ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 9, pp. 1535-1545 © The Author(s) 2024. This article is an open access publication.
1535
REVIEW
Universal Adapter Protein Bag3
and Small Heat Shock Proteins
Maria A. Zamotina
1
, Lidia K. Muranova
1
, Artur I. Zabolotskii
1
,
Pyotr A. Tyurin-Kuzmin
2
, Konstantin Yu. Kulebyakin
2
, and Nikolai B. Gusev
1,2,a
*
1
Department of Biochemistry, Faculty of Biology, Lomonosov Moscow State University,
119991 Moscow, Russia
2
Department of Biochemistry and Regenerative Biomedicine, Faculty of Fundamental Medicine,
Lomonosov Moscow State University, 119991 Moscow, Russia
a
e-mail: nbgusev@mail.ru
Received April 26, 2024
Revised May 20, 2024
Accepted May 24, 2024
Abstract— Bag3 (Bcl-2-associated athanogene3) protein contains a number of functional domains and interacts
with a wide range of different partner proteins, including small heat shock proteins (sHsps) and heat shock
protein Hsp70. The ternary Bag3–sHsp–and Hsp70 complex binds denatured proteins and transports them to
phagosomes, thus playing a key role in the chaperone-assisted selective autophagy (CASA). This complex also
participates in the control of formation and disassembly of stress granules (granulostasis) and cytoskeleton reg-
ulation. As Bag3 and sHsps participate in multiple cellular processes, mutations in these proteins are often asso-
ciated with neurodegenerative diseases and cardiomyopathy. The review discusses the role of sHsps in different
processes regulated by Bag3.
DOI: 10.1134/S0006297924090013
Keywords: Bag3, Hsp70, small heat shock proteins, selective autophagy, proteostasis
Abbreviations: CASA, chaperone-assisted selective autopha-
gy; Hsps, heat shock proteins; sHsp, small heat shock pro-
tein; TSC, tuberous sclerosis complex.
* To whom correspondence should be addressed.
INTRODUCTION
At present, the interests of many scientists are fo-
cused on adapter proteins that play a role of “hubs”
coordinating interaction and functioning of many pro-
teins. After binding with the adapter protein, the part-
ner proteins can interact with their targets, thus form-
ing protein ensembles, multiple components of which
regulate each other’s activity and form a pipeline pro-
ducing certain metabolites or providing efficient trans-
mission of various signals. Bag3 is one of these adapt-
er proteins. Bag family proteins are involved in the
regulation of various intracellular pathways, including
apoptosis, regulation of protein synthesis, quality con-
trol of protein folding, hormonal signal transduction,
and many other vital processes.
DOMAIN STRUCTURE
AND SOME PROPERTIES OF Bag3
Human genome contains six genes coding for Bag
family proteins [1]. The name Bag is an abbreviation
of Bcl-2-associated athanogene, because Bag proteins
had been originally considered as partners of the im-
portant antiapoptotic protein Bcl-2 [1, 2]. All Bag fam-
ily members contain one or several conserved Bag
domain(s) consisting of 80-90 residues that provide
interaction of these molecules with the Hsp70/Hsc70
heat shock proteins (Hsps). Bag proteins differ in their
size and can include various functional domains that
determine their activity mechanisms and processes
they regulate [2]. In this review, we limited ourselves
to discussing only one member of the Bag family, Bag3,
and only briefly mentioned another member of this
family, Bag1.
Human Bag3 contains 575 residues and is located
predominantly in the cytosol [1], although a fraction
of Bag3 molecules co-localizes with the contractile
ZAMOTINA et al.1536
BIOCHEMISTRY (Moscow) Vol. 89 No. 9 2024
Fig. 1. In silico model of the 3D structure (PMDB ID: PM0083680)(a) and functional domains(b) of Bag3. a) Dark blue, WWdo-
main; light blue, IPV domain; lemon yellow, short M domain; brown, long PXXP domain; red, Bag domain; green, parts ofmol-
ecule not included in the functional domains. The main functional domains of Bag3 together with amino acids numbers are
presented on the panel(b). b)The WW domain interacts with proteins containing poly-Pro sites. This domain interacts with
synaptopodin (SYNPO2) and LATS1/2 protein kinases that regulate intracellular location of the transcription co-activators YAP
and TAZ. The IPV domain contains two sites of small heat shock protein (sHsp) interaction and two sites of phosphorylation.
