARP-L
iso-forms, spleen showed the highest level (3.89 ratio)
fol-lowed by lung, lymph node and kidney Interestingly,
muscle and brain were almost negative, indicating that
there is a differential expression of these transcripts
within different human tissues Interestingly,
MASK-BP3 exhibits a different profile confirming the
pres-ence of these variants at different levels in multiple
tissues
Next, we tested several human primary and
esta-blished cell lines of different lineages for the presence
of the above three isoforms (Fig 3B) Results
indica-ted that VBARP-L is present in most of the tesindica-ted cell
lines with varying amounts with the highest expression
level in dendritic cells (ratio of 8), followed by PBMC
and PBL It is interesting to note that among the
dif-ferent cell lines tested, primary lymphocytes (PBL,
PBMC) express higher level compared with the
immor-talized T-cell line CEMx174 However, we have been
unable to identify a cell line that is negative for
VBARP-L transcript
Expression and biochemical characterization
of VBARP The predicted sizes of the proteins encoded by VBARP-L and VBARP-S are 69 and 49 kDa, respec-tively To test this, C-terminal V5-tagged VBARP-L and VBARP-S constructs were translated in vitro, immunoprecipitated with anti-V5 IgG, and the protein products were resolved on an 8% SDS⁄ PAGE gel (Fig 4A) The VBARP constructs expressed the pre-dicted molecular mass protein, however VBARP-S expressed an additional, equally intense band slightly higher than the predicted 49 kDa band The addi-tional protein resolved at 55 kDa, suggesting that
VBARP-S might be modified post-translationally Next, the expression of the VBARP clones was tested
in vivo using HEK293T cells HEK293T cells were transfected with VBARP-L, VBARP-S or pcDNA3.1 vector plasmids using calcium phosphate Forty-eight hours post transfection, cells were lysed and subjected
to western blot analysis using anti-V5 IgG, and devel-oped using the ECL kit (Amersham Biosciences, Piscataway, NJ) (Fig 4B) Results indicated that both VBARP-L and VBARP-S expressed the predicted molecular mass of protein similar to the in vitro
B
Fig 3 (A) RNA expression of VBARP in various human tissues using real-time RT-PCR Total RNA was extracted from different human tis-sues and reverse transcribed Real-time PCR was carried out in triplicate The expression level of VBARP was normalized to the level of RPLPO control for each sample (B) mRNA expression of VBARP in various cell lines using real-time RT-PCR Total RNA was extracted from different human cell lines and reverse transcribed Real-time PCR was carried out in triplicate The expression level of VBARP was normal-ized to that of RPLPO control for each sample Each panel represents primers and probes that were used to specifically detect ANKHD1, VBARP-L and MASK-BP3 by Applied Biosystems All analyses were performed in triplicate.
Trang 6translated product Furthermore, VBARP-S exhibited
protein products of 49 and 55 kDa, further confirming
the possibility of post-translational modifications
and⁄ or splice variants
Subcellular distribution of VBARP
To determine the subcellular localization of VBARP
isoforms, His-tagged VBARP plasmids were
transfect-ed into HeLa cells, and the distribution was assesstransfect-ed
by indirect immunofluorescence using anti-His IgG
(Fig 5) Both VBARP-L and VBARP-S exhibited a distinct cytoplasmic pattern upon expression Similar cytoplasmic distribution was observed in 293 and A172 cells upon transfection with VBARP constructs (data not shown) This result was in agreement with the structure prediction analysis that indicated that VBARP is a cytoplasmic protein Together, the transi-ently expressed VBARP exhibited a distinct cytoplas-mic arrangement, supporting the lack of any predicted nuclear localization signal (NLS) sequences or trans-membrane domains in VBARP Also, the use of unsynchronized cells in these analyses further con-firmed that the cytoplasmic distribution of VBARP is independent of the cycling stage of the cells
Identification of biological function(s) of VBARP using a siRNA assay
To examine the role of endogenous VBARP in the regulation of normal cellular events such as cell cycle and apoptosis, RNA interference studies were per-formed using VBARP siRNA and control siRNA Sev-eral VBARP siRNA duplexes were synthesized and purified using the Qiagen siRNA Tool Kit Based on the initial results, siRNA spanning nucleotides 143–154 (from the ATG) was identified that blocked the VBARP RNA synthesis in tested cell lines and this siRNA was used in subsequent assays Following transfection of VBARP siRNA into HeLa and NT2 cells, physical observation revealed that cell death occurred in a dose- and cell-dependent manner (data not shown) To further quantitate this effect, NT2 cells were transfected with different concentrations of VBARP siRNA or control siRNA and assayed for functional effects First, to confirm that VBARP
Fig 4 Expression of VBARP isoforms in vitro and in vivo: (A).
