Yong-Woo Jun

Intracellular Membrane Association of the Aplysia cAMP Phosphodiesterase Long and Short Forms via Different Targeting Mechanisms

Background: Phosphodiesterases play a role in cAMP regulation through specific targeting. Results: Membrane targeting of the Aplysia phosphodiesterase long and short forms is mediated hydrophobically and electrostatically, respectively. Conclusion: The Aplysia phosphodiesterase long and short forms are targeted to the intracellular membranes by different mechanisms. Significance: This is the first report demonstrating that phosphodiesterase is targeted to the membranes by hydrophobic or electrostatic interactions. Phosphodiesterases (PDEs) play key roles in cAMP compartmentalization, which is required for intracellular signaling processes, through specific subcellular targeting. Previously, we showed that the long and short forms of Aplysia PDE4 (ApPDE4), which are localized to the membranes of distinct subcellular organelles, play key roles in 5-hydroxytryptamine-induced synaptic facilitation in Aplysia sensory and motor synapses. However, the molecular mechanism of the isoform-specific distinct membrane targeting was not clear. In this study, we further investigated the molecular mechanism of the membrane targeting of the ApPDE4 long and short forms. We found that the membrane targeting of the long form was mediated by hydrophobic interactions, mainly via 16 amino acids at the N-terminal region, whereas the short form was targeted solely to the plasma membrane, mainly by nonspecific electrostatic interactions between their N termini and the negatively charged lipids such as the phosphatidylinositol polyphosphates PI4P and PI(4,5)P2, which are embedded in the inner leaflet of the plasma membrane. Moreover, oligomerization of the long or short form by interaction of their respective upstream conserved region domains, UCR1 and UCR2, enhanced their plasma membrane targeting. These results suggest that the long and short forms of ApPDE4 are distinctly targeted to intracellular membranes through their direct association with the membranes via hydrophobic and electrostatic interactions, respectively.

Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy

Macroautophagy allows for bulk degradation of cytosolic components in lysosomes. Overexpression of GFP/RFP‐LC3/GABARAP is commonly used to monitor autophagosomes, a hallmark of autophagy, despite artifacts related to their overexpression. Here, we developed new sensors that detect endogenous LC3/GABARAP proteins at the autophagosome using an LC3‐interacting region (LIR) and a short hydrophobic domain (HyD). Among HyD‐LIR‐GFP sensors harboring LIR motifs of 34 known LC3‐binding proteins, HyD‐LIR(TP)‐GFP using the LIR motif from TP53INP2 allowed detection of all LC3/GABARAPs‐positive autophagosomes. However, HyD‐LIR(TP)‐GFP preferentially localized to GABARAP/GABARAPL1‐positive autophagosomes in a LIR‐dependent manner. In contrast, HyD‐LIR(Fy)‐GFP using the LIR motif from FYCO1 specifically detected LC3A/B‐positive autophagosomes. HyD‐LIR(TP)‐GFP and HyD‐LIR(Fy)‐GFP efficiently localized to autophagosomes in the presence of endogenous LC3/GABARAP levels and without affecting autophagic flux. Both sensors also efficiently localized to MitoTracker‐positive damaged mitochondria upon mitophagy induction. HyD‐LIR(TP)‐GFP allowed live‐imaging of dynamic autophagosomes upon autophagy induction. These novel autophagosome sensors can thus be widely used in autophagy research.

Analysis of phosphoinositide-binding properties and subcellular localization of GFP-fusion proteins.

Specific protein-phosphoinositide (PI) interactions are known to play a key role in the targeting of proteins to specific cellular membranes. Investigation of these interactions would be greatly facilitated if GFP-fusion proteins expressed in mammalian cells and used for their subcellular localization could also be employed for in vitro lipid binding. In this study, we found that lysates of cells overexpressing GFP-fusion proteins could be used for in vitro protein-PI binding assays. We applied this approach to examine the PI-binding properties of Aplysia Sec7 protein (ApSec7) and its isoform ApSec7(VPKIS), in which a VPKIS sequence is inserted into the PH domain of ApSec7. EGFP-ApSec7 but not EGFP-ApSec7(VPKIS) did specifically bind to PI(3,4,5)P3 in an in vitro lipid-coated bead assay. Overexpression of EGFP-ApSec7 but not EGFP-ApSec7(VPKIS) did induce neurite outgrowth in Aplysia sensory neurons. Structure modeling analysis revealed that the inserted VPKIS caused misfolding around the PI(3,4,5)P3-binding pocket of ApSec7 and disturbed the binding of PI(3,4,5)P3 to the pleckstrin homology (PH) domain. Our data indicate that plasma membrane localization of EGFP-ApSec7 via the interaction between its PH domain and PI(3,4,5)P3 might play a key role in neurite outgrowth in Aplysia.

Sequestration of PRMT1 and Nd1-L mRNA into ALS-linked FUS mutant R521C-positive aggregates contributes to neurite degeneration upon oxidative stress.