After phosphorylation, Bag3 acquires the ability to interact with 14-3-3 protein and dynein (via 14-3-3). The M domain provides
interaction with protein kinases p38 and JNK. The PXXP domain interacts with SH2 domain-containing proteins (Src and Yes).
The Bag domain interacts with the Bcl family antiapoptotic proteins and Hsps of the Hsp70/Hsc70 family; it activates the ADP/
ATP exchange in the nucleotide-binding site of Hsc70/Hsp70. Ubiquitin ligase STUB1 binds to Hsp70 and ubiquitinates protein
substrates bound to this proteins complex. Bag3 also interacts with the autophagy receptor p62/SQSTM, which binds both
ubiquitinated target proteins and LC3 protein located on the phagosome membrane.
apparatus in the region of Z-discs in skeletal and car-
diac muscles [3]. The data on the 3D structure of the
full-size Bag3 are lacking, although, the structure of
conserved Bag domain (homologous for all Bag family
members) of Bag1 in a complex with Hsc70 has been
resolved by X-ray crystallography [4]. The 3D struc-
ture of the full-size Bag3 (PMDB ID: PM0083680) was
modeled insilico [5] (Fig.1a). According to this model,
the most part of the Bag3 molecule is predominant-
ly unfolded with a limited number of β-strands. Only
the Bag domain has an ordered structure and includes
three α-helices; there is also an additional α-helices at
the C-terminus of Bag3 [5]. The theoretical model [5]
and the data obtained by X-ray crystallography for
the isolated Bag domain in a complex with Hsc70 [4]
correlate well. Both theoretical and experimental data
indicate that Bag3 has a predominantly unfolded struc-
ture and, therefore, belongs to the so-called intrinsi-
cally disordered proteins, which makes difficult its
crystallization and analysis by X-ray crystallography,
as well as by other methods.
Bag3 contains several functional domains, each re-
sponsible for the interaction with specific protein tar-
gets and capable of affecting the functioning of neigh-
boring domains (Fig. 1b). The WW domain is located
at the N-terminus of Bag3 (the name comes from Trp
residues separated by 20-22 amino acids). This domain
interacts with the poly-Pro motifs (PPPY, PPSY) found
in certain proteins, such as synaptopodin (SYNPO2),
protein kinases LATS1/2, and AMOTL1/2 adaptor pro-
teins [6, 7]. Synaptopodin interacts with the autophagy
proteins VPS16, VPS18, and ATG16 [7, 8]. Through syn-
aptopodin, Bag3 can interact with proteins involved in
the phagophore formation, which might be the reason
why downregulation of synaptopodin synthesis inhib-
its chaperone-assisted selective autophagy (CASA) and
suppresses elimination of phosphorylated tau protein
in mature neurons [9]. Protein kinases LATS1/2, which
also contain poly-Pro motifs in their structure, phos-
phorylate YAP and TAZ transcription co-activators
[10, 11]. Under normal conditions the WW domain of
Bag3 binds the AMOTL1/2 adapter proteins, LATS1/2
proteins kinases, and YAP/TAZ transcription co-acti-
vators forming a tight complex that makes possible
phosphorylation of YAP/TAZ by LATS1/2. Phosphorylat-
ed YAP/TAZ proteins interact with the adapter protein
14-3-3 and either undergo proteolytic degradation or
remain in cytosol [12]. In any case, they fail to reach
the nucleus and to activate TEAD1-4 transcription fac-
tors. Proteotoxic stress leading to the accumulation
of ubiquitinated proteins results in the LATS–YAP/
TAZ–AMOTL complex dissociation, which prevents YAP/
TAZ phosphorylation. YAP/TAZ activators move to the
nucleus, activate TEAD1-4, and initiate transcription
of certain genes [12, 13]. This regulatory mechanism
consisting of activation/inactivation of LATS protein
kinases followed by phosphorylation of transcription
activators is similar to that occurring during activa-
tion of the HYPPO regulatory complex involved in cell
differentiation, hormonal signal transduction, and cell–
cell interactions [11, 12, 14]. The signaling through the
HYPPO pathway can be regulated by mechanical stress
induced by the contractile activity or cell translocation
in viscous media. Hence, it was suggested that Bag3
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BIOCHEMISTRY (Moscow) Vol. 89 No. 9 2024
acts as a mechanosensor that transduces mechanical
signal using the above-described mechanism based on
phosphorylation and translocation of YAP/TAZ in the
cell [15].