In vitro transcription ⁄ translation of VBARP-L and VBARP-S One
microgram of VBARP-L, VBARP-S and vector plasmid was in vitro
transcribed ⁄ translated using 35
S-methionine as described in Experi-mental procedures In vitro translated products were
immunopre-cipitated with anti-V5 IgG, resolved in an 8% SDS ⁄ PAGE and
autoradiographed (B) Expression of VBARP using transient
trans-fection system: HEK293T cells were transfected with 5 lg of
VBARP-L, VBARP-S and vector plasmids and immunoblotted with
anti-V5 IgG Lanes are represented with the respective plasmid
used on the top and the markers are labeled on the left Arrows
indicate specific gene products.
Fig 5 Subcellular distribution of VBARP-L and VBARP-S: HeLa cells were transiently transfected with His-tagged VBARP-L and VBARP-S Post transfection cells were stained with anti-His IgG and detected by Alexaflour 594 (Red) Nuclei were stained with Dapi (Blue) All images were captured
at 60· magnification using a Nikon micro-scope.
Trang 7expression is specifically blocked by treatment with
VBARP siRNA, siRNA transfections were performed
and total RNA was isolated from the cells, 36 h post
transfection, and used to amplify VBARP and b-actin
by RT-PCR (Fig 6A) Results indicated that
treat-ment of VBARP siRNA inhibited the synthesis of
VBARP RNA in a dose dependent manner, whereas
the b-actin control was not altered, suggesting that
VBARP treatment is specific and does not alter the
global cellular transcription Effect of VBARP siRNA
on cell viability was tested by the trypan blue exclusion
assay and cell viability assay The cell viability results
of VBARP and control siRNA, compared with the
oligofectamine control (considered to be 100%), are
presented in Fig 6B Results indicated that NT2 cells
treated with 10 nm of VBARP siRNA exhibited 50%
cell death, whereas control siRNA at the same
concen-tration did not affect cell viability However, at a
con-centration of 100 nm the percentage of cell viability in
VBARP and control siRNA-treated cells was 20 and
75, respectively The number of viable cells in the
VBARP siRNA-treated group was reduced in a
dose-dependent manner At the highest concentration
(200 nm), both control and VABRP siRNA complexes
became toxic to NT2 cells Similar results were
observed in HeLa cells, also indicating that VBARP
might perform similar functions in cells of different
lineages
Caspases involved in VBARP-mediated cell survival
Caspases, a family of cysteine acid proteases, are cen-tral regulators of apoptosis [12,13] Caspases are rou-tinely used as a measure of apoptosis, in contrast to necrosis Caspase 3 activation occurs at the intersec-tion of all caspase-dependent pathways and is, there-fore, an excellent marker of caspase-dependent apoptotic death We sought to identify whether caspase 3 is activated during siRNA-mediated blocking
of VBARP gene expression Cells treated with increas-ing concentrations of VBARP or control siRNA were assessed for caspase activity as described in Experi-mental Procedures, and the results are presented in Fig 7 Results indicated that cells treated with VBARP siRNA exhibit a higher amount of caspase 3⁄ 7 activity when compared with the control siRNA-treated cells in a dose-dependent manner Also, the effect of the siRNA was also measured against time following transfection and the results indicated that the effect of VBARP siRNA was also time dependent (data not shown) Taken together, these results suggest that VBARP isoforms may possess an antiapoptotic effect and protect cells during normal cell proliferation Furthermore, the regulation of casp-ases may be one of the pathway(s) by which VBARP regulates cell survival Further study is warranted to
B
Fig 6 Functional analysis of VBARP using siRNA: (A) siRNA-specific knockdown of VBARP RNA Cells (NT2) cells were transfected with VBARP-specific siRNA or control siRNA Thirty-six hours post transfection, total RNA was isolated from the cells and amplified with VBARP
or actin specific primers by RT-PCR M, represents DNA marker, L, represents the lipofectamine control Different concentrations of VBARP and control siRNA used are indicated at the bottom of the respective lanes (B) Effect of siRNA on cell viability HeLa 1 and NT2 2 cells were transfected with the various concentration of VBARP or control siRNA in triplicate Forty-eight hours post transfection, cells were assayed for cell viability Percentage of viable cells in mock transfected (oligofectamine) was considered as 100% Results represent an average of three independent experiments.
