From the Departamento de Bioquímica y
Biología Molecular and
Area de Farmacología,
Facultad de Medicina, Instituto Universitario de Oncología,
Universidad de Oviedo, 33006-Oviedo, Spain
Received for publication, August 12, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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We have cloned four human cDNAs encoding
putative cysteine proteinases that have been tentatively called
autophagins. These proteins are similar to Apg4/Aut2, a yeast enzyme
involved in the activation of Apg8/Aut7 during the process of
autophagy. The identified proteins ranging in length from 393 to 474 amino acids also contain several structural features characteristic of
cysteine proteinases including a conserved cysteine residue that is
essential for the catalytic properties of these enzymes. Northern blot
analysis demonstrated that autophagins are broadly distributed in human tissues, being especially abundant in skeletal muscle. Functional and
morphological analysis in autophagy-defective yeast strains lacking
Apg4/Aut2 revealed that human autophagins-1 and -3 were able to
complement the deficiency in the yeast protease, restoring the
phenotypic and biochemical characteristics of autophagic cells. Enzymatic studies performed with autophagin-3, the most widely expressed human autophagin, revealed that the recombinant protein hydrolyzed the synthetic substrate
Mca-Thr-Phe-Gly-Met-Dpa-NH2 whose sequence derives
from that present around the Apg4 cleavage site in yeast Apg8/Aut7.
This proteolytic activity was diminished by
N-ethylmaleimide, an inhibitor of cysteine proteases
including yeast Apg4/Aut2. These results provide additional evidence
that the autophagic process widely studied in yeast can also be fully reconstituted in human tissues and open the possibility to explore the
relevance of the autophagin-based proteolytic system in the induction,
regulation, and execution of autophagy.
Proteolytic enzymes, through their ability to catalyze
irreversible hydrolytic reactions, play crucial roles in the
development and maintenance of all living organisms (1). Proteases were initially characterized as nonspecific degradative enzymes associated with protein catabolism, but recent studies have demonstrated that they
influence a wide range of cellular functions by processing multiple
bioactive molecules. These essential processes initiated, regulated, or
terminated by proteases include DNA replication, cell-cycle
progression, cell proliferation, differentiation and migration,
morphogenesis and tissue remodeling, and angiogenesis and apoptosis
(1). An additional process in which proteolytic enzymes have also been
recently implicated is autophagy (2-4).
Autophagy is a biological process involved in the intracellular
destruction of endogenous proteins and the removal of damaged organelles and has been suggested to be essential for cell homeostasis as well as for cell remodeling during differentiation, metamorphosis, non-apoptotic cell death, and aging (3-6). In addition, autophagy has
also been associated with diverse pathological conditions. Thus, the
reduced levels of autophagy have been described in some malignant
tumors, and a role for autophagy in controlling the unregulated cell
growth linked to cancer has been proposed (7). A deficiency in
autophagy has also been found in heart diseases such as Danon
cardiomyopathy (8). By contrast, elevated levels of autophagy have also
been reported in other human pathologies, especially in
neurodegenerative diseases (9). There are four distinct
autophagy-related mechanisms: macroautophagy, microautophagy, crinophagy, and chaperone-mediated autophagy (3-6, 10, 11). Macroautophagy, the most widely studied mechanism in this regard and
usually referred to as simply as autophagy, is a nutritionally and
developmentally regulated process by which a portion of the cytosol is
sequestered by an isolation membrane (3-6). This results in the
formation of a structure known as autophagosome containing a double
membrane, which subsequently fuses with the lysosome/vacuole. The inner
membrane of the autophagosome called the autophagic body and its
protein and organelle contents are then degraded by lysosomal/vacuolar
proteases and recycled.
The knowledge of the molecular mechanisms underlying autophagy has
considerably improved after the isolation and characterization of
autophagy-defective mutants in the yeast Saccharomyces
cerevisiae (12, 13). These mutants were derived from screening for
starvation-sensitive yeast strains (apg mutants) or for
strains defective in the degradation of specific cytosolic proteins
(aut mutants). These mutants partially overlap with those
isolated in genetic screens for yeast strains defective in the
cytoplasm to vacuole-targeting pathway (cvt mutants), a
process that shares significant morphological and mechanistic similarities with autophagy (14). A series of elegant studies directed
to the functional characterization of these autophagy mutants has
revealed that two ubiquitin-like conjugation systems are required for
yeast autophagy (15, 16). The first one is initiated by Apg12, a
modifier protein whose C-terminal Gly residue forms a covalent
isopeptide bond with a Lys residue from Apg5. This conjugation process
involves an activating
E11-like enzyme called Apg7
and a conjugating E2-like enzyme named Apg10 (17, 18). The second
ubiquitin-like system requires the participation of Apg8/Aut7
synthesized as a precursor protein, which is cleaved after a Gly
residue by Apg4/Aut2, a recently described cysteine proteinase (2, 19,
20). This Gly-terminal residue from the modifier Apg8/Aut7 is also
activated by Apg7, but then the modifier protein is transferred
to Apg3 and finally conjugated with membrane-bound
phosphatidylethanolamine (PE) through an amide bond (16). The complex
Apg8·PE is also deconjugated by the protease Apg4/Aut7,
leading to the release of Apg8/Aut7 from membranes. These modification
systems are essential components of the membrane rearrangement dynamics
taking place during the formation of autophagosomes and execution of
autophagy. Recent studies (21-26) have shown that these ubiquitin-like
conjugation systems associated with autophagy in yeast are conserved in
higher eukaryotes. In fact, proteins structurally and functionally
related with the diverse yeast Apg/Aut proteins have been described in mammalian cells, and their roles in the process of autophagy have been elucidated in some cases. However, to date, very little is known
regarding the putative mammalian homologues of Apg4/Aut2, the yeast
cysteine proteinase essential for the proteolytic activation, and
subsequent lipidation and delipidation processes of Apg8/Aut7 (2, 19,
20).
