2. The Relationship between the UPS and Ca2+ Signaling
2.1. UPS-Mediated Degradation of Ca2+-Related Proteins
The UPS and Ca2+ signaling are interconnected, since each affects the other. The interconnected nature of these signals plays a critical role in regulating cellular functions. The UPS regulates Ca2+ signaling through the degradation of Ca2+-related proteins. Ca2+ channels and transporters are distributed on the membranes of intracellular organelles or the plasma membrane. In this section, researchers will discuss the relationship between these systems and how this affects protein degradation.
The endoplasmic reticulum (ER) is a major intracellular Ca
2+ store. On the ER membrane, inositol 1,4,5-trisphosphate receptor (IP
3R) releases Ca
2+ to the cytosol via the binding of released IP
3 from phosphatidylinositol 4,5-bisphosphate (PIP
2)
[38][39]. Generally, PIP
2 is hydrolyzed to IP
3 by phospholipase C (PLC), which is stimulated by the G-protein-coupled receptor, and IP
3 subsequently activates IP
3R to release ER Ca
2+ [38][39]. Ubiquitin ligase ring finger protein 170 (RNF170), which has three membrane-spanning helices, is localized to the ER membrane and binds to IP
3R
[40]. Ubiquitin ligase RNF170-induced UPS downregulates IP
3R in rat pancreas cells and CHO cells
[41][42][43]. A deletion of endogenous RNF170 increased the expression of IP
3R1
[40]. In other words, the knock-down of RNF170 inhibits IP
3R ubiquitination and degradation
[40]. In addition, reactive oxygen species are involved in the proteasome-associated degradation of IP
3R. H
2O
2 treatment enhances the proteasome-induced degradation of IP
3R in vascular smooth muscle cells
[44]. The treatment of MG-132 recovers the H
2O
2-induced degradation of IP
3Rs
[44]. Other ER resident proteins: sarco-/endoplasmic reticulum Ca
2+-ATPase (SERCAs) and ryanodine receptors (RyRs) are also degraded by the UPS
[23][45]. SERCAs are family to the ER-localized P-type cation ATPase that transports cytosolic Ca
2+ to the ER
[46][47]. Inhibition of the UPS with MG-132 increases SERCA expression
[23]. Type-2 RyR (RyR2), which contributes to cardiac excitation–contraction coupling, is degraded by the UPS
[45]. In the simulated ischemia–reperfusion of mouse cardiomyocytes, RyR2 is degraded by the UPS following the activation of the Ca
2+-dependent cysteine protease calpain, which is activated during ischemia/reperfusion
[45]. Although the studies of the relationship between the UPS and intracellular organelle-releasing Ca
2+ have been well-developed in the ER, it is meaningful to investigate the effect of the UPS on Ca
2+ channels and transporters on other intracellular organelles, including the mitochondria and Golgi apparatus.
Regulatory channels of Ca
2+ signaling, store-operated Ca
2+ channels (SOCCs), are stimulated by changes in the Ca
2+ store levels. When the concentration of ER Ca
2+ is depleted, stromal interaction molecule (STIM) senses the depleted ER, elicits oligomerization, and forms a complex with the Orai channels to induce Ca
2+ influx
[48]. The overexpression of E3 ubiquitin ligase reduces the surface expression of STIM1, and the treatment with MG-132 increases the store-operated Ca
2+ entry (SOCE), a Ca
2+ homeostatic process to regulate cellular functions, by rescuing the STIM1 expression
[49], whereas the inhibition of the proteasome degrades STIM1 and STIM2 through the complementary activation of autophagy
[50]. To maintain the cellular activity by the degradation of proteins, autophagy and the UPS are known to communicate with each other
[5]. If one is inhibited, the other is activated to degrade proteins
[5]. Thus, the inhibition of the UPS complementally stimulates autophagic flux to maintain the [Ca
2+]
i level.
The N-type Ca
2+ channel voltage-gated calcium channel (Ca
V)2.2, which induces peripheral neuron neurotransmission
[51], is degraded via the UPS to maintain the precise modulation of its expression
[52][53]. For example, the overexpression of Parkin, which is an E3 ligase, decreases the current of Ca
V2.2 through proteasome-induced degradation
[52], and proteasome inhibition through MG-132 increases the current of Ca
V2.2
[53]. The degradation of Ca
V2.2 is induced by the light chain of microtubule-associated protein B through ubiquitin-conjugating enzyme E2 L3 (UBE2L3)-mediated ubiquitination
[54]. UBE2L3 is an E2-type ubiquitin ligase that is related to the occurrence of various diseases, including rheumatoid arthritis, celiac disease, and Crohn’s disease
[55]. The β-subunit of Ca
V2.2 protects against the excessive degradation of Ca
V2.2 and even the formation of polyubiquitin chains but not from the binds of one to four ubiquitins
[56][57]. Ca
V1.2 is expressed in the brain, cardiomyocytes, pancreas, adrenal medulla, and bladder smooth muscle
[58] and specifically initiates cardiac excitation-contraction coupling
[59] and triggers smooth muscle contractions
[60]. Similar to Ca
V2.2, the β-subunit of Ca
V1.2 promotes the trafficking of Ca
V1.2 to the plasma membrane to avoid the UPS
[61]. The aberrant splicing variant form of the Ca
V1.2 β-subunit increases the UPS-induced degradation of Ca
V1.2, which triggers cardiac hypertrophy
[62]. Galectin-1 (Gal-1), which reduces the current density of Ca
V1.2
[63], induces the proteasome-induced degradation of Ca
V1.2 by disrupting the Ca
V1.2 β-subunit in HEK 293 cells
[64]. Coupling between Ca
V1.2 and Gal-1 regulates the blood pressure, and Gal-1 deficiency triggers hypertension by activating Ca
V1.2 in spontaneously hypertensive rats
[64]. In conclusion, adjustment of the UPS with the scope of the UPS to regulate Ca
2+ signaling is proposed as a therapeutic strategy for Ca
2+ channel-associated diseases, including cardiac hypertrophy and ischemia–reperfusion injury.