The WW domain also interacts with tuberous
sclerosis complex (TSC) through the PY motifs in TSC1
protein. In muscles, Bag3 localizes to the contractile
apparatus, where it binds TSC1 which activates GTPase
of the small G protein RHEB. RHEB with GDP in its
active site inhibits mTORC1 and, therefore, suppresses
protein synthesis in the vicinity of contractile appara-
tus. Simultaneously, this leads to the activation of CASA
and promotes selective proteolysis of certain proteins,
for instance, filamin. At the same time, TSC1 binding to
the contractile apparatus reduces its concentration in
the cytosol, thus leading to the activation of mTORC1
in the cytosol and activation of protein synthesis [16].
The WW domain is followed by the IPV domain
(Fig. 1b). According to computer predictions, this do-
main has an unordered structure and contains only
short β-strands [5]. This domain was named so because
it includes two short sequences (residues 87-101 and
200-213) containing the IPIPV and IPVI motifs, respec-
tively. These sequences are similar to the correspond-
ing sequences in the C-terminal domains of some sHsps
(HspB1, HspB4, and HspB5). The (I/V)P(I/V) motifs of
sHsps interact with the hydrophobic groove formed
by the β4 and β8 strands in the α-crystallin domain
of neighboring subunits and, hence, play an import-
ant role in the formation of large oligomers of sHsps
[17-19]. It was suggested that the IPV motifs of Bag3
can interact with the same hydrophobic β4-β8 groove
in sHsps and form a tight complex with these proteins.
We will discuss details of this interaction below.
The sequence between the two IPV motifs con-
tains two Ser residues (Ser136 and Ser173) which,
when phosphorylated, ensure Bag3 binding with 14-3-3
[20,21]. This protein provides interaction of Bag3 with
dynein. Therefore, Bag3 can bind dynein through the
14-3-3 protein and its own PXXP domain [20]. Such in-
teraction with dynein at two different sites provides
for the microtubule-associated transport of Bag3 and
all bound proteins to aggresomes, which are intracel-
lular centers of accumulation of aggregation-prone de-
natured proteins. Therefore, the IPV domain contains
important elements of Bag3 structure and plays im-
portant role in the functioning of this protein.
The M and PXXP domains are located in the center
of Bag3 molecule [6] (Fig. 1b). These domains contain
the binding sites for different protein kinases. It is be-
lieved that the M domain binds p38 and ERK protein
kinases, whereas the PXXP domain binds Yes and Src
tyrosine kinases. Bag3 controls interaction of ERK with
Dusp6 protein phosphatase; deletion of Bag3 results
in the dissociation of Dusp6 leading to the constitu-
tive ERK activation [22]. Non-receptor tyrosine kinas-
es interact with Bag3 through their SH3 domains [23].
Thebinding of tyrosine kinases with the PXXP domain
is dependent on Hsp70, which binds to the conserved
neighboring Bag domain [23]. In the case of massive
proteotoxic stress, i.e., upon accumulation of large
quantities of denatured proteins, Hsp70 interacts with
these denatured proteins and dissociates from Bag3,
which affects interaction of non-receptor protein ki-
nases with Bag3 and influences their intracellular lo-
cation and activity.
By interacting with various protein kinases, Bag3
can regulate translocation of transcription activators
from the cytosol and to the nucleus. As mention above,
proteotoxic stress and, presumably, mechanical stress
can lead to the dissociation of protein kinases LATS1/2
from the YAP/TAZ transcription activators bound to the
WW domain of Bag3. As a result, the kinases cannot
phosphorylate YAP at Ser127, which prevents the in-
teraction between YAP/TAZ and 14-3-3, so that YAP and
TAZ are no more retained in the cytosol and move to
the nucleus [6]. Simultaneously, tyrosine kinases bound
to the PXXP domain phosphorylate YAP at Tyr357, thus
inhibiting its nuclear export and promoting its tran-
scriptional activity [24].
To summarize the above, the M and PXXP do-
mains of Bag3 interact with different protein kinases
and affect their activity, making Bag3 an important
participant in the transmission of hormonal signals.
It should also be mentioned that the above-described
HYPPO pathway is very complex and involves multiple
protein kinases. For instance, the activity of LATS1/2
is regulated by proteins kinase MST1 and Salvado 1
complex (SAV1). The activity of MST1 depends on its
phosphorylation by MAP kinases (MAP4K) which are
under the hormonal control [10]. Therefore, Bag3 can
indirectly participate in the transduction of hormonal
signals by regulating the activity of protein kinases.