In this work, we report the identification and characterization of four
human proteins closely related to yeast Apg4/Aut2. We also report the
tissue distribution and a preliminary analysis of the enzymatic
properties of these proteins that we have tentatively called
autophagins. Finally, we demonstrate that human autophagins-1 and -3 are able to complement the autophagy defect observed in yeast strains
defective in Apg4/Aut2, providing further evidence on the functional
conservation in higher eukaryotes of essential components of the
autophagy pathway in yeast.
Materials--
The S. cerevisiae strains
aut2 and aut7, defective in the process of
autophagy, were kindly provided by Dr. M. Thumm (University of
Stuttgart, Stuttgart, Germany). Yeast strains were grown in either YPD
(1% yeast extract, 2% peptone, and 2% glucose) or synthetic medium
(0.67% yeast nitrogen base without amino acids, 2% glucose, and
auxotrophic amino acids as needed). Escherichia coli strain XL-Blue was used for subcloning experiments. The yeast expression vector pGAD424 was obtained from Clontech (Palo
Alto, CA). Restriction endonucleases and other reagents used for
molecular cloning were from Roche Molecular Biochemicals.
Oligonucleotides were synthesized in an Applied Biosystems (Foster
City, CA) model 392A DNA synthesizer. Double-stranded DNA probes were
radiolabeled with [ Bioinformatic Screening of the Human Genome and cDNA
Cloning--
The advanced BLAST program from the National Center for
Biotechnology Information was used to search human genome databases looking for regions encoding putative proteins with sequence similarity to yeast Apg4/Aut7. This computer search led us to identify DNA contigs
in chromosomes 1, 2, 19, and X containing regions with significant
sequence similarity to Apg4/Aut7. To obtain full-length cDNA
sequences corresponding to the putative proteins encoded by these DNA
contigs, we designed specific oligonucleotides for each of them and
performed PCR experiments using a panel of commercially available
cDNA libraries (Clontech) and the Expand High
Fidelity PCR system (Roche Molecular Biochemicals). All PCR assays were carried out in a GeneAmp 9600 PCR system (PerkinElmer Life Sciences) for 40 cycles of denaturation (94 °C, 15 s), annealing
(64 °C, 15 s), and extension (68 °C, 60 s). Full-length
cDNAs were cloned into pBluesScript vector and characterized by
nucleotide sequencing.
Nucleotide Sequence Analysis--
Full-length cDNAs were
sequenced by the dideoxy chain termination method using the Sequenase
version 2.0 kit (U.S. Biochemicals, Cleveland, OH) and the ABI-Prism
310 DNA sequencer (Applied Biosystems). All of the nucleotides were
identified in both strands. Computer analysis of DNA and protein
sequences was performed with the GCG software package of the University
of Wisconsin Genetics Computer Group.
Northern Blot Analysis--
Nylon filters containing 2 µg of
poly(A)+ RNA of a wide variety of human tissues and cancer
cell lines were prehybridized at 42 °C for 3 h in 50%
formamide, 5× saline/sodium phosphate/EDTA (1× saline/sodium
phosphate/EDTA = 150 mM NaCl, 10 mM
NaH2PO4, 1 mM EDTA, pH 7,4), 10×
Denhardt's solution, 2% SDS, and 100 µg/ml of denatured herring
sperm DNA and then hybridized with radiolabeled probes for each
full-length clone cDNA. Hybridization was performed for 20 h
under the same conditions. Filters were washed with 0.1× SSC and 0.1%
SDS for 2 h at 50 °C and exposed to autoradiography. RNA
integrity and equal loading were assessed by hybridization with an
actin probe.
Transformation of Yeast Strains with Plasmid DNA--
5 ml of
overnight culture of yeast cells were added to 45 ml of fresh medium
and incubated for 4 h at 30 °C with shaking. The cells were
collected, washed with 50 mM Tris and 1 mM
EDTA, and resuspended in 2 ml of 0.1 M lithium acetate.
Approximately 1 µg of plasmid DNA was added to a 200-µl aliquot of
cells and incubated for 10 min at 30 °C. Heat shock treatments were
done at 42 °C for 5 min, and then cells were washed with 1 M sorbitol, were spread on selective plates, and incubated
for 48 h at 28 °C.
Immunoblotting and Complementation Studies--
Cultures of
parental and autophagin-transformed yeast cells were grown in complete
YPD or selective medium (synthetic medium/Leu Autophagin-3 Expression and Purification--
Autophagin-3
cDNA was cloned in the expression vector pCEP-Pu provided by Dr.
