1
July 26, 2024
Supplementary
Information
(SI)
for
Defining and naming N
-degron pathways
Alexander Varshavsky
Division of Biology and Biological Engineering, California Institute of Technology,
Pasadena, CA 91125
Email: avarsh@caltech.edu
This PDF includes:
Fig
ures
S1
and S2, and their legends
.
References for SI citations
.
2
Fig. S1
.
The fMet/N
-degron pathway
and the Leu/N
-degron pathway. For designations,
see the legend to the main Fig. 1. (
A
) The eukaryotic (
S. cerevisiae
) fMet/N
-degron
pathway
(1). 10
-fTHF
, 10
-formyltetrahydrofolate
. (
B
) The b
acterial (
Vibrio vulnificus
)
Leu
/N-degron pathway
(2)
. M
ost Gram
-negative
bacteria contain both Aat and Bpt
; some
bacteria, including
E. coli
, lack Bpt (3
-5).
3
Fig. S
2.
Calpain
-generated C
-terminal (Ct) fragments of mammalian proteins that are
either identified or predic
ted substrates of the Arg/N
-degron pathway.
E ntries whose
numbers are colored in green are the experimentally identified subs
trates of the
4
Arg/N
-degron pathway
(6)
. E
ntries whose numbers are color
ed in black are predicted
(putative)
Arg/N
-degron
substrates. Each entry cites a calpain
-generated Ct
-fragment of a
protein and the fragment’s Nt
-residue (in red, using three
-letter abbreviations for amino
acids), followed by a brief description of a
full
-length (
uncleaved
) precursor protein. A
n
experimentally determined
calpain cleavage site,
indicated
by an arrowhead, is shown
using single
-letter abbreviations for amino acids. An enlarged P1’ residue (in red) becomes
N-terminal upon the cleavage. The indicated residue numbers are the number of the first
shown residue of a full
-length protein and the number of its last residue, respectively. All
entries are mouse proteins
(denoted with the first letter uppercase)
, save for
#14 and #27,
which are human proteins
(denoted with all letters uppercase)
.
#1.
Glu
-Bak is the proapoptotic Ct
-fragment of t
he apoptotic regulator BAK.
Glu
-BAK is generated by calpain
-1 in vitro and is apparently formed in vivo as well (7).
#2.
Arg
-Bid. Bid is a 22 kDa member of the BCL
-2 family of apoptosis regulators (8
-10)
.
Although full
-length Bid is a
lready a proa
poptotic protein, its Ct
-fragments, which can be
naturally produced by activated caspases, calpains or granzyme B, can be even more active
than inta
ct Bid as proapoptotic protein fragments
(11)
. The cleavage of Bid by calpains
produces the 14 kDa
Arg
71
-Bid fragment (11
-14)
that has been shown to be a short
-lived
substrate of the Arg/N
-degron pathway (15). #3.
Asp
-Bcl
XL
. Bcl
XL
is a 26 kDa
antiapoptotic regulatory protein (8, 16)
. Under conditions that include glucose and oxygen
deprivation, Bcl
XL
can be cleaved by activated calpain
-1, resulting in the 21 kDa Asp
61
-
Bcl
XL
Ct
-fragment. In contrast to full
-length Bcl
XL
,
Asp
61
-Bcl
XL
has proapoptotic activity
(17)
, and has been shown to be a short
-lived substrate of the
Arg/N
-degron
pathway (15)
.
#4.
Arg
-c-Fos is the Ct
-fragment of the c
-Fos transcriptional regulator. c
-Fos is targeted for
conditional degradation through more than one degron, including the path that includes the
cleavage by calpains (18)
. #5.
Glu
-
IκBα is the Ct
-
fragment of the IκBα subunit of the
NFκB
-
IκBα complex in which the NFκB transcriptional regulator is inhibited by IκBα. The
IκBα subunit is targeted for degradation either through a conditional phosphodegron or
through a specific calpain
-mediated cleavage (19)
. #6.
Arg
-Igf
bp2 is the calpain-
generated
Ct-fragment of the insulin-
like growth factor binding protein-
2 (20)
. #7.