The conserved Bag domain contains three ex-
tended α-helices. It is located at the C-terminus of
Bag3 (Fig. 1a) and is found in all Bag family mem-
bers. Itprovides for the interaction with a number of
protein partners. One of the most important partners
is the heat shock proteins belonging to Hsp70/Hsc70
family. The Bag domain of Bag1 was co-crystallized
with Hsc70, which allowed to demonstrate how this
domain catalyzes exchange of adenine nucleotides in
the active site of Hsc70 [4]. Bag1 and Bag3 can com-
pete for Hsp70 binding [20, 25, 26], causing the switch
between the pathways involved in the elimination of
denatured proteins. As a rule, the complex formed
by Bag1 and Hsp70 directs denatured proteins to the
degradation in proteasomes, whereas the complex of
Bag3 and Hsp70 usually provides degradation of de-
natured proteins in autophagosomes. Typically, indi-
vidual proteins or small protein complexes are degrad-
ed in proteasomes, whereas large protein aggregates
ZAMOTINA et al.1538
BIOCHEMISTRY (Moscow) Vol. 89 No. 9 2024
Fig. 2. Chaperone assisted selective autophagy (CASA) com-
plex. Protein substrate (light blue cylinder) binds to HspB8
(two red circles) and the substrate-binding site of Hsp70 (dark
blue). Hsp70 binds ubiquitin ligase STUB1 (light green oval)
that transfer ubiquitin (small orange circles) onto the protein
substrate. Bag3 (green crescent) interacts with the phagosome
receptor p62 (yellow crescent) and tethers the protein com-
plex to LC3 (brown cylinder) located on the phagophore mem-
brane. Bag3 through its WW domain interacts with SYNPO2
(light blue oval), thus providing additional interaction with
the ATG16 and VPS18/16 proteins of the autophagosomal
complex and the Z-disk of myofibrils.
and organelles are degraded in autophagosomes [27].
The proteasomal and autophagosomal systems inter-
act with each other in a quite complicated manner, as
each of these systems can degrade the key components
of the other one [27]. However, as a rule, inhibition
of any of these systems results in the compensatory
increase in the other system’s activity. Aging or prote-
asome overload lead to the upregulation of Bag3 syn-
thesis and promote selective degradation of denatured
proteins in autophagosomes [20, 25]. The exchange of
adenine nucleotides in the active site is necessary for
proper Hsp70 functioning. Therefore, by acting as a
nucleotide exchange factor, Bag3 plays a critical role
in the regulation of Hsp70 activity. Because Bag3 forms
tight complexes with Hsp70, a significant increase in
its content can result in the inhibition (instead of acti-
vation) of the chaperone activity of Hsp70. This can be
explained by the fact that chronic stress upregulates
the synthesis of Bag3, which forms tight complexes
with Hsp70, making its normal functioning impossible
and decreasing its chaperone activity [28, 29]. Since
the Bag domain plays a crucial role in the activity of
Bag3, mutations in this domain are often associated
with various inherited diseases, including different
types of cardiomyopathies [13, 30].
Bag3 acts as a scaffold in the formation of com-
plexes including sHsps bound to the IPV motifs and
Hsp70 bound to the Bag domain, in which sHsps in-
teract with denatured protein substrates. Hsp70 bound
to Bag3 interacts with the E3 ubiquitin ligase STUB1
through tetratricopeptides in STUB1 [13], resulting in
the formation of a complex consisting of Bag3, sHsps,
Hsp70, STUB1, and denatured proteins that are ubiq-
uitinated by STUB1. This multicomponent complex
interacts with dynein and is transported to the mi-
crotubule organizing center (MTOC) close to nucle-
ar envelope. The phagophore receptor protein p62/
SQSTM1 (sequestosome 1) is added to this complex.
This protein interacts with LC3 and participates in the
phagophore formation [7, 31-33]. The fusion with the
lysosome finishes the process of CASA (Fig.2).
The Bag domain is not involved only in the bind-
ing of Hsp70, but also forms the contacts with the anti-
apoptotic proteins of the Bcl family (Bcl1, Bcl-x, Mcl-1)
[34]. It is believed that Bag3 stabilizes Bcl family pro-
teins and ensures their high antiapoptotic effect [35].