E. Kohfeldt (Max-Plank-Institut fur Biochemie, Martinsried, Germany)
downstream of the BM40 signal peptide to allow protein secretion to the
culture medium (27), and a His6 tag was placed at the
C-terminal end to facilitate purification. The resulting plasmid,
pCEP-Autophagin3-His, was transiently transfected into 293EBNA cells
(Invitrogen), and 24 h after transfection, culture medium was
replaced by serum-free medium. Cells were allowed to secrete the
recombinant protein for 48 h, and autophagin-3 was purified from
the conditioned medium using a nitrilotriacetic acid-agarose column
(Qiagen). Purified His6-tagged autophagin-3 concentration
was determined by Western blot using known amounts of
His6-tagged MMP-26 as standard and analyzed with the
software package Phoretix 1D Advanced version 5.10 (Nonlinear
Dynamics). Protein purity was confirmed by SDS-PAGE followed by silver staining.
Enzymatic Assays--
Enzymatic activity of recombinant
autophagin-3 was tested using the fluorogenic peptide
Mca-Thr-Phe-Gly-Met-Dpa-NH2 synthesized by Dr. C. G. Knight (University of Cambridge, Cambridge, United Kingdom). Enzyme
assays were performed with the purified protein at 30 °C and at
substrate concentration of 5 µM in an assay buffer of 50 mM Tris/HCl, pH 7.5, 125 mM NaCl, 1 mM dithiothreitol, 10 mM EDTA, and 2 mM AEBSF. The fluorometric measurements were made in a LS
50-B PerkinElmer spectrofluorometer ( Identification and Cloning of cDNAs Encoding Four Novel Human
Proteins Similar to Yeast Apg4/Aut2 Cysteine
Proteinase--
To identify human proteins related to the yeast
Apg4/Aut2 protease involved in autophagic processes, we used the BLAST
algorithm to screen the human genome databases looking for DNA
sequences encoding putative proteins similar to the S. cerevisiae protease. This search allowed us to identify five DNA
contigs in chromosomes 1p31.3, 2q37, 19p13.2, Xq13, and Xq22, which
contained coding information for putative cysteine proteases related to
yeast Apg4/Aut2. Preliminary analysis of the DNA contig in chromosome
Xq13 revealed the presence of several stop codons in different regions
of the putative protease coding region present in this contig,
indicating that it corresponds to a pseudogene unable to encode a
functional enzyme. To generate cDNA clones for the remaining four
genes, we carried out PCR amplifications using a panel of human
cDNA libraries and specific oligonucleotides derived from the
identified genomic sequences. DNA fragments large enough to encode
complete Apg4/Aut2-like proteins (~1.4 kb) and containing in-frame
initiator and stop codons were amplified from cDNA libraries
prepared from human testis, liver, ovary, and brain. After cloning and
sequencing the PCR-amplified products, we concluded that the isolated
cDNAs coded for proteins of 393, 398, 458, and 474 amino acids
(Fig. 1) (GenBankTM accession
numbers AJ504652, AJ504651, AJ312234, and AJ312332). Further structural
analysis of these amino acid sequences confirmed that they were
closely related to yeast Apg4/Aut2 with the percentage of identities
ranging from 32 to 25%. Because of the relevance of this yeast
protease during the process of autophagy, we have tentatively called
autophagins to this family of Apg4/Aut2-related human proteins.
Autophagins-1 and -2 matched perfectly with sequences originally
predicted from human ESTs and identified because of their homology with
yeast Apg4/Aut2 (2). cDNA sequences closely related or identical to
those here identified for human autophagins and derived in most cases
of large scale sequencing projects have also been recently deposited in
databases (GenBankTM accession numbers AL080168, KIAA0943,
and AB066215 for autophagin-1; GenBankTM accession numbers
AJ320508 and AB066214 for autophagin-2; GenBankTM accession
numbers AJ320169, BC008395, and BC033024 for autophagin-3; and
GenBankTM accession numbers NM_032885 and AK056210 for
autophagin-4). These proteins have also been annotated in the protease
data base MEROPS (www.merops.ac.uk) as members of the C-54
family of cysteine proteases (C54.003, C54.002, C54.004, and C54.005,
respectively). However, no report describing the cloning and
characterization of any of these human cDNAs or of their encoded
proteins has been published yet.
An alignment of the deduced amino acid sequence for human autophagins
confirmed that they maintain a significant sequence similarity with
yeast Apg4/Aut2 along the entire protein sequence with the exception of
notable divergences in both the N- and C-terminal ends of the diverse
proteins (Fig. 1). Human autophagins also exhibit the structural
features characteristic of the yeast protease including a putative
active site Cys residue at positions 74, 77, 110, and 134 in
autophagins 1-4, respectively (Fig. 1). The amino acid sequences
surrounding this Cys residue are highly conserved between human and
yeast proteins and include a Gln residue (positions 80, 83, 116, and
140 in autophagins 1-4), which can be part of the oxanion hole present
in the structure of cysteine proteases (29). Human autophagins also
contain some conserved His and Asp or Asn residues (Fig. 1) that can
correspond to the equivalent residues present in other cysteine
proteases and found to be essential in the catalytic process (29). The
absence in autophagins of a recognizable hydrophobic signal sequence
close to the initiator methionine is also remarkable. This indicates
that these proteins are cytoplasmic enzymes and therefore distinct from
members of the large papain family of secreted cysteine proteases
(30). All of these structural features are also absolutely
conserved in the amino acid sequence of the mouse orthologues of the
four human autophagins whose sequence was deduced from information derived from publicly available ESTs (Fig.