Trang 8understand the pathway(s) and mechanism(s) involved
in VBARP and its regulation apoptosis
Discussion
We identified and functionally characterized VBARP,
a novel splice variant of ANKHD1 Human
ANKHD1 gene is a large transcript containing 2
mul-tiple ankyrin repeat motif domains and a single KH
domain similar to the MASK gene found in
Drosophila Drosophila MASK (dMASK) has been
implicated in cell survival and may play a role in
promoting proliferation and preventing apoptosis [8]
dMASK mediates receptor tyrosine kinase (RTK)
sign-aling, independent of MAP kinase (MAPK) by either
functioning downstream of MAPK or by defining a
new pathway of RTK signaling [8] RTKs play
import-ant roles in cell signaling during cell proliferation,
apoptosis, and cell survival upon stress [14–16]
Despite the fact that a homolog of dMASK is present
in the human genome, neither its function nor its
involvement in RTK signaling is established for
hMASK (ANKHDI) Poulin et al [17] identified a
gene fusion between MASK and 4E-BP3 that occurs
rarely in the human genome Although 4E-BP3 is a
member of the eukaryotic initiation factor family, the
role of this 4E-BP3–MASK fusion in transcription
regulation has yet to be defined
VBARP, identified as an HIV-1 Vpr-interacting
pro-tein through yeast two-hybrid system analysis is a
dis-tinct splice variant of ANKHD1 blast search analysis
revealed that VBARP is located on chromosome
5q31.3, which has no known biological function(s)
Unlike hMASK, VBARP does not contain a KH
domain RNA analysis and blast analysis indicated
that homologs and orthologs of VBARP exist,
indica-ting the presence of VBARP in diverse phyla such as
plants, yeast, and eukaryotes These results suggest that VBARP might be evolutionarily conserved, impli-cating its involvement in basic cellular function(s) Also, VBARP appears to be ubiquitously expressed in multiple human tissues and primary and secondary cell lines of various lineages Taqman analysis further con-firmed that VBARP is expressed at varying levels in different tissue types such as spleen, cervix, heart, brain, lung, liver, and skeletal muscle Although VBARP appears to be present in all the tested tissues, the various expression levels and transcripts suggest that differential or alternate splicing might be taking place in these tissues These findings were consistent with a recent study by Poulin et al [17], introducing a novel 8.0 kb transcript called human MASK which contains part of VBARP Stringent real-time RT-PCR analysis, using various cellular subsets, revealed expres-sion of a range of ANKHD1 isoform 1 (human MASK) among different cell types This suggests a specific role or requirement for VBARP in cells of many different types Because a large portion of VBARP is present within ANKHD1 (hMASK), a simi-lar conclusion can be drawn for VBARP using these data Therefore, owing to its ubiquitous expression, it
is possible that VBARP plays an important role in the life cycle of various tissues in multiple organisms Functional analysis supports predictions that ubiqui-tously expressed VBARP appears to play a role in cell survival, because blocking the expression of VBARP resulted in apoptosis Human cells of different lineages exposed to VBARP siRNA resulted in apoptosis in a dose- and time-dependent manner when compared with control siRNA-treated cells, confirming an important role for VBARP in cell survival and antia-poptotic pathway(s) However, our analysis focuses on human cell types, and it was not extended to other species Based on the presence of VBARP in many eukaryotes and the high level of homology, it is pos-sible to propose that a similar phenomenon might occur in cells of other species In Drosophila, MASK is shown to be critical for photoreceptor differentiation, cell survival and proliferation [8] Further studies are
in progress to address these pathways
Several ankyrin repeat proteins are associated with cell survival and are antiapoptotic Interfering with the normal expression of these proteins leads to cell death and lethality during development [18] These proteins are also proposed to play important roles in neuronal degeneration and apoptosis induced by chemical toxins via degradation [19] It is not clear whether VBARP has a similar functional phenotype Our results indi-cate that expression of VBARP is essential for normal cell function and knockout of VBARP expression leads
Fig 7 VBARP siRNA induced caspase activity: NT2 cells (triplicate
wells) were transfected with VBARP or control siRNA Thirty-six
hours post transfection cells were lyzed and assessed for
caspase 3 ⁄ 7 activity Caspase activity was measured and
represen-ted in relative light units (RLU) Figure represents one of three
inde-pendent experiments.
