GRP78 Overexpression Triggers PINK1-IP3R-Mediated Neuroprotective Mitophagy Part 3

3.1.3. Mitophagy Induction by GRP78 Overexpression Mediates Neuroprotection

We hypothesized that GRP78-mediated neuroprotection may be through inducing selective autophagy of mitochondria to accelerate the removal of defective organelles. 

In recent years, more and more studies have shown that mitochondria play an important role in the formation and maintenance of memory. Mitochondria are an important organ in cells that are responsible for producing energy. In addition, mitochondria are also involved in the regulation of the entire cell metabolism and the balance of redox reactions. Recent studies have also found that mitochondria can also affect the solidification of long-term memory by regulating changes in neuronal excitability.

Mitochondria are responsible for oxidative metabolism in cells, and the energy of cells is mainly transmitted through adenosine triphosphate (ATP) in mitochondria. Neurons, as information transmission units of the human body, have a particularly high energy demand, so the normal function of their healthy mitochondria is essential for the physiological characteristics of neurons and the normal function of the nervous system. Researchers have found that in the state of disease or aging, the health of mitochondria will be damaged and their function will decline, resulting in a decline in neuronal function and affected memory ability.

The formation and maintenance of memory requires complex coordination between neurons, synapses, and mitochondria at the same time. At the synapses at the end of neurons, mitochondria are located between neurons and glial cells that support neurons. These synapses present a specific structure and are highly plastic, which can be adjusted according to the needs of memory and learning. Mitochondria are like the "production center" of synapses, providing neurons and synapses with the energy and substances they need, which can quickly regulate the excitability of neurons and maintain the long-term plasticity and stability of synapses.

Another research direction related to mitochondria and memory is to treat cells through transformation-related factors (a type of protein). This treatment can enhance mitochondrial function, increase metabolic rate, and protect the health of neurons and synapses. Based on these studies, it is suggested that by strengthening the health of mitochondria, we can enhance our memory and intellectual ability while reducing the risk of Alzheimer's and other neurological diseases.

In summary, mitochondria are an integral part of memory formation and maintenance. The health of mitochondria is essential for the normal function of neurons and synapses and for maintaining long-term plasticity and stability. We should pay attention to diet, exercise, and lifestyle habits to maintain the health of mitochondria, reduce the risk of Alzheimer's and other neurological diseases, and enhance our intelligence and memory. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because it can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche deserticola can improve blood flow and promote oxygen delivery, which can ensure that the brain obtains adequate nutrition and energy, thereby improving brain vitality and endurance.

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In support of this hypothesis, GRP78 has been proven to promote macroautophagy [4] (hereafter referred to as autophagy). 

We sought to determine whether autophagy was necessary for GRP78-induced neuroprotection using several well-characterized autophagy modulators: rapamycin, which activates autophagy by inhibiting mTORC1, and two inhibitors of the autophagy flux: 3-methyladenine (3-MA), an inhibitor of class III phosphatidylinositol kinase PI3K, and LY294002, an inhibitor of class I PI3K. 

All three autophagy modulators decreased survival in both GFP and GRP78 groups in Tun-treated cells compared to vehicle (Figure 4A). These results suggested that the correct initiation of autophagy, through the PI3K-Beclin1 pathway, and late flux was necessary, although not sufficient, for the neuroprotective effects of GRP78 overexpression in ER-stressed cells.

Figure 3. GRP78 overexpression rescues mitochondrial dysfunction in stressed NSC34 cells. (A) Immunoblot and bar graph showing the levels of GRP78 in NSC34 cells nucleofected with plasmid vector to overexpress GRP78 or a non-related protein as control (GFP). (B) Percentage of survival of GFP- or GRP78-expressing NSC34 cells (means ± SEM) after 24 h in a medium containing 1 µg/mL tunicamycin (Tun) (left) or 10 µM nocodazole (right) determined using an MTT assay (n = 4; * p < 0.005, ** p < 0.001 vs. GFP, Student's t-test). (C) Quantitative analysis of MitoSOX fluorescence in cells overexpressing GFP or GRP78 and treated with Tun or vehicle (left) or efavirenz (EFV) as control or vehicle (right). (D) Quantitative analysis of TMRM fluorescence in cells overexpressing GFP or GRP78 and treated with Tun or vehicle (left) or EFV or vehicle (right) (GFP-Veh is the control group). (E) Left: Representative analysis of O2 concentration (using a Clark-type O2 electrode) as a function of time in cells overexpressing GFP or GRP78 and treated with Tun. Right: O2 consumptions after 5 h of Tun treatment in cells overexpressing GFP or GRP78 (GFP-Veh is the control group) (n = 4; * p < 0.05 vs. Veh-GFP, # p < 0.05 vs. GRP78, one-way ANOVA).