To summarize, Bag3 is the multidomain protein
interacting with many different protein partners and
participating in various cellular processes. Below, we
will analyze the structure and properties of the IPV
domain and discuss the role of this domain in the in-
teraction with sHsps and other processes controlled
byBag3.
SMALL HEAT SHOCK PROTEINS AND Bag3
Human genome contains ten genes coding for
sHsps [36-38]. sHsp monomers have a conserved struc-
ture and contain the α-crystallin domain (80-100 amino
acids) flanked by the N- and C-terminal domains vary-
ing in size and structure. Some sHsps (HspB6, HspB7,
and HspB8) exist as monomers or form small oligo-
mers (dimers or tetramers) [39-41], whereas other
sHsps form large oligomers of more than 20 subunits
[42, 43]. sHsps prone to the formation of large oligo-
mers contain in their C-terminal domains the so-called
IPV motifs with the (I/V)P(I/V) sequence [18]. Resi-
dues of the IPV motif interact with the β4-β8 hydro-
phobic groove of the neighboring monomers, which
stabilizes large sHsp oligomers [17, 19]. As already
mentioned, Bag3 contains two IPV motifs (IPIPV and
IPVI) in the fragments 87-101 and 200-213, respec-
tively. It was reasonable to suggest that these motifs
would also interact with the corresponding groove
in the α-crystallin domain of sHsps. This hypothesis
was checked experimentally. Using mutant forms of
HspB6 and HspB8, Fuchs et al. [44] found that dele-
tion of either IPV motifs had no effect on the Bag3
interaction with sHsps, whereas deletion of both mo-
tifs completely prevented the binding of HspB6 and
HspB8 to Bag3. Moreover, mutations in the hydropho-
bic β4-β8 affected the interaction of sHsps with Bag3.
Inthis first publication, the binding between Bag3 and
sHsps was demonstrated only for HspB6 and HspB8.
Later studies have thoroughly investigated the binding
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ofother sHsps with Bag3. Thus, the interaction of Bag3
with HspB1, HspB5, HspB6, and HspB8 was studied
by the isothermal titration calorimetry method [45].
The highest affinity (K
d
~ 1.2 µM) was found for the
Bag3 binding with HspB6 and HspB8, whereas the af-
finity of HspB1 was lower (K
d
~ 5-9 µM). Surprisingly,
the binding stoichiometry (2 moles of sHsp per mole
of Bag3) was the same for sHsps forming small (HspB6,
HspB8) and large (HspB1, HspB5) oligomers [45]. This
can be possible only if Bag3 possesses a very high af-
finity for sHsp monomers and, therefore, is capable to
induce dissociation of large oligomers resulting in the
extraction of monomers (or dimers) from oligomeric
complexes. Later, this effect was described by Rauch
etal. [45] who analyzed the interactions between Bag3
and HspB1. Similar effect was observed in the exper-
iments on the Bag3 binding with HspB8 [46]. At high
concentrations, HspB8 tended to form large complexes,
in which monomers interacted weakly with each other.
Affinity of Bag3 toward HspB8 monomers was higher
than the affinity between HspB8 monomers. Therefore,
Bag3 was able to extract HspB8 monomers from their
large complexes. It should be mentioned that the K
d
of the HspB8–Bag3 complex determined by the surface
plasmon resonance varied between 2.4 and 4.6nM and
was significantly lower than that determined by iso-
thermal titration calorimetry.
Several mutations of Pro209 located in the sec-
ond IPV motif of Bag3 have been described. Thus, the
P209L mutation is associated with myofibrillar myop-
athy, sensorimotor axonal neuropathy, axonal neurop-
athy, Charcot–Marie–Tooth disease of the second type,
and cardiomyopathy. The P209Q mutation correlates
with myofibrillar myopathy and sensorimotor axonal
neuropathy. Finally, the P209S mutation was found
in patients with the Charcot–Marie–Tooth disease of
the second type [30, 47]. The P209L mutation results
in approximately three-fold increase (from 0.45 up
to 1.8 µM) in the K
d
value for the Bag3 binding with
the α-crystallin domain of HspB1 and decrease in the
stoichiometry from ~2 to 1 moles of HspB1 α-crystal-
lin domain per mole of Bag3 [48]. Pro209 mutations
(P209/L,S,Q) reduce the binding of Bag3 with sHsps,
but do not affect the first stages of CASA complex
formation. Such defective complex is transported by
dynein to the MTOC. At the final stage of CASA, the
phagophore fuses with the lysosome, thus finishing the
process of autophagy. In the case of Pro209 mutants,
the formed protein complexes interact with histone
deacetylase (HDAC6) and vimentin and form unusual-
ly highly stable aggresomes that contain immobilized
Hsp70 and other proteins involved in CASA. This blocks
selective autophagy and induces various pathological
processes [3, 49]. Thus, mutations in the second IPV
motif affect Bag3 interactions with sHsps, but have no
effect on the initial stages of CASA complex formation
and dynein-dependent transportation of CASA com-
plex to aggresomes. At the same time, mutations in
the second IPV motif block the final stages of autoph-
agy and immobilize CASA components in aggresomes
which are highly resistant to dissociation. Therefore,
the second IPV motif not only provides the interaction
between Bag3 and sHsps, but plays an important role
in thenormal functioning of the entire CASA complex.