Trang 9infected cells alone cannot account for this severe loss
of T cells Several viral and host cellular proteins are
known to play important roles in cell depletion in vivo
[20,21] One of the HIV-1 virion-associated proteins,
Vpr, has been shown to dysregulate several host
cellu-lar functions including apoptosis in infected and
unin-fected bystander target cells through its interaction
with host cellular proteins [22–25] However, it is not
clear at this point how interaction of Vpr and
VBARP leads to apoptosis but several scenarios exist
One possibility is that redistribution or degradation of
VBARP in the presence of Vpr could abolish the
antiapoptotic function of VBARP or alter it from its
normal cell functions Using these potential
mecha-nism(s), Vpr could exploit this pathway to induce
apop-tosis in the bystander-uninfected population, given the
fact that VBARP is abundantly present in many of
the tested human primary cells Understanding the
functions and identifying the other regulatory proteins
involved in antiapoptotic functions regulated by
VBARP will shed new light on the function of this
novel protein as well as developing additional
thera-peutics for HIV-1
Experimental procedures
Cell culture
Established cell lines HeLa, NT2, 293, HEK293T, and
CEMx174 were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% fetal bovine serum
(FBS) and 1% penicillin–streptomycin solution Normal
human primary PBMC, PBL, macrophages and dendritic
cells were isolated from heparinized blood using the
Ficoll-Hypaque method PBMC and PBL were blasted with
PHA-P (5 lgÆmL)1, Sigma, St Louis, MO) for 3 days and
cultured in RPMI containing 10% FBS, 1% penicillin–
streptomycin and interleukin-2 growth factor Monocytes
To identify Vpr-interacting host cellular protein(s), we used
a human cDNA library as described [9] Briefly, full-length Vpr was fused in-frame with a Gal4 DNA-binding domain and used as bait in the yeast expression vector pGBT9 The GAL-4 activation domain tagged brain cDNA library (gift from Dr Srinivasan, Thomas Jefferson University, PA) was used as prey To eliminate the false-positive clones, cDNA clones alone were transformed in yeast and screened on high-stringency plates Sequencing the remaining seven clones identified a 915-bp fragment multiple times Interac-tion of this 915-bp fragment was further confirmed by yeast two-hybrid system, as well as by using a Checkmate mam-malian hybrid system as suggested by the manufacturer (Promega, Madison, WI)
Construction of VBARP and Vpr expression plasmids
Upon completion of the human genome project, the VBARP cDNA construct was available through the Ameri-can Tissue Type Collection (ATCC) as an IMAGE clone PCR primers were designed to amplify the original 915-bp Vpr-interacting fragment, as well as the two VBARP iso-forms, VBARP-L (1.9 kb) and VBARP-S (1.3 kb) The PCR products (Table 1 for details on PCR primers) were amplified and cloned into pcDNA3.1 CMV⁄ T7 TOPO vec-tor with a V5⁄ His epitope as per the manufacturer’s instructions (Invitrogen) for use in further eukaryotic expression studies Positive clones were sequenced to verify nucleotide integrity at the University of Pittsburgh Genom-ics and ProteomGenom-ics Core Laboratory The resulting plas-mids were designated as pVBARP-L and pVBARP-S Vpr expression clones were constructed as described previously [9]
GenBank BLASTand computer analyses
The 915-bp fragment recognized as the Vpr-binding domain was sequenced The UCSC Genome Bioinformatics Blast
Table 1 Primers used to construct VBARP expression plasmids.