Figure 4. Mitochondria tagging for mitophagy is privileged by GRP78 overexpression. (A) Bar graph of the percentage of viable NSC34 cells (mean ± SEM) nucleofected with either GFP or GRP78 plasmids and treated with Tun or combination of Tun with autophagy modulators: rapamycin (RAPA), 3MA, or LY-902, concerning control vehicle (Veh), determined by MTT assay 24 h after treatment (GFP-Veh is the control group) (n = 4–8, * p < 0.05 vs. 

Veh-GFP; # p < 0.05 vs. Tun-GFP). (B) Representative confocal images of cells nucleofected with PARK2-HA plasmid alone (top, middle panels, control) or with GRP78 plasmid (bottom panels) treated with either vehicle (Veh) or Tun (5 h) and immunostained for HA (green) and HSP60 (red); scale bar = 10 µm. (C) Average ± SEM of Pearson's correlation coefficient of Parkin and Hsp60 co-localization from Veh, Tun, and Tun+GRP78 groups (n = 4; * p < 0.05 vs. Parkin-Veh, # p < 0.05 vs. 

Parkin-Tun, one-way ANOVA). (D) Left, Western blots for indicated proteins in the mitochondrial (mito) and cytosolic (cyto) pooled fractions from GFP- or GRP78-overexpressing cells treated with vehicle or Tun for 5 h. The proteins analyzed are OPA-1, CV-β, PDI, GRP78, Ubiquitinated residues, LC3, and actin. Right, bar graphs of the average fold change of GRP78 in both pooled fractions and LC3-II in the mitochondrial fraction relative to actin (cytosol) or the beta subunit of the complex V(CV-B) (mitochondria) in the GFP group. (E) Bar graph of the percentage of viable cells (means ± SEM) overexpressing GFP or GRP78 proteins and treated with Tun alone or in combination with CCCP, CSA, or BAPTA-AM, determined by MTT assay 24 h after treatment concerning control vehicle-treated cells. (F) Similar bar graph of cell viability for cells treated with thapsigargin (THA) with or without CCCP (GFP-Veh is the control group) (n = 4–8, in 3 different experiments, * p < 0.05 vs. control-Veh; # p < 0.05 vs. GRP78-Tun).

Parkin does not always participate in mitophagy since it can also be triggered through alternative mechanisms [46]. To ascertain whether Parkin is involved in the GRP78-mediated effect, we determined the subcellular location of HA-tagged Parkin expressed in NSC34 cells with and without ER stress. 

By confocal microscopy, we observed that Parkin was distributed throughout the NSC34 cell after 5 h of vehicle or Tun treatment (Figure 4B). In contrast, in cells overexpressing GRP78, there was an increase in the co-localization of Parkin with HSP60, a mitochondrial protein, suggesting targeting of this organelle for mitophagy (Figure 4B, C). 

In addition, we isolated the mitochondrial and cytosolic fractions from cells that overexpressed either GFP or GRP78 and were treated with Tun or vehicle as control at 5 h after the insult. The purity of pooled mitochondrial fractions was confirmed by analyzing the presence of OPA1 and CVβ, and the absence of an ER-resident protein, the protein disulfide isomerase (PDI) (Figure 4D). 

Interestingly, we also observed a tendency to increase GRP78 abundance in the pooled mitochondrial fractions of ER-stressed cells compared to the control. Note that although Tun stimulus might also induce an increase in GRP78 levels in cytosol, this was not seen at the mitochondrial fraction. 

Forced overexpression of GRP78 promoted a tendency to increase both PINK1 and Parkin accumulation in the mitochondrial fraction (Figure 4D and Figure S4). It is reported that the presence of Parkin amplifies the accumulation of Ub and enhances mitophagy compared to the presence of PINK alone [42,47]. Accordingly, Ubiquitin (Ub) immunoblotting revealed differences in its profile in mitochondrial fractions from GRP78-expressing cells compared to the GFP group (Figure 4D). 