2) (GenBankTM accession
numbers AJ504653, AJ504654, AJ312233, and AJ312333 for mouse
autophagin-1-4, respectively) as well as in the autophagin-like
proteins present in other organisms such as Drosophila
melanogaster and Caenorhabditis elegans (2, 31). To
further explore the evolutionary and structural relationships between
human autophagins and related proteins, we next performed a
computational phylogenetic tree analysis (Fig. 2). This analysis revealed that the autophagins can be classified into two subfamilies of
closely related members. Autophagins-1 and -2 should be grouped together with CeApg4-2 and DmApg4-2, whereas
autophagins-3 and -4 should be closer to CeApg4-1 and
DmApg4-1.
In summary and according to this structural analysis, we can conclude
that the four identified and cloned human cDNAs encode members of a
novel family of intracellular proteins are closely related to the
founding member of this family, the yeast cysteine protease
Apg4/Aut2.
Expression Analysis of Autophagins in Human Tissues and Cancer Cell
Lines--
As a preliminary step to study the physiological role of
autophagins in human tissues, we examined by Northern blot analysis the
expression pattern of these genes in a variety of tissues including
colon, small intestine, ovary, testis, prostate, thymus, spleen,
pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, and
heart. The filters containing poly(A)+ RNA from these
tissues were sequentially hybridized with radiolabeled full-length
cDNA probes for autophagins-1-4, and the results obtained are
shown in Fig. 3. A single transcript of
~4.5 kb was mainly detected in skeletal muscle after hybridization
with a probe for autophagin-1 with some expression being also apparent
in heart, liver, and pancreas. Autophagin-2 transcripts of ~3.2 kb
were detected in several tissues, the strongest signal corresponding to
skeletal muscle. A major 3.5-kb autophagin-3 transcript was detected in
many tissues with the highest expression levels found in skeletal
muscle, heart, liver, and testis. Additional autophagin-3 transcripts
of 3.0 kb could be also observed in the same tissues. Finally, a unique
autophagin-4 transcript of ~2.4 kb was detected in skeletal muscle
and at lower levels in the testis.
We also evaluated the expression of autophagins in human fetal tissues
with the finding that autophagin-2 is detected in fetal liver. No
significant levels of autophagins-1, -3, and -4 expression were
detected in the examined fetal tissues (Fig. 3). Finally, we also
addressed the possibility that autophagins could be expressed by human
cancer cell lines from different sources. For this purpose, we
hybridized a Northern blot containing poly(A)+ RNAs
extracted from different cell lines (HL-60, HeLa, K-562, MOLT-4,
Burkitt's lymphoma Raji, colorectal adenocarcinoma SW480, lung
carcinoma A549, and melanoma G361) with probes for the different autophagins. As shown in Fig. 3, autophagin-1 was widely expressed in
these cells with the strongest signal observed in A549 and Raji cells.
Autophagin-3 was also detected in diverse cell lines such as HeLa,
MOLT-4, and Raji, whereas autophagin-4 was strongly expressed in the
chronic myelogenous leukemia cell line K-562 and at lower levels in
SW480 and HeLa cells.
Complementation Studies with Human Autophagins in
Autophagy-defective Yeast Strains--
To study the putative
implication of the identified proteins in the process of autophagy, we
cloned the full-length cDNAs for the four human autophagins in the
yeast expression vector pGAD424 (32) under the control of the
constitutive ADH1 gene promoter, obtaining four new plasmid constructs
pGAD-Autophagin1, pGAD-Autophagin2, pGAD-Autophagin3, and
pGAD-Autophagin4. These plasmid constructs were used to transform the
S. cerevisiae autophagy-defective mutants strains aut2 and
aut7, and the properties of the transformed yeasts in terms of
restoration of biochemical and morphological markers of autophagy were analyzed.
For this purpose, we first examined the processing of the vacuolar
hydrolase API from its inactive precursor, a process that is defective
in autophagy yeast mutants. Thus, wild-type cells and aut2
mutants carrying the autophagin cDNAs were grown overnight in YPD
or selective medium (synthetic medium/Leu
We also tested the ability of the autophagy-defective yeast mutants
carrying autophagin-1 or -3 to accumulate autophagic bodies in the
vacuole, a characteristic feature of the autophagic degradation pathway. To do that, wild-type, aut2, and aut7
mutants transformed or not with autophagin-1 or -3 cDNA were grown
in YPD or selective medium overnight and then transferred to a medium
containing 1 mM PMSF. This serine proteinase inhibitor
blocks the activity of vacuolar proteases, thereby hampering the
autophagosome degradation and leading to the accumulation of these
structures inside the vacuole. After 4 h of incubation, cells were
washed, fixed, and photographed. In agreement with the results observed
in the maturation of proAPI, only aut2 mutant cells carrying
autophagin-1 or -3 cDNA were able to restore the process of
autophagy completing the transport of vesicles to the vacuole (Fig.
4B).
Enzymatic Assays of Human Autophagin-3--
To test the enzymatic
activity of human autophagin-3, the most widely distributed family
member in human tissues, we produced recombinant autophagin-3 in an
eukaryotic expression system. For that purpose, we first cloned the
full-length cDNA for human autophagin-3 into the expression vector
pCEP-Pu containing the BM40 signal peptide to allow protein secretion
to the culture medium as well as a His6 tag at the
C-terminal end to facilitate detection and purification (27). The
resulting plasmid, pCEP-Autophagin3-His, was transfected into 293EBNA
cells, and the secreted protein was detected by Western blot using a
monoclonal antibody against the His6 epitope (Fig.