Mutations I94F and R90XD in the first IPV motif
have also been reported [30]. However, the data on
the effect of these mutation on Bag3 functioning are
lacking so far. In mouse Bag3, Ile81 is located close to
the first IPV motif. In experiments conducted in mice,
ischemia of limb arteries led to the ischemic myopa-
thy, whose manifestations (e.g., limb necrosis) were
more severe in the animals injected with an adeno-
virus coding for Bag3 with the Ile81Met mutations
than in the mice injected with the adenovirus coding
for the normal protein. The wild-type Bag3 with Ile at
position 81 demonstrated better HspB8 binding than
the Ile81Met mutant. The autophagic flux was more
pronounced in the muscle cells of animals expressing
the wild-type Bag3 vs. the mutant protein [50]. These
data suggest that the IPV motives play an important
role in the functioning Bag3. This agrees with the fact
that point mutations in the IPV domain of human Bag3
(residues 55-213) are associated with dilated cardiomy-
opathy[13, 30].
Which sHsps interact with the IPV domain of
Bag3? According to the initial point of view, the IPV
motives in Bag3 interact only with the hydrophobic
β4-β8 groove of the α-crystallin domain in Hsps [44].
If this hypothesis is correct, then taking into account
a high homology of α-crystallin domains, all sHsps
should interact with Bag3 with a similar affinity. How-
ever, the initial investigations [44] have shown that
HspB8 and HspB6 interact with Bag3 with high affin-
ity, whereas HspB1 had only very low affinity. At the
same time later investigations have shown that the
isolated α-crystallin domain of HspB1 has the highest
affinity [45]. It was suggested that the specificity of in-
teraction is determined by the hydrophobicity of the
β4-β8 groove in the α-crystallin domain of sHsp [44],
but this hypothesis has not received an experimental
verification.
The K141E mutation in HspB8 is associated with
distal motor neuropathy. The mutant protein was found
in autophagosomes; however, these autophagosomes
did not co-localize with the lysosomes. Atthe same time,
the wild type HspB8 was observed in autophagosomes
that co-localized with lysosomes [51]. This can indicate
that mutations in the β6 strand of HspB8, i.e., relative-
ly far from the β4 and β8 strands, can affect the af-
finity of HspB8 and its interaction with Bag3 [52, 53].
Thus, not only the β4 and β8 strands, but other sites
of HspB8 as well can affect its interaction with Bag3.
ZAMOTINA et al.1540
BIOCHEMISTRY (Moscow) Vol. 89 No. 9 2024
Recently, four new HspB8 mutants were described
with the shift in the reading frame starting from the
residues 170, 173, 176, and 194 and leading to a signif-
icant increase in the length of the C-terminal peptide
[54]. These mutants efficiently interacted with Bag3,
but had a lower solubility in the isolated state or in a
complex with Bag3 compared to the wild-type protein.
This led to the suppression of CASA and development
of distal myopathy with rimmed vacuoles (DMRV) [54].
There were attempts to elucidate the role of
HspB8 in autophagy at the cellular level. For this, the
cells were treated with a proteasome inhibitor and
analyzed for the formation and accumulation of ag-
gresomes. It was found that HspB8 affected interaction
of Bag3 with p62/SQSTM1 presumably via regulation
of p62 phosphorylation or oligomerization [55]. In any
case, HspB8 affected the early stages of micro aggre-
gate formation that later enter aggresomes and are
subjected to autophagy. Hence, Bag3 and HspB8 coop-
erate in the processes of autophagy.