Under stressful conditions, mitochondrial protein ubiquitylation leads to the recruitment of autophagosome machinery components that begins with the accumulation of the lipidated isoform of LC3, and LC3II [42,48]. 

Abundant LC3II protein was observed in the mitochondrial fraction with a tendency to increase higher presence in the stressed cells that overexpress GRP78 concerning those with GFP at similar conditions (Figure 4D). Taken together, these data suggested that the forced expression of GRP78 could facilitate its mitochondrial translocation and tagging for mitophagy. 

To determine the possible relevance of mitophagy induction to the neuroprotective effect mediated by GRP78 overexpression, we assessed the alterations induced by activators and inhibitors of mitophagy. We used carbonyl cyanide m-chlorophenyl hydrazine (CCCP) to chemically uncouple oxidative phosphorylation and induce mitophagy by collapsing the mitochondrial membrane potential [49,50], as well as two mitophagy inhibitors: BAPTA-AM, a cell-permeant chelator of intracellular Ca2+ and cyclosporin A (CSA), the mitochondrial permeability transition pore (mPTP) inhibitor that blocks Ca2+ efflux from mitochondria [51]. 

Although CCCP treatment in NSC34 was insufficient to reduce notably viability, as widely reported, we found that the same treatment on ER-stressed cells increased its survival, regardless of whether or not GRP78 was overexpressed (Figure 4E). 

Importantly, the neuroprotective effect promoted by GRP78 overexpression on ER-stressed cells was abolished in the presence of either CSA or BAPTA-AM (Figure 4E). These findings indicate that mitophagy induction allows cells to cope with ER stress and Ca2+-flux is important for the neuroprotective effect promoted by GRP78. 

One possibility for GRP78 to induce mitophagy might be facilitating the necessary Ca2+-mediated action, perhaps through the action of the ER-resident inositol 1,4,5 triphosphate receptor (IP3R) whose opening allows Ca2+ flow from the ER to the mitochondria. We wanted to explore this possibility by impeding the activation of IP3R using thapsigargin (THA), a Ca2+ ATPase inhibitor [52]. 

Thapsigargin is also known to produce ER stress in the cells since this treatment depletes Ca2+ stores from ER, and therefore increases GRP78 expression as part of the canonical unfolded protein response [53]. Figure 4F shows that thapsigargin affects cell viability similarly to tunicamycin treatment in GFP-control cells and that CCCP concomitant treatment prevents cell death as observed with Tun. 

However, forced overexpressing of GRP78 was not capable of blocking the detrimental thapsigargin effect as it did with the tunicamycin insult. This observation confirms the necessary involvement of Ca2+ flux in the neuroprotective effect of GRP78, probably through IP3R.

3.1.4. Neuroprotection Mediated by GRP78 Depends on PINK1 and IP3R

To initiate the identification of the mediators of the GRP78 neuroprotective effect, we used shRNA technology. First, we verified that the shRNA chosen reduced the expression of GRP78, PINK1, or IP3R, respectively (Figure S4A–C) and that they did not compromise the viability of control cells (Figure S4D). We first confirmed that the expression of GRP78 itself was necessary for both GRP78- and CCCP-mediated neuroprotection to face Tun-induced ER stress (Figure 5A). 

Next, silencing either PINK1 or IP3R blocked the neuroprotective effect promoted by GRP78 overexpression or CCCP treatment (Figure 5A). Furthermore, we found the GRP78 shift, normally distributed in a smooth spotty distribution around the nucleus, to be co-localized to IP3R in speckle foci when it was overexpressed (Figure 5B).

Together these results suggested that GRP78 modulates protective mitophagy in our in vitro model, and it exerts this neuroprotection in a PINK1- and IP3R-dependent manner.

4. Discussion

Boosting the endogenous mechanisms of neuroprotection may yield efficient therapeutic tools to prevent neurodegenerative processes after traumatic lesions or disease [3,54,55]. The ER-resident chaperone GRP78 is at the crossroads of several of these mechanisms promoting neuroprotection when overexpressed in several disease models [4]. 