2.2. Ca2+ Signaling and Ca2+-Related Proteins Regulate UPS Acitivity
Ca
2+ signaling regulates numerous cellular functions. In this section, researchers will elucidate Ca
2+ signaling to regulate the UPS. For example, treatment with a Ca
2+ ionophore (A23187) activates the proteasome within 10 min in ascidian and
Xenopus eggs
[65][66]. Increased proteasome activation is attenuated by the Ca
2+-chelating agent 1,2-bis(o-aminophenoxy) ethane-N,N,N′,N′-tetra acetic acid (BAPTA)-AM
[65][66]. Furthermore, A23187-induced Ca
2+ increasingly activates the UPS to degrade the signaling proteins, including cyclooxygenase-1 and islet-brain1/JNK interacting protein 1
[67][68]. In neuronal membrane proteasome-inhibited neurons, Ca
2+ signaling is dominantly attenuated
[69]. Similarly, [Ca
2+]
i increases by the constitutive activation of the epithelial sodium channel, which induces the aggregation and activation of caspase-8 to inhibit the proteasome, and activated caspase-8 induces cellular apoptosis
[70].
The ER is a major source of increased [Ca
2+]
i that regulates the UPS. Acute ER stress increases the degradation of the amyloid precursor protein, a diagnostic marker of AD
[71]. In contrast, human islet amyloid polypeptide aggregation induces ER stress and subsequently impairs the UPS
[72]. Aggravated oxidative stress and ER stress produce misfolded proteins in pancreatic β cells and subsequently impair the β-cell function
[73]. In summary, the mechanisms by which Ca
2+-related proteins regulate the UPS can be used to elucidate the interplay between Ca
2+ signaling and the UPS with the scope of Ca
2+ signaling to regulate the UPS and may provide dynamic tools for potential therapeutic applications.
2.2.1. Membrane-Bound Proteins and the UPS
Membrane-bound Ca
2+ channels are categorized into various subfamilies. In this section, researchers will discuss the membrane-associated Ca
2+ channels, which regulate the UPS. First, the UPS is regulated by Ca
2+ signaling from intracellular organelles, including the mitochondria and ER. For example, the treatment with curcumin, which may have anticancer properties
[74][75], induces a mitochondrial Ca
2+ increase, which inhibits the UPS and induces severe vacuolation, which is a marker of paraptosis, along with apoptotic signals, including cellular shrinkage and the generation of apoptotic bodies
[76]. Plasma membrane-bound VGCCs are categorized into several subtypes, including L-, N-, P/Q-, R-, and T-type channels
[77]. The T-type Ca
2+ channel inhibitor NNC 55-0396 blocks angiogenesis in human umbilical vein endothelial cells through hypoxia-inducible factor-1 (HIF-1) degradation
[78]. Under hypoxic conditions, NNC 55-0396 treatment induces the ubiquitination of HIF-1 and subsequent UPS degradation
[78]. Thus, modulation of the T-type Ca
2+ channels and the subsequent UPS may have therapeutic potential in treating cancer by inhibiting angiogenesis. Transient receptor potential (TRP) channels are nonselective Ca
2+ channels with various functions and subtypes
[79][80] that also regulate the UPS. In oxidative stress induced by ultraviolet irradiation, TRP vanilloid (TRPV)1 is activated and induces an increase in the Ca
2+ levels in human dermal fibroblasts
[81]. The activation of TRPV1 induces the ubiquitination of nuclear factor erythroid 2-related factor 2 (Nrf2), which is a key factor in oxidative stress
[81]. In addition, the overexpression of TRPV1 increases the ubiquitination of the epidermal growth factor receptor (EGFR) to reduce EGFR expression
[82]. Another plasma membrane channel, the Ca
2+-sensing receptor (CaSR), which maintains Ca
2+ homeostasis, also induces proteasome-induced degradation
[83]. CaSR inhibits the TGF-beta-dependent phosphorylation of Smad2, which increases its proliferative effect in human embryonic kidney (HEK) 293 cells
[83]. The mechanisms by which Ca
2+ channels and transporters are activated are diverse, and their roles in regulating ubiquitination and the UPS should be studied in further detail. Although the importance of Ca
2+ channels and transporters is being magnified, the study of the UPS for Ca
2+ channels and transporters is still attractive.