To understand the mechanisms of processes in-
volving Bag3, it is necessary to identify its protein
partners simultaneously interacting with this adapter
protein. In vitro experiments have demonstrated that
Bag3 can simultaneously bind both Hsp70 and sHsps,
thus forming the ternary complex Hsp70–Bag3–sHsp.
Moreover, HspB8 was found to affect the ability of
Bag3 to regulate the ATPase of activity Hsp70 and to
increase its chaperone activity, which was demonstrat-
ed using luciferase as a model substrate [45]. Thus, the
representatives of two Hsp families (Hsp70 and sHsps)
can simultaneously interact with Bag3 and influence
each other’s activity. This agrees with the fact that the
knockout of Bag3 or expression of its E455K mutant
(which does not interact with Hsp70) decreased the
level of sHsps, especially, HspB6 and HspB8, which in-
teract with Bag3 most efficiently [56, 57]. At the same
time, the content of sHsps (HspB1 and HspB5) in the
heart of transgenic mice overexpressing Bag3 was de-
creased [58]. The authors explained this fact by an
increased turnover of sHsps due to the autophagy up-
regulation resulting in the sHsp degradation. It is prob-
ably impossible to combine and to compare the data
obtained on different small heat shock proteins. For in-
stance, overexpression of HspB5 in the primary culture
of astrocytes or glioblastoma cells was accompanied by
the increased synuclein aggregation [59]. The authors
explain this unexpected effect by the competition be-
tween overexpressed HspB5 and HspB8 for Bag3, lead-
ing to the inhibition of autophagy and accumulation of
synuclein aggregates. Therefore, it is impossible to ex-
clude the probability that different sHsps can compete
with each other and affect differently the function-
ing of Bag3. This conclusion agrees with the experi-
mental results on the aggregation of huntingtin frag-
ment coded by DNA containing 43 CAG repeats [60].
Aggregation of this fragment was effectively prevented
by the Bag3–HspB8 complex, whereas HspB1 was un-
able to replace HspB8. Interestingly, the prevention of
aggregation did not depend on the presence of Hsp70.
It was concluded that for some substrates, the process
of autophagy can be provided by Bag3 and HspB8
without participation of Hsp70 [60, 61].
Amyotrophic lateral sclerosis (ALS) is associated
with the accumulation of protein aggregates. Among
these proteins, there is a product of the C9ORF72 gene.
The ATG-independent translation of this gene produces
a protein containing at the N-terminus short amino acid
repeats coded by the G
4
C
2
hexanucleotide sequence.
These repeats (poly-PA, poly-GP, poly-GR, or poly-PR
sequences) cause aggregation of the mutant proteins
[62]. Cristofani etal. [62] showed that in immortalized
NSC32 motoneurons, the modified proteins were ac-
cumulated in aggregates containing p62/SQSTM1 that
slowly underwent autophagy, while overexpression of
HspB8 significantly decreased formation of all possible
types of aggregates. ALS can be also associated with
the aggregation of certain RNA/DNA-binding proteins.
TAR-DNA binding protein (TDP43) is located in the nu-
cleus and can undergo proteolysis with the formation
of TDP25 and TDP35 fragments. Because of the loss of
nuclear localization signals (NLSs), these fragments are
translocated to the cytosol where they aggregate and
accumulate, leading to the ALS development. Overex-
pression of HspB8 prevented TDP25 and TDP35 aggre-
gation in immortalized motoneurons [63]. The data of
Italian scientists indicate that upregulation of HspB8
expression slowed down (or completely prevented)
aggregation of TDP25, TDP43, mutant superoxide dis-
mutase, and proteins containing short repeated dipep-
tides [64, 65]. Hence, HspB8 in a complex with other
CASA system components can efficiently protect mo-
toneurons from accumulation of detrimental protein
aggregates. Therefore, development of small-molecu-
lar-weight compounds capable of upregulating HspB8
synthesis can be a promising approach in the treat-
ment of many neurodegenerative diseases [64].