5A). A single band of the
expected molecular mass (54 kDa) was detected, confirming that the
protein was expressed and correctly secreted to the culture medium.
Recombinant autophagin-3 was purified from the conditioned medium using
a nitrilotriacetic acid-agarose column. The purified protein was
recovered at very low levels, and it was necessary to use Western blot
analysis to clearly detect its presence. Nevertheless, no evidence of
additional proteins co-purifying with autophagin-3 was observed. This
recombinant autophagin-3 was used to assay its proteolytic activity
using as a substrate the fluorescent peptide
Mca-Thr-Phe-Gly-Met-Dpa-NH2. To design this substrate, we
first took into account the amino acid sequence of Apg8/Aut7 in the
region that is cleaved by Apg4/Aut2:Thr-Phe-Gly
After synthesizing the fluorogenic substrate
Mca-Thr-Phe-Gly-Met-Dpa-NH2, it was incubated with 10 ng of
purified autophagin-3 and the hydrolyzing activity of the protease was
followed by fluorometric measurements. As shown in Fig. 5B,
autophagin-3 exhibited significant proteolytic activity against the
synthetic substrate. This proteolytic activity was diminished by
N-ethylmaleimide, an inhibitor of cysteine proteases
including yeast Apg4/Aut2, providing additional evidence that the human
autophagin-3 is a functional homologue of this yeast cysteine protease.
The presence in the enzyme assay of inhibitors against serine
proteinase or metalloproteinases did not have any effect on the
autophagin-3 proteolytic activity against the fluorogenic substrate. As
an initial attempt to determine the catalytic activity of this
protease, a kinetic study was performed using the fluorogenic substrate
described above. For this study, autophagin-3 (34 nM in 2 ml of reaction buffer) was incubated with different concentrations of
substrate, and the kcat/Km
was deduced as described by Northrop (28). The
kcat/Km was determined to be 3.06 × 104 M Because of the expanding roles for proteolytic enzymes in the
cellular control of multiple biological processes, there has been an
increasing interest in the identification and functional characterization of the human degradome, the complete set of proteases produced by human tissues (1). In this work, we describe a new family
of human proteases called autophagins because of their structural and
functional similarity with a yeast cysteine protease involved in the
development of autophagy. The approach followed to identify human
autophagins was first based on a computer search of the human genome
sequence databases looking for regions with similarity to yeast
Apg4/Aut2. After identification of several DNA sequences encoding
proteins related to this yeast protease and PCR amplification
experiments using human cDNA libraries as template, full-length
cDNAs coding for four distinct proteins were finally isolated and
characterized. A structural analysis of the identified sequences
confirmed the close relationship of these four human proteins with
their yeast counterpart including an absolutely conserved cysteine
residue probably corresponding to the active site residue of cysteine proteases.
Consistent with these structural characteristics, functional analysis
of the recombinant autophagin-3 produced in a mammalian expression
system revealed that it is a catalytically active cysteine proteinase.
In fact, the recombinant human protein exhibits a significant
proteolytic activity against a fluorogenic substrate designed in this
work to specifically analyze the activity of Apg4/Aut2-related
proteases. This fluorogenic peptide contains the sequence around the
Apg4/Aut2-cleavage site of Apg8/Aut7, the natural substrate of this
yeast protease. Furthermore, this sequence is absolutely conserved in
two human proteins, MAP-LC3 and GATE-16, proposed to play equivalent
roles to yeast Apg8/Aut7 in the conjugation cascade associated with
autophagy (22, 24-26). The finding that autophagin-3 hydrolyzes the
peptide containing the sequence present in these two human proteins, is
consistent with the possibility that MAP-LC3 and GATE-16 are bona
fide substrates for human autophagins. It is also remarkable that
this degrading activity was diminished by N-ethylmaleimide,
an inhibitor of Apg4/Aut2p that also blocks the process of autophagy in yeast.
With the exception of autophagins-1 and -3, which complement the
autophagy defect in Apg4/Aut2-deficient yeast strains, we do not have
evidence yet that the two other autophagins described herein are
related to autophagy in human. One possibility is that autophagins-1
and -3 are closely related in functional terms to their yeast
homologue, whereas the remaining human autophagins have diverged
considerably or possess specific structural or functional constraints
because of the need to target different substrates. In fact, the
finding that the mammalian autophagin-based proteolytic system is
composed of four distinct proteases that may target at least three
putative specific substrates compared with the simplified yeast system
involving a single protease with a specific substrate clearly indicates
that this conjugation system has acquired a high degree of complexity
during eukaryote evolution. Therefore, the observation that
autophagins-2 and -4 do not complement the autophagy defect in
Apg4/Aut2-deficient yeast strains should not be used to rule out their
relevance in this process. Interestingly, hApg5 and hApg12, the human
homologues of two yeast proteins essential for autophagy, do not
complement the autophagy deficiency in Apg5 or Apg12 mutant yeasts
(21), providing additional evidence that the complementation
experiments may have limitations to extrapolate functional roles from
yeast proteins to their human counterparts. It is also remarkable that
GABARAP, the third human homologue of Apg8/Aut7, has a sequence around
the putative cleavage site by autophagins, which markedly deviates from
the consensus sequence found in Apg8/Aut7 as well as in the other human
homologues of this yeast protein (33).