To determine the underlying mechanisms, we used a model of spinal root avulsion [55] that disrupts motoneuron connectivity causing a retrograde neurodegenerative process [20]. 

Motoneuron death in this model is characterized by ER stress, a blocked autophagy flux, and is nonapoptotic since no active forms of caspase 3 or 12 were observed, indicating that apoptosis is not the final executor of neuronal demise [20]. In accordance, we previously verified with proteomics approaches that anti-apoptotic features occur in parallel to apoptotic ones after RA, blocking an effective apoptosis execution [25]. 

In that neurodegenerative context, the overexpression of GRP78 exerted motor neuroprotection [20,26,27]. Here, initially, we used unbiased comparative and quantitative analysis of the proteome to uncover the primary basis for this neuroprotection. 

Unexpectedly, mitochondria were the main target, with a reduced organelle protein content accompanied by decorations of engulfed mitochondria into vesicles within damaged motoneurons that overexpressed GRP78. 

These observations suggested the presence of mitophagy as a key element for neuroprotection. Deeper in vitro experiments validated this hypothesis since GRP78 mediated neuroprotection by (i) restoring mitochondria respiration and ROS levels, (ii) stimulating PINK1/PARKIN mitochondria translocation, tagging the organelle for mitophagy, and (iii) depending on IP3R function as an essential mediator. 

To our knowledge, this is the first study showing that GRP78 overexpression promotes protective mitophagy. In addition to the GRP78 role in chaperoning misfolding proteins and other moonlighting functions as being a calcium-binding protein [4,56], we added its pro-active action to stimulate mitophagy. Moreover, our study joins a set of recent studies linking ER stress to mitochondrial dysfunction [57–59]. 

We found hallmarks for mitophagy in vivo when GRP78 was overexpressed and promoted neuroprotection. The same was observed using in vitro models that mimic some traits of the neurodegenerative process that occurs after RA such as ER stress [20,26]. 

Quality control of mitochondria by mitophagy is crucial to monitor the mitochondrial content and metabolism homeostasis. In the literature, it is still controverted whether mitophagy induction promotes survival or cell death in several diseases due to its extensive crosstalk with apoptosis signaling [60], although it has been described that the clearance of damaged mitochondria has a fundamental role in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, or in aging [61]. 

Previously, it has been reported that GRP78 inhibits apoptosis triggered by ER stress by preventing CHOP induction [62,63], the activation of caspase 7 [5], or by activating the PI3K-AKT-mTOR signaling axis [4]. It has also been demonstrated to maintain low levels of oxidative stress and DNA damage [4,64]. In particular, in cancer cells, it has been demonstrated that GRP78 attenuates ROS by activating protein kinase RNA-like endoplasmic reticulum kinase (PERK)-NRF2 signals [65,66], leading to upregulation of antioxidant-related genes as well as enhancing the protein levels of glycolytic enzymes [67]. 

Instead, we have found that GRP78 overexpression downregulated glycolytic enzymes (e.g., ENO1 and ENO2) (Supplemental tables). These results may suggest that in our model, the observed attenuation of ROS might be rather related to increased mitophagy, although details of the mechanism involved should be further investigated. Mitochondrial dysfunction is a common condition in neurodegenerative diseases [68], and mitophagy has been involved in Parkinson's [49,69], Huntington's [70,71], and Alzheimer's diseases and Tauopathies [72]. 

Several authors pointed out that by proper regulation of mitophagy pathways, the body can avoid harmful oxidative species, and regulate the redox balance and homeostasis [73]. Thus, GRP78 overexpression through this controlled mitophagy induction might be considered a neuroprotective strategy in these diseases. Neuroprotection mediated by GRP78 overexpression appears to be dependent on a Ca2+ flux since it was blocked by BAPTA-AM. BAPTA-AM chelating of Ca2+ might intervene at several different steps along the autophagic flux pathway, blocking not only the triggering of autophagy but also inhibiting steps in the formation and processing of autophagosomes [74]. 

In contrast, the use of another agent affecting Ca2+ flux, such as thapsigargin, yielded surprising results. Thapsigargin blocks sarco-endoplasmic reticulum Ca2+ ATPases (SERCAs), sustaining cytosolic Ca2+ elevation, but it also triggers ER stress through chronic depletion of intracellular Ca2+ stores and the accumulation of unfolded proteins. 