Asp
-Capns1 is the
Ct-fragment of the calpain regulatory subunit that is cleaved by activated calpains (21, 22)
.
#8.
Arg
-Atp2b2 is the Ct
-fragment of the transmembrane Atp2b2 plasma membrane Ca
2+
pump (PMCA) that ejects Ca
2+
from cells. This pump is activated either by the binding of
Ca
2+
/calmodulin or by the calpain-
mediated truncation of Atp2b2 that generates the
Arg
-
Atp2b2 fragment and thereby activates the pump (23)
. #9.
Leu
-Capn1 is the auto-
gen
erated, catalytically active Ct
-fragment of calpain
-1 (24, 25)
. #10.
Arg
-Ankrd2. Ankrd2
(Marp2, Arpp), a member of the MARP (muscle ankyrin repeat protein) family, functions
as a negative regulator of muscle differentiation (26). Calpains can produce the 30 kDa
Arg
103
-Ankrd2 Ct
-fragment (27)
. #11.
Tyr
-Grm1. Grm1 is the Ct
-fragment of the
mGluR1α transmembrane metabotropic glutamate receptor
(28)
. Receptors containing the
calpain
-
truncated mGluR1α
Ct
-fragment
can
elevate cytosolic Ca
2+
but cannot
activate
PI
3
K-Akt signaling pathways, in contrast to uncleaved receptors
(28, 29)
. #12.
Lys
-Ica512.
Ica512 (Ptprn) is a member of the transmembrane receptor protein phosphatase family
(30)
. The 43 kDa calpain-
generated mouse
Lys
609
-Ica512 Ct
-fragment enters the nucleus
and acts as a transcriptional regulator (30, 31)
. #13.
Phe
-GluN2a. GluN2a (NMDA-
R2a) is
a subunit of the N
-methyl-
D-aspartate receptor (NMDAR), a glutamate receptor that can
function as a ligand-
gated Ca
2+
channel (32, 33)
. The GluN2b subunit of NMDAR can also
be cleaved by calpains (34). Ct
-fragments of NR2A and NR2B contain domains required
for the association of these subunits with other synaptic proteins. NMDAR receptors
5
lacking a Ct
-region of GluN2a could function as glutamate
-gated Ca
2+
channels but the
intracellular traffic of cleaved receptors and their electrophysiological properties were
altered (35). #14.
Asn
-DSCR1 (Rca
n1) is the calpain
-generated Ct
-fragment of the Down
syndrome critical region 1 protein Dscr1, which binds to Raf1, inhibits the phosphatase
activity of calcineurin, and enhances its degradation. The
Asn
-DSCR1 fragment does not
bind to the Raf1 kinase (36)
. #15.
Arg
-Glyt1a is the Ct-
fragment of the transmembrane
Glyt1a glycine transporter (37)
. Another Gly transporter, Glyt1b, is also cleaved by
calpains, yielding the
Arg
-Glyt1b fragment (37). These Ct fragments are still active as
transporters but are impaired in their ability to remove Gly (an inhibitory neurotransmitter)
from synaptic clefts (37)
. #16.
Asn
-Ca
v
1.1 is the Ct fragment of the voltage
-gated
transmembrane Ca
2+
channel. This (apparently) calpain-
generated fragment is
noncovalently associated with the rest of the channel and can inhibit its activity. Upon
dissociation from the channel, the
Asn
-Ca
v
1.1 fragment enters the nucleus and functions as
a transcriptional regulator (38
-41)
. #17.
Lys
-cortactin is the Ct-
fragment of cortactin, an
actin
-binding protein that regulates actin polymerization (42)
. #18.
Asn
-Bfl
-1. Bfl
-1 is
antiapoptotic regulatory protein whose cleavage by calpain-
1 generates the
Asn
72-
Bfl
-1
proapoptotic Ct
-fragment (43)
. #19.
Arg
-dystrophin is the calpain
-generated Ct
-fragment
of a major cytoskeletal protein in the skeletal muscle (44)
. #20.
Gln
-Ryr1 is the Ct
-
fragment of the Ryr1 ryanodine receptor, a Ca
2+
channel in the ER (45)
that mediates the
efflux of Ca
2+
from the ER into the cytosol. Calpain-
mediated cleavage of Ryr1 increases
Ca
2+
efflux (46)
. #21.