In this work and as a previous step to elucidate the physiological role
of human autophagins, we have also examined the tissue distribution of
these proteins. Similar to other cysteine proteases involved in general
degradative processes, the expression of autophagins is detected in a
wide variety of human tissues, albeit at low levels in most cases. This
finding is consistent with the idea that autophagy is a mechanism for
bulk degradation of cytosolic proteins and organelles that takes place
in all cells at basal levels (3-6). Nevertheless, the observation of
high expression levels of most human autophagins in skeletal muscle
suggests that autophagic activity may be especially relevant in this
tissue. This finding is also of particular interest in light of
previous data reporting the association of autophagy abnormalities in
pathological conditions involving skeletal muscle including some forms
of muscular dystrophy (8, 34, 35). These putative associations between autophagins and skeletal muscle diseases may also imply the possibility that inherited alterations in these genes could be linked to familial forms of these pathologies. Chromosomal location analysis of autophagin genes indicate that they are not clustered in the human genome mapping
to chromosomes 1p31.3 (autophagin-3), 2q37 (autophagin-1), 19p13.2
(autophagin-4), and Xq22 (autophagin-2). Genetic lesions in these
regions have been linked to several diseases including muscular
disorders whose responsible genes remain to be characterized. Of
special interest is the finding of an autosomal dominant vacuolar neuromyopathy, which exhibits a muscle pathology with features of
autophagic diseases and which is linked to 19p13 (36), the region where
the autophagin-4 gene is located. The X-linked vacuolar myopathies
distinct from Danon disease caused by mutations in LAMP-2
(lysosome-associated membrane protein-2) at Xq24 (8) have also been
reported (35). It will be of future interest to examine the possibility
that the autophagin genes could be a target of some of these genetic
abnormalities. Likewise, the identification in this work of the
putative murine orthologues of human autophagins opens the possibility
to generate mice deficient in these genes that could contribute to
clarifying the role of this proteolytic system in physiological and
pathological conditions including its specific functions in skeletal muscle.
Previous studies have also shown that the process of autophagy may be
of great relevance in cancer. Thus, the finding that the tumor
suppressor beclin 1 (Apg6) is an inducer of autophagy has demonstrated
that components of the autophagy machinery may play a fundamental role
in the control of the unregulated cell growth associated with tumor
development (7). Autophagy is also linked with type II (non-apoptotic)
programmed cell death and may contribute to death in cells in which
caspase activity is blocked (37). These findings together with the
multiple observations indicating that expression and activity of many
proteolytic enzymes are profoundly deregulated in cancer suggest that
specific alterations in autophagin-mediated pathways may also be linked
to tumor development. As a preliminary step to evaluate this question,
we have performed an analysis of autophagin expression levels in human
cancer cell lines. The results obtained in these experiments indicate
that these proteases are overexpressed in some cancer cells, whereas they appear to be completely absent in other tumor cells. It is also
worthwhile mentioning that the regions containing the autophagin genes
are frequently altered in several human tumors (38-40). It will be of
great interest to examine the possibility that autophagins may play
specific roles in tumorigenesis in a similar way to that reported for
other cysteine proteases, such as Unp, HAUSP, Tre-2/USP6, Dub-1,
BAP1, and ubiquitin C-terminal hydrolase 1, associated with protein
modification pathways that are related to those mediated by autophagins
in autophagy and whose unregulated expression or activity has been
linked to cancer (41-46).
Finally, we would like to emphasize that the description of four
distinct human and mouse autophagins confirms and extends previous
findings proposing the widespread occurrence of this proteolytic system
originally described in yeast but also found in mammals, insects,
nematodes, and plants (2). Nevertheless, the complexity of the human
autophagin system compared with that present in other eukaryotes
provides an additional example of the impressive diversity of cysteine
proteases mediating a variety of modification reactions in human
tissues. To date, four different families of enzymes capable of
conjugate/deconjugate protein or lipid adducts through cleavage
adjacent to the C terminus of a Gly residue have been described (47,
48). These cysteine proteases include ubiquitin C-terminal hydrolases,
ubiquitin-specific processing proteases (USPs or UBPs),
sentrin/sumo-specific processing proteases or SUSPs, and now
autophagins. According to our most recent estimations derived from
human genome sequence analysis, at least 5 ubiquitin C-terminal
hydrolases, 50 USPs, 7 sentrin/sumo-specific processing proteases, and
4 autophagins are produced by human tissues. Interestingly, a novel
family of metalloproteases with deubiquitinating properties has also
been identified recently (49, 50). The large and growing number of
human proteases belonging to these different families underscores the
relevance of conjugation/deconjugation systems for the regulation of
multiple biological processes (51-53). Further studies directed to
clarify the functional roles of autophagins will be very useful in
establishing their relative importance in the context of the diverse
ubiquitin-related modification systems occurring in human tissues.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-32P]dCTP (3000 Ci/mmol) from
Amersham Biosciences using a commercial random-priming kit purchased
from the same company. cDNA libraries from human tissues and nylon
filters containing polyadenylated RNAs from different fetal and adult
human tissues were from Clontech. All of the media
and supplements for cell culture were obtained from Sigma with the
except of calf serum, which was from Roche Molecular Biochemicals.