2.2.2. Cytosolic Ca2+-Binding Proteins and the UPS
Cytosolic Ca
2+-binding proteins involved with the UPS have emerged in various studies. In this section, researchers will discuss the accumulating evidence of the role of Ca
2+-binding/related proteins in the modulation of the UPS. The secondary messenger Ca
2+ delivers signals through Ca
2+-binding proteins, such as calmodulin (CaM)
[84]. CaM is stimulated by the binding of Ca
2+ and activates Ca
2+/calmodulin-dependent protein kinases (CaMK) to regulate a variety of physiological functions, including smooth muscle contraction
[85], the activation of phosphorylase kinase
[86], and activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
[87]. In addition, CaM and CaMK regulate the UPS. E3 ligase, mahogunin ring finger 1 (MGRN1), and glycoprotein 78 (GP78) bind CaM under high [Ca
2+]
i, and the treatment with BAPTA attenuates the ubiquitination of MGRN1 and GP78
[88]. CaM bound to MGRN1 and GP78 activates the translocation of GP78 onto the ER membrane to induce ER-associated protein degradation
[88]. In hippocampal neurons, the UPS induces an action potential that is inhibited by MG-132
[89]. The treatment with the Ca
2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor AIPII reduces the rate of protein degradation, while overexpression of the constitutively active form of CaMKII increases the protein degradation
[89]. A recent study addressed a new T-type channel enhancer, ethyl-8-methyl-2,4-dioxo-2-(piperidin-1-yl)-2H-spiro[cyclopentane-1,3-imidazo [1,2-a]pyridin]-2-ene-3-carboxylate (SAK3), which has potential therapeutic effects against AD
[90]. CaMKII is a scaffold protein that phosphorylates the proteasome subunit Rpt6
[91]. The administration of SAK3 increases CaMKII-Rpt6 signaling, which enhances the proteasome activity in dendritic cells
[90]. In myotubules, the Ca
2+ ionophore A23187 induces the UPS, while CaMKII inhibitors KN-62 and KN-93 dominantly attenuate the proteasome activity
[92].
During muscle wasting caused by cachexia, the Ca
2+-binding protein calpain induces Ca
2+-dependent proteolysis and the breakdown of myofibrillar proteins
[93]. The calpains activate ER-bound transcription factor 11 (TCF11)/Nrf1, which activates the 26S proteasome subunit genes
[94]. Calpain-1 cleaves TCF11/Nrf1 to generate the active form, and the inhibition of calpain-1 slows down the degradation of TCF11/Nrf1
[94]. Another ER-related protein RNF122 interacts with Ca
2+ to modulate the cyclophilin ligand (CAML) to stabilize RNF122, thus inhibiting the ubiquitination of RNF122
[91]. The lectin chaperone calreticulin, which maintains [Ca
2+]
i homeostasis, regulates the proteasome activity
[95]. In calreticulin-deficient cells, the number of ubiquitinated proteins and proteasome activity are increased
[95]. In addition, the Ca
2+-binding protein S100, which regulates the tumor cell viability
[96], interacts with the E3 ubiquitin ligase C-terminus of the Hsc70-interacting protein (CHIP) to inhibit ubiquitination and the proteasome system
[97]. The current understanding of Ca
2+ signaling and its associated proteins in the UPS is summarized in
Table 1.
Table 1. The effect of Ca2+ signaling on the UPS.
Related Signaling |
Effect on UPS |
Details |
Ref |
Mitochondrial Ca2+ release |
Inhibition |
Curcumin inhibits the UPS to induce paraptosis. |
[76] |
T-type Ca2+ channel |
Inhibition |
NNC 55-0396 inhibits T-type Ca2+ channels to attenuate cancer angiogenesis. |
[78] |
TRPV1 |
Activation |
Activation of TRPV1 induces the ubiquitination of Nrf2. |
[81] |
Overexpressed TRPV1 increases the ubiquitination of EGFR to induce the UPS. |
[82] |
CaSR |
Activation |
CaSR maintains Ca2+ homeostasis through the UPS. |
[83] |
CaM |
Activation |
CaM induces the translocation of GP78 for ER-associated UPS. |
[88] |
CaMKII |
Activation |
Phosphorylation of Rpt6 through CaMKII enhances the UPS. |
[90] |
Calpain |
Activation |
Calpain-induced activation of Nrf1 stimulates the 26S proteasome subunit gene. |
[94] |
CAML |
Inhibition |
CAML stabilizes RNF122. |
[91] |
Calreticulin |
Inhibition |
Deficiency of calreticulin increases the UPS. |
[95] |
S100 |
Inhibition |
Inhibition of the E3 ubiquitin ligase. |
[97] |