TAAGCTACTACGTAAAGAATATATC (Reverse)
CATATATTCTTTACGTAGTAGCTTA (Reverse)
Trang 10Like Alignment Tool (http://www.genome.ucsc.edu/cgi-bin/
hgBlat) was used to find the location of this nucleotide
sequence in the human genome and also to identify the
intron⁄ exon boundaries of its genomic sequence spidey
software (http://www.ncbi.nlm.nih.gov/) was also used to
generate the intron⁄ exon maps The nucleotide and deduced
amino acid sequences were subjected to homology searches
using the NCBI blast program (http://www.ncbi.nlm.nih
gov/BLAST) in order to identify homologous sequences
present in the sequence databases [26,27] Invitrogen’s
alignx software was used for multiple sequence alignment
of VBARP homologous proteins Databases of protein
families and domains including pfam (http://www.sanger
ac.uk/Software/Pfam/) prosite (http://lsexpasy.org/
prosite/) and NCBI’s Conserved Domain Database (CDD;
http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) were
searched to identify the presence of conserved motifs and
domains [28,29] Several sequence analysis programs
avail-able from the Expert Protein Analysis System (expasy;
http://lsexpasy.org/tools) proteomics server of the Swiss
Institute of Bioinformatics were used for in sillico
character-ization of VBARP, including prediction of its subcellular
localization (psort: http://www.psort.org), prediction of
post-translational modification (netphos: http://www.cbs
dtu.dk/services/NetPhos), prediction of its topology and the
primary and secondary structure analysis (predictprotein:
http://www.cubic.bioc.columbia.edu/predictprotein)
RNA extraction and quantitative real-time
RT-PCR
Total RNA was extracted from human tissues and human
primary cells using Qiagen RNA purification kit (Qiagen,
Valencia, CA) and used in real-time PCR analysis for
var-ious VBARP isoforms Two-step RT-PCR was performed
as follows: RNA (0.2–0.5 lg) was reverse transcribed using
Taqman reverse transcription reagents (Applied Biosystems,
Foster City, CA) Real-time PCR was carried out in
tripli-cate using an ABI Prism 7000 Detection System and
ana-lyzed using the included sequence detector software
Commercially available primer⁄ probe sets specific for the
ANKHD1⁄ VBARP isoforms and the ribosomal large
pro-tein (RPLPO) were used (Applied Biosystems, San Diego,
CA) to identify the different variants The comparative CT
method was used to determine the relative ratio of
tran-script between different samples as described [30] RNA
levels were normalized to RPLPO
Protein expression analysis
In vitro T7 transcription⁄ translation
In vitro transcription⁄ translation of VBARP plasmids was
accomplished using the TnT Quick Coupled System as
per the manufacturer’s instructions (Promega) Briefly,
1–2 lg of VBARP-His were combined with 40 lL of TnT Master mix containing rabbit reticulocyte lysates,
2 lL of [35S]-methionine (MP Biomedicals, Irvine, CA) and nuclease-free water to a total volume of 50 lL The reaction was incubated at 30C for 90 min and used in subsequent experiments For transient expression studies, HEK293T cells (1· 106
) were transfected with control vec-tor, VBARP-L or VBARP-S expression constructs using calcium phosphate as described previously [31] Forty-eight hours post transfection, cells were washed with cold NaCl⁄ Piand collected Cell pellets were subsequently lysed with RIPA buffer containing 50 mm Tris (pH 7.5), 150 mm NaCl, 1% Triton X-100, 1 mm sodium orthovanadate,
10 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluo-ride 0.05% deoxycholate, 10% SDS, trypsin inhibitor (0.07 unitÆmL)1) aprotinin, and protease inhibitors leupep-tin, chymostaleupep-tin, and pepstatin (1 lgÆmL)1; Sigma) as des-cribed [14] for 15 min in ice Cell lysates were centrifuged
at 22 000 g for 10 min at 4C to remove cell debris Pro-tein estimation of the cell lysates was carried out using Bradford reagent (Bio-Rad Laboratories, Hercules, CA) Total cell lysates (50 lg) were separated on an 8–12% SDS⁄ PAGE gel, transferred to poly(vinylidene difluoride) membrane and immunoblotted with antialpha-Tubulin (1 : 500) (NeoMarkers, Fremont, CA) and mouse mono-clonal anti-His IgG (1 : 200) (Abcam, MA) followed by horseradish peroxidase-conjugated goat anti-mouse IgG (1 : 10000) and blots were developed using the ECL chemi-luminescence detection kit (Amersham Biosciences)
Subcellular localization studies by indirect immunofluorescence
HeLa cells (1· 104) were seeded in a four-well chamber slide (Falcon, Franklin Lakes, NJ) and transfected with VBARP-L and VBARP-S expression plasmid DNA using Lipofectamine (Invitrogen) according to the manufacturer’s instructions Forty-eight hours post transfection, cells were washed with 1· NaCl ⁄ Pi, fixed in 2% paraformaldehyde at room temperature for 10 min, then washed and permeabi-lized with 0.05% Triton X-100 for 10 min After washing three times with 1· NaCl ⁄ Pi, fixed cells were incubated at room temperature for 90 min with anti-His IgG (1 : 200) (Abcam), washed three times with 1· NaCl ⁄ Pi, and then incubated with goat anti-(mouse epitope) IgG Alexafluor
594 (1 : 400) (Molecular Probes, Eugene, OR) for 60 min
at room temperature Following several washes with 1· NaCl ⁄ Pi, cells were dried and mounted with
Laboratories, Burlingame, CA) Immunofluorescence was detected using a fluorescence microscope with Nikon SPOT camera (Fryer, Huntley, IL) and images were processed using metamorph software (Universal Imaging Corp., Downington, PA)