Arg
-Mef2d is the Ct
-fragment of the Mef2d myocyte enhancer factor
2d, a transcriptional regulator that contributes to neuronal survival, development, and
synaptic plasticity (47)
. #22.
Gln
-talin is the calpain
-generated Ct
-fragment of talin, an
adaptor protein that interacts with the integrin family of cell adhesion transmembrane
proteins (23, 48, 49)
. #23.
Arg
-p39 is the calpain-
generated Ct
-fragment of the p39
activator of the Cdk5 protein kinase (50)
. The indicated cleavage site is located
immediately downstream of two
other closely spaced (and strongly conserved) calpain
cleavage sites in p39. A cleavage at any one of these sit
es yields a predicted Arg/N
-degron
substrate. #24.
Gln
-Egfr
is one of calpain
-generated Ct
-fragments of the transmembrane
epidermal growth factor (EGF) receptor protein kinase (51)
. Remarkably, all 7 calpain
cleavage sites in the cytosol
-exposed domain of the 170-
kDa EGFR contain P1’ residues
that ar
e recognized as destabilizing by the Arg/N
-degron pathway (51)
. #25.
Leu
-
β
-catenin
is the cal
pain
-generated Ct
-
fragment of β
-catenin, a conditionally short
-lived cytoskeletal
protein and transcriptional regulator. The
Leu
-
β
-catenin fragment is a nuclear protein that
activates specific genes in conjunction with other transcription factors (52)
. #26.
Leu
-NF2
is the calpain
-generated Ct
-fragment of NF2 (merlin), a tumor suppressor and cytoskeletal
protein. Loss of function NF2 mutants result in autosomal
-dominant neurofibromatosis, a
predisposition to specific kinds of brain tumors
(53). #27.
Arg
-caspase-
9 is
the Ct
-fragment
of caspase-
9, which can be inactivated by calpains (54)
, followed by the (predicted)
degradation of the Arg-
caspase-
9 Ct
-fragment by the Arg/N
-degron pathway. #28.
Leu
-troponin T2 is the Ct
-fragment of the cardiac troponin T that is produced by calpain-
1
in the troponin-
containing cardiac myofibril complex (55)
. #29.
Lys
-Pk
Cα is the cal
pain
-
generated Ct
-fragment of Pk
Cα, a broadly expressed Ser/Thr kinase of the PKC family
(56)
. Being catalytically active but no longer controlled by the regulatory Nt
-domain of the
full
-
length PkCα, the Lys
-Pk
Cα fragment can be toxic, for ex
ample, upon its formation in
an ischemic heart (57)
. #30.
Leu
-Ra
d21 is the calpain-
generated Ct
-fragment of the
Scc1/Rad21 subunit of the chromosome
-associated cohesin complex (58)
. The calpain
-
mediated generation of
Leu
-Rad21 contributes to the control of chromosome
6
cohesion/segregation, together with proc
esses that include the separase
-mediated cleavage
of the same Rad21 subunit
of cohesin (58
-61)
. #31.
Phe
-Pk
Cγ is the cal
pain
-generated Ct
-
fragment of Pk
Cγ, a Ser/Thr kinase of the PKC family
(56)
. The
Phe
-Pk
Cγ fragment is
constitutively active as a kinase, bec
ause it lacks the regulatory Nt
-domain of the
full
-length Pk
Cγ kinase
(56)
. #32.
Leu
-STEP
33
is the Ct
-fragment of the striatal-
enriched
STEP
61
phosphatase, a brain-
specific Tyr
-phosphatase whose substrates inc
lude the
MAPK-
family kinases Erk
1/2 and p38. The calpain-
generated
Leu
-STEP
33
fragment lacks
phosphatase activity (62)
. #33.
Leu
-Camk
-IV is the calpain
-generated Ct
-fragment of the
Ca
2+
/calmodulin
-dependent kinase
-IV. This fragment lacks kinase activity (63)
. #34.
Leu
-vimen
tin is the calpain
-generated Ct
-fragment of vimentin, a component of
intermediate filaments (64).
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