Antiserum against proaminopeptidase-I (proAPI) was kindly provided by
Dr. M. J. Mazón (Instituto de Investigaciones
Biomédicas, Madrid, Spain). Penta-His monoclonal antibody against
His5 tag was purchased from Qiagen (Valencia, CA).
) for
16 h at 28 °C. For immunoblotting studies,
A600 was adjusted to 3.0 and extracts were
obtained by lysis with 1.85 M NaOH and 7.4%
-mercaptoethanol. Proteins were precipitated with 25%
trichloroacetic acid followed by centrifugation at 14,000 × g. The pellets were resuspended in urea buffer (5% SDS,
8 M urea, 200 mM Tris/HCl pH 6.8, 0.1 mM EDTA, and bromphenol blue), incubated for 10 min at
60 °C, and loaded onto 13% SDS-PAGE gels. Western blots were blocked in 5% milk in PBT (PBS containing 0.1% Tween 20) and then incubated for 1 h with rabbit antiserum against API dilution of 1:1000 in PBT. After three washes in PBT, blots were incubated for
1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG at
a 1:20,000 dilution and developed with the Renaissance chemoluminescence kit (PerkinElmer Life Sciences). For complementation studies, cells grown overnight were subjected to starvation for 4 h in a medium containing 0.1% potassium acetate and 1 mM
PMSF, collected, fixed, and visualized in a phase-contrast microscope.
ex = 328 nm and
em = 393 nm, where "ex" stands for excitation and
"em" for emission). For the pH profile, assays were performed in
assay buffer as noted above but using 50 mM bisTris, pH
6-9, 50 mM sodium acetate, pH 4.5-5.5, or 50 mM glycine, pH 10, as buffer for the indicated pH range and
containing 2.5 µM fluorogenic substrate and 10 ng of
enzyme. Kinetic studies were carried out using different concentrations of the fluorogenic peptide (0.5-5 µM) in 2 ml of assay
buffer containing 10 ng of autophagin-3, and peptide hydrolysis was
measured as the increase in fluorescence at 25 °C for 15 min.
Initial velocities were calculated using the analysis package FL WinLab
2.01 (PerkinElmer), and
kcat/Km ratio was calculated
as described previously (28). For inhibition experiments, reaction
mixture was preincubated for 30 min at 20 °C with 1 mM
N-ethylmaleimide (Sigma).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
View larger version (74K):
[in a new window]
Fig. 1.
Amino acid sequence of human and mouse
autophagins and comparison with yeast Apg4/Aut2. The multiple
alignment was performed with the ClustalX program. Gaps are indicated
by hyphens. Common residues to all sequences are
shaded. The Cys residues characteristic of cysteine
proteinases are shown with an asterisk.
View larger version (10K):
[in a new window]
Fig. 2.
Phylogenetic tree of the autophagin
family. Amino acid sequences of the different human and mouse
autophagins and of related proteins identified in Arabidopsis
thaliana, C. elegans, and D. melanogaster were aligned
using the Phylip program package (version 3.6). Numbers represent
reliability values after bootstrapping the data. GenBankTM
accession numbers for A. thaliana, C. elegans, and D. melanogaster autophagins are BAB88384, BAB88383, Z68302, AL110500,
CG6194, and CG4428.
View larger version (53K):
[in a new window]
Fig. 3.
Analysis of expression of autophagins in
human tissues and cell lines. Filters containing ~2 µg of
polyadenylated RNAs from the indicated adult and fetal tissues, and
cancer cell lines were hybridized with the full-length cDNAs
isolated for human autophagins. RNA sizes are indicated. Filters were
subsequently hybridized with a human actin probe to ascertain the
differences in RNA loading among the different samples.
). The cells
were collected, lysed in SDS-PAGE sample buffer, and analyzed by
Western blot with a polyclonal rabbit antiserum against API.
Transformed aut2 mutant cells lacking the endogenous Apg4/Aut2 activity but carrying the autophagin-1 or the autophagin-3 cDNAs were able to complete the processing of proAPI (Fig.
4A). By contrast, the parental
aut2 mutant cells were unable to perform the processing of
this marker that signals the integrity of the autophagic process. When
the same experiments were performed with autophagin-2 and -4 cDNAs,
no obvious processing of proAPI was observed, indicating that these
human enzymes do not behave as autophagin-1 and -3 in their ability to
restore the autophagy deficiency in aut2 mutant yeasts (data
not shown). To rule out the possibility that the expression of
autophagin-1 or -3 in aut2 cells could complement their
autophagy deficiency through a nonspecific effect, aut7
mutant cells that lack the Apg4/Aut2 substrate and are also deficient
in autophagy were transformed with autophagin-1 or -3 cDNA and
analyzed as above. As shown in Fig. 4A, these transformed yeast cells were unable to complete the proAPI processing, confirming that these human autophagins specifically complement the autophagy deficiency derived from absence of the yeast protease.
View larger version (77K):
[in a new window]
Fig. 4.
Complementation studies with human
autophagins in autophagy-defective yeast strains.
A, Western blot analysis of aminopeptidase I (API) in
Aut2 and Aut7 cells transformed with
pGAD-Autophagin1 or pGAD-Autophagin3 or left nontransformed. Indicated
cells were lysed as described under "Experimental Procedures," and
API processing was analyzed using a rabbit serum anti-API. proAPI and
mature aminopeptidase I (mAPI) are indicated by
arrowheads. Wild-type (WT) cells were included as
a positive control. B, Aut4 yeast mutants were
transformed with pGAD-Autophagin1 (aut2/Autophagin-1) or
pGAD-Autophagin3 (aut2/Autophagin-3) or left nontransformed
(aut2). Micrographs of cells were taken after 4 h of
starvation in the presence of 1 mM PMSF to examine the
accumulation of autophagic bodies. WT cells were included as a positive
control.