In this situation, we observed that GRP78 overexpression could not rescue dying cells, and we suspected that it might be because thapsigargin also impedes IP3R action [52]. Basal IP3R activity and continuous low-level Ca2+ flux from ER to mitochondria are essential to promote mitochondrial respiration and cell bioenergetics [75]. 

GRP78 interacts at MAMs with a complex formed by the sigma-1 receptor (SIGR1) and IP3R [76,77]. Upon ER stress, the SIGR1-GRP78 interaction decreases caused by Ca2+ depletion subsequently boosting Ca2+ flux from the ER to mitochondria through IP3Rs [76]. In addition, it was also demonstrated that IP3R was necessary for Parkin-induced mitophagy in particular to allow mitochondrial clustering downstream Parkin recruitment [78]. 

Our results agree with that since the reduction of IP3R by shRNA technology abolished neuroprotection exerted by GRP78 overexpression in ER-stressed cells. The observation that GRP78 might increase at the mitochondria fraction itself is interesting as well. GRP78 has been detected within the inner membrane intermediate space and matrix of the mitochondria, although its function there remains to be elucidated [79]. 

Nevertheless, and regarding its several functions, we speculate that it can be buffering Ca2+. If this was the case, its increased presence within mitochondria in ER-stressed cells when overexpressed might attenuate massive Ca2+ influx to avoid driving apoptosis. Further experimentation on that would be very valuable.

Study Limitations and Future Research

This study paves the way for future analyses on the neuroprotective role of GRP78-dependent mitophagy in neuronal death after neurotrauma. The first limitation of this study is that mitophagy was analyzed in a specific time window after RA, and since mitophagy is a dynamic process, it would be interesting to decipher its flow and which molecules are involved. 

Thus, further experiments modulating mitophagy (through pharmacological or genetic approaches) are needed to decipher whether the effects observed in vivo are purely dependent on mitophagy. 

This will allow us to confirm that overexpression of GRP78 promotes MN survival through mitophagy. Since there is a well-described crosstalk between mitochondria and apoptosis, further in vitro and in vivo manipulations are essential to deciphering which death mechanism triggers MN death in our model. This will help to elucidate whether GRP78 directly modulates that cell death mechanism by blocking it or whether its presence allows MNs to cope with it and survive.

5. Conclusions

The present study was the first to describe a novel role for GRP78 in modulating mitophagy to achieve motoneuronal protection. Our results reveal that GRP78 could drive mitophagy to promote neuroprotection of degenerating motor neurons following severe traumatic nerve injury, restoring damaged mitochondrial function in neuronal cells. Moreover, this GRP78-mediated neuroprotection is dependent on PINK1 and IP3R. Therefore, the activation of fine-tuned mitophagy, through gene therapy with GPR78 or other candidates, may be used as a novel therapeutic approach for traumatic injuries of the nervous system.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biomedicines9081039/s1. Figure S1: Bioinformatic analysis. Figure S2: Endogenous GRP78 levels after Tun treatment. Figure S3: Tun and EFV cause mitochondrial dysfunction in NSC34 cells. Figure S4: No title. Figure S5: Cell viability analysis after shRNA nucleofection The supplementary materials also include the tables with all the proteins detected in the proteomic analysis.

Author Contributions: T.L.-R. and D.R.-G. designed and performed the experiments, analyzed the results, and wrote part of the manuscript. M.H.-G. and J.F. helped with in vivo experiments. P.M.-G. and J.M.L. performed the mitochondrial purifications. M.P., C.B., and N.A. helped with the mitochondria functional analysis. V.P. and A.B. prepared all the GRP78 plasmids and viral vectors used. C.C. conceived, designed, supervised, and analyzed all the experiments and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding: This work was mainly supported by the Ministerio de Economía y Competitividad of Spain (#SAF 2014-59701) and by the Marató de TV3 (#201607.10). We are also grateful for support from CIBERNED and Generalitat de Catalunya funding. CB is a recipient of Miguel Servet's contract (CP19/00077) from Carlos III Health Institute.

Institutional Review Board Statement: All the procedures that involved animals were approved by the Universitat Autònoma de Barcelona and Generalitat de Catalunya and followed the European Community Council Directive 2010/63/EU.

Informed Consent Statement: Not applicable.