Stress can induce formation of the so-called stress
granules. These granules are formed by RNA and
RNA-binding proteins (FUS, TIA01, and hnRNPA1) and
accumulate in the cytosol. They can undergo phase
separation and form specific drops lacking a mem-
brane. In the norm, stress granules disassemble after
stress and release RNA. Under pathological conditions,
stress granules can bind misfolded proteins or prod-
ucts of incomplete protein synthesis, which affects
their structure and makes them more rigid. As a re-
sult, the granules do not dissociate and form aggre-
gates detrimental for the cell functioning. Accumula-
tion of such aggregates can lead to the development
of neurodegenerative diseases, such ALS or fronto-
temporal dementia[66]. Immediately after the stress,
PROTEIN Bag3 AND SMALL HEAT SHOCK PROTEINS 1541
BIOCHEMISTRY (Moscow) Vol. 89 No. 9 2024
HspB8 dissociates from the complex with Bag3-Hsp70
and migrates to stress granules containing incomplete-
ly folded or denatures proteins [66]. When bound to
the granules, HspB8 recruits the Bag3–Hsp70 complex,
thus initiating and activating CASA. This mechanism
is specific for HspB8 only and cannot be realized by
other sHsps, e.g., HspB1 [66].
Mitosis is accompanied by the rearrangement of
the entire cytoskeleton and actin filaments in partic-
ular. Bag3 is phosphorylated by the cyclin-dependent
protein kinase 1 [67] and in a complex with HspB8 and
p62/SQSTM1 binds the Arp2/3 protein complex, which
controls polymerization and branching of actin fila-
ments, as well as histone deacetylase 6 (HDAC6), that
regulates another actin-binding protein, cortactin [68].
A decrease in the levels of HspB8 or/and Bag3 is ac-
companied by mitosis impairment and appearance of
cells with two or more nuclei [69]. It was suggested
that multifunctional Bag3 interacts with many protein
partners and thus, regulates many intracellular pro-
cesses. Interaction with HspB8 can switch the activity
of Bag3 from the control of autophagy to the regula-
tion of cytoskeleton [69]. Therefore, the protein quality
control system not only prevents accumulation of pro-
tein aggregates and ensures their selective proteolysis,
but can participate in the regulation of actin polymer-
ization/depolymerization and even orientation of the
mitotic spindle [70]. Interestingly, this activity depends
on HspB8 and p62/SQSTM1, but not on Hsp70 [70].
CONCLUSION
Bag3 is a universal adapter protein capable of in-
teracting with multiple protein partners. For instance,
it can simultaneously bind Hsp70/Hsc70 and different
sHsps. sHsps recognize and bind misfolded and de-
natured proteins that are then transferred to Hsp70
and undergo ubiquitination by ubiquitin ligase STUB1
bound to Hsp70. The complex composed of ubiquiti-
nated protein substrate, Bag3, Hsp70, and sHsps is
transferred by dynein to the MTOC, where it interacts
with the autophagy receptors p72/SQSTM1 and LC3
on the phagophores. Later, the phagophores fuse with
lysosomes in the process of CASA. The composition
of such complex can vary and include different sets
of components depending on the nature of the rec-
ognized protein substrate. Rather contradictory data
indicate that all sHsps interact with Bag3. However,
there is a consensus that HsB8 is a predominant Bag3
partner. In the future, it would be important to ana-
lyze the interactions of different sHsps with Bag3 and
their involvement in the processes controlled by Bag3.
Mutations in HspB8 and Bag3 fragments responsible
for sHsp binding are often associated with neurode-
generative diseases and cardiomyopathies and most-
ly cause disturbance in the functioning of the CASA
system. The CASA system is not only involved in the
selective degradation of denatured proteins, but also
plays an important role in granulostasis and cytoskel-
eton regulation.
Contributions. M.A.Z. collected the published
data; L.K.M. and A.I.Z. analyzed the collected data and
edited the manuscript, P.A.T.-K. and K.Yu.K. provid-
ed additional information on the interaction of Bag3
with protein kinases; N.B.G. wrote and edited the final
version of manuscript.
Funding. The study was supported by the Russian
Science Foundation (no. 24-74-10008) and Lomonosov
Moscow State University Program of Development
(project23-SCH04-11).
Ethics declarations. This work does not contain
any studies involving human and animal subjects.
Theauthors of this work declare that they have nocon-
flicts of interest.
Open access. This article is licensed under a Cre-
ative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution,
and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s)
and the source, provide a link to the Creative Com-
mons license, and indicate if changes were made.
Theimages or other third party material in this article
are included in the article’s Creative Commons license,
unless indicated otherwise in a credit line to the mate-
rial. If material is not included in the article’s Creative
Commons license and your intended use is not permit-
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use, you will need to obtain permission directly from
the copyright holder. To view a copy of this license,
visit http://creativecommons.org/licenses/by/4.0/.
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