Arg. This
sequence is conserved in the equivalent region of two human proteins
MAP-LC3 (microtubule-associated protein light chain 3) and GATE-16
(Golgi-associated ATPase enhancer of 16 kDa) proposed to be functional
homologues of yeast Apg8/Aut7 (22, 24-26). The sequences for MAP-LC3
and GATE-16 are Thr-Phe-Gly
Met and
Thr-Phe-Gly
Phe, respectively. The sequence of GABARAP
(
-aminobutyric acid receptor-associated protein), another Apg8/Aut7
homologue present in mammalian tissues, is only partially conserved in
the putative cleavage region (Val-Tyr-Gly
Leu), and it
was not considered for the purpose of synthesis of a consensus peptide
substrate for Apg4/Aut2-related proteases. On the other hand and
despite the fact that residue located after the cleaved Gly residue
does not appear to have influence in the proteolytic cleavage of the
susceptible bond (2), we introduced a Met residue at that position of
the synthetic peptide since this is the residue present in human
MAP-LC3 (22). In addition, a Apg8/Aut7 mutant with a Met at that
position instead of the naturally occurring Arg residue is perfectly
cleaved by Apg4/Aut2 (2).
View larger version (12K):
[in a new window]
Fig. 5.
Production of recombinant autophagin-3 in
mammalian cells and enzymatic analysis. A, 50 µl of
conditioned medium from 293EBNA cells transfected with
pCEP-Autophagin3-His (lane 1) and with pCEP-Pu (lane
2) were analyzed by Western blot using the penta-His monoclonal
antibody (Qiagen). The sizes of the molecular mass markers are shown on
the left. B, fluorogenic peptide
Mca-Thr-Phe-Gly-Met-Dpa-NH2 (5 µM) was
incubated with 10 ng of purified autophagin-3 in the absence ( ) or
presence (
) of 1 mM NEM in 50 mM Tris/HCl,
pH 7.5, 125 mM NaCl, 1 mM dithiothreitol, 10 mM EDTA, and 2 mM AEBSF. The fluorometric
measurements were made at
ex = 328 nm and at
em = 393 nm. Activity is expressed as nanomoles of
cleaved substrate per microgram of autophagin-3. C, rate
assays were performed using Mca-Thr-Phe-Gly-Met-Dpa-NH2 as
substrate and 50 mM bisTris, sodium acetate, or glycine as
buffer (see "Experimental Procedures").
1
s
1. To further characterize the enzymatic activity of
this protease, the pH profile was determined using the fluorogenic
substrate Mca-Thr-Phe-Gly-Met-Dpa-NH2. As shown in Fig.
5C, recombinant autophagin-3 exhibited a pH optimum of 7.5, which is identical to that used for analysis of proteolytic activity of
yeast Apg4/Aut2 (2). Studies aimed at producing the remaining human
autophagin family members in this expression system are currently in
progress. The future availability of these recombinant proteins will
allow us to evaluate the similarities or differences in the pattern of
substrates targeted by these enzymes.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Drs. G. Velasco, M. Balbín, A. M. Pendás, L. M. Sánchez, and J. M. P. Freije for helpful comments; Drs. M. Thumm, E. Kohfeldt, and M. J. Mazón for providing reagents; and M. Fernández for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported by grants from Comisión Interministerial de Ciencia y Tecnología-Spain (SAF00-0217) and Gobierno del Principado de Asturias-Spain and European Union (QLG1-CT-2000-01131). The Instituto Universitario de Oncología is supported by Obra Social Cajastur-Asturias.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AJ312234, AJ312332, AJ504651, AJ504652, AJ312233, AJ312333, AJ504653, and AJ504654.
§ Recipients of fellowships from Ministerio de Ciencia y Tecnología, Madrid, Spain.
¶ Both authors contributed equally to this work.
** To whom correspondence should be addressed: Dept. de Bioquímica y Biología Molecular Facultad de Medicina, Universidad de Oviedo 33006-Oviedo, Spain. Tel.: 34-985-104201; Fax: 34-985-103564; E-mail: CLO@correo.uniovi.es.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M208247200
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ABBREVIATIONS |
---|
The abbreviations used are:
E1, ubiquitin-activating enzyme;
E2, ubiquitin carrier protein;
kb, kilobase;
AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride
hydrochloride;
apg/aut and APG/AUT, yeast
autophagy mutant and wild-type genes;
Apg, expression products of APG
genes;
Dpa, N-3-(2,4-dinitrophenyl)-L-,
-diaminopropionyl];
GABARAP,
-aminobutyric acid receptor-associated protein;
GATE-16, Golgi-associated ATPase enhancer of 16 kDa;
MAP-LC3, microtubule-associated protein light chain 3;
Mca, 7-(methoxycoumarin-4-yl)acetyl;
NEM, N-ethylmaleimide;
proAPI, proaminopeptidase I;
PMSF, phenylmethylsulfonyl fluoride;
USP, ubiquitin-specific processing proteases.
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