Data Availability Statement: All data generated or analyzed during this study are included in this published article and its supplementary information files.

Acknowledgments: We thank Marta Monserrat, Ariadna Aransanz, Miguel Chillón, Julia Lorenzo, Sara Marmolejo, and the Neuroplasticity and Regeneration Group at the UAB. We extend special thanks to Carlos Guillem (University Complutense de Madrid) for kindly providing the PARK2-HA plasmid and to Elena Galea and Esther Dalfo at the UAB for antibodies.

Conflicts of Interest: The authors declare no conflict of interest.

Special Statement: The authors wish to dedicate a special mention in memory of Caty Casas, an influential scientist, and a wonderful and brave person, who died prematurely at the age of 54 years. It has been an honor and a privilege to share part of her life, her professional capacity, and her ethical and human values.

References

1. Conforti, L.; Gilley, J.; Coleman, M.P. Wallerian degeneration: An emerging axon death pathway linking injury and disease. Nat. Rev. Neurosci. 2014, 15, 394–409. [CrossRef] 

2. Romeo-Guitart, D.; Forés, J.; Herrando-Grabulosa, M.; Valls, R.; Leiva-Rodríguez, T.; Galea, E.; González-Pérez, F.; Navarro, X.; Petegnief, V.; Bosch, A.; et al. Neuroprotective Drug for Nerve Trauma Revealed Using Artificial Intelligence. Sci. Rep. 2018, 8, 1879. [CrossRef] 

3. Romeo-Guitart, D.; Forés, J.; Navarro, X.; Casas, C. Boosted Regeneration and Reduced Denervated Muscle Atrophy by NeuroHeal in a Pre-clinical Model of Lumbar Root Avulsion with Delayed Reimplantation. Sci. Rep. 2017, 7, 12028. [CrossRef] 

4. Casas, C. Grp78 at the Centre of the Stage in Neurodegeneration and Cancer. Front. Neurosci. 2017, 11, 1–15. [CrossRef] 

5. Reddy, R.K.; Mao, C.; Baumeister, P.; Austin, R.C.; Kaufman, R.J.; Lee, A.S. Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors. Role of ATP binding site in suppression of caspase-7 activation. J. Biol. Chem. 2003, 278, 20915–20924. [CrossRef] [PubMed] 

6. Wang, M.; Kaufman, R.J. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature 2016, 529, 326–335. [CrossRef] [PubMed] 7. Printsev, I.; Curiel, D.; Carraway, K.L. Membrane Protein Quantity Control at the Endoplasmic Reticulum. J. Membr. Biol. 2016, 250, 1–14. [CrossRef] [PubMed] 

8. Shu, C.-W.; Sun, F.-C.; Cho, J.-H.; Lin, C.-C.; Liu, P.-F.; Chen, P.-Y.; Chang, M.D.-T.; Fu, H.-W.; Lai, Y.-K. GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis. J. Cell. Physiol. 2008, 215, 627–635. [CrossRef] 

9. Cook, K.L.; Clarke, R. Heat shock 70 kDa protein 5/glucose-regulated protein 78 "AMP" ing up autophagy. Autophagy 2012, 8, 1827–1829. [CrossRef] 

10. Li, Z.; Wang, Y.; Newton, I.P.; Zhang, L.; Ji, P.; Li, Z. GRP78 is implicated in the modulation of tumor aerobic glycolysis by promoting autophagic degradation of IKKβ. Cell. Signal. 2015, 27, 1237–1245. [CrossRef] 

11. Cha-Molstad, H.; Yu, J.E.; Lee, S.H.; Kim, J.G.; Sung, K.S.; Hwang, J.; Yoo, Y.D.; Lee, Y.J.; Kim, S.T.; Lee, D.H.; et al. Modulation of SQSTM1/p62 activity by N-terminal arginylation of the endoplasmic reticulum chaperone HSPA5/GRP78/BiP. Autophagy 2016, 12, 426–428. [CrossRef] 

12. Cha-Molstad, H.; Sung, K.S.; Hwang, J.; Kim, K.A.; Yu, J.E.; Yoo, Y.D.; Jang, J.M.; Han, D.H.; Molstad, M.; Kim, J.G.; et al. Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding. Nat. Cell Biol. 2015, 17, 917–929. [CrossRef] [PubMed]

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