Mutations in fused in sarcoma (FUS), a DNA/RNA binding protein, are associated with familial amyotrophic lateral sclerosis (ALS). However, little is known about how ALS-causing mutations alter protein-protein and protein-RNA complexes and contribute to neurodegeneration. In this study, we identified protein arginine methyltransferase 1 (PRMT1) as a protein that more avidly associates with ALS-linked FUS-R521C than with FUS-WT (wild type) or FUS-P525L using co-immunoprecipitation and LC-MS analysis. Abnormal association between FUS-R521C and PRMT1 requires RNA, but not methyltransferase activity. PRMT1 was sequestered into cytosolic FUS-R521C-positive stress granule aggregates. Overexpression of PRMT1 rescued neurite degeneration caused by FUS-R521C upon oxidative stress, while loss of PRMT1 further accumulated FUS-positive aggregates and enhanced neurite degeneration. Furthermore, the mRNA of Nd1-L, an actin-stabilizing protein, was sequestered into the FUS-R521C/PRMT1 complex. Nd1-L overexpression rescued neurite shortening caused by FUS-R521C upon oxidative stress, while loss of Nd1-L further exacerbated neurite shortening. Altogether, these data suggest that the abnormal stable complex of FUS-R521C/PRMT1/Nd1-L mRNA could contribute to neurodegeneration upon oxidative stress. Overall, our study provides a novel pathogenic mechanism of the FUS mutation associated with abnormal protein-RNA complexes upon oxidative stress in ALS and provides insight into possible therapeutic targets for this pathology.

ApCPEB4, a non-prion domain containing homolog of ApCPEB, is involved in the initiation of long-term facilitation

Two pharmacologically distinct types of local protein synthesis are required for synapse- specific long-term synaptic facilitation (LTF) in Aplysia: one for initiation and the other for maintenance. ApCPEB, a rapamycin sensitive prion-like molecule regulates a form of local protein synthesis that is specifically required for the maintenance of the LTF. However, the molecular component of the local protein synthesis that is required for the initiation of LTF and that is sensitive to emetine is not known. Here, we identify a homolog of ApCPEB responsible for the initiation of LTF. ApCPEB4 which we have named after its mammalian CPEB4-like homolog lacks a prion-like domain, is responsive to 5-hydroxytryptamine, and is translated (but not transcribed) in an emetine-sensitive, rapamycin-insensitive, and PKA-dependent manner. The ApCPEB4 binds to different target RNAs than does ApCPEB. Knock-down of ApCPEB4 blocked the induction of LTF, whereas overexpression of ApCPEB4 reduces the threshold of the formation of LTF. Thus, our findings suggest that the two different forms of CPEBs play distinct roles in LTF; ApCPEB is required for maintenance of LTF, whereas the ApCPEB4, which lacks a prion-like domain, is required for the initiation of LTF.

Dual roles of the N-terminal coiled-coil domain of an Aplysia sec7 protein: homodimer formation and nuclear export.

Cytohesin family proteins act as guanine nucleotide exchange factors (GEFs) for the ADP‐ribosylation factor family of small GTP‐binding proteins. Aplysia Sec7 (ApSec7), a member of the cytohesin family in Aplysia, plays key roles in neurite outgrowth in Aplysia neurons. Although ApSec7 has a conserved coiled‐coil (CC) domain, its role was not clear. In this study, we found that the CC domain of ApSec7 and ARNO/cytohesin 2 are involved in homodimer formation, leading to efficient plasma membrane targeting of ApSec7 and ARNO/cytohesin 2 in HEK293T cells. Therefore, deletion of the CC domain of ApSec7 and ARNO/cytohesin 2 may result in a loss of dimerization and reduce plasma membrane localization. In addition, the CC domains of ApSec7 and ARNO/cytohesin 2 have partially or fully CRM1‐dependent nuclear export signals, respectively. Taken together, our results suggest that the CC domain of cytohesin family proteins, including ApSec7 and ARNO/cytohesin 2, has dual roles in intracellular targeting: increased plasma membrane targeting through homodimer formation and nuclear exclusion through either a CRM1‐dependent or a CRM1‐independent pathway.

D-AKAP1a is a signal-anchored protein in the mitochondrial outer membrane

Dual A‐kinase anchoring protein 1a (D‐AKAP1a, AKAP1) regulates cAMP signaling in mitochondria. However, it is not clear how D‐AKAP1a is associated with mitochondria. In this study, we show that D‐AKAP1a is a transmembrane protein in the mitochondrial outer membrane (MOM). We revealed that the N‐terminus of D‐AKAP1a is exposed to the intermembrane space of mitochondria and that its C‐terminus is located on the cytoplasmic side of the MOM. Moderate hydrophobicity and the positively charged flanking residues of the transmembrane domain of D‐AKAP1a were important for targeting. Taken together, D‐AKAP1a can be classified as a signal‐anchored protein in the MOM. Our topological study provides valuable information about the molecular and cellular mechanisms of mitochondrial targeting of AKAP1.

Activation of Aplysia ARF6 induces neurite outgrowth and is sequestered by the overexpression of the PH domain of Aplysia Sec7 proteins

HighlightsActivation of ARF6 signaling induces neurite outgrowth in Aplysia neurons.Binding to the PH domain of ApSec7 sequesters active ARF6.ARF6 may be a downstream signaling target of Aplysia Sec7‐mediated neurite outgrowth. Abstract ADP‐ribosylation factors (ARFs) are small guanosine triphosphatases of the Ras superfamily involved in membrane trafficking and regulation of the actin cytoskeleton. Aplysia Sec7 protein (ApSec7), a guanine nucleotide exchange factor for ARF1 and ARF6, induces neurite outgrowth and plays a key role in 5‐hydroxyltryptamine‐induced neurite growth and synaptic facilitation in Aplysia sensory‐motor synapses. However, the specific role of ARF6 signaling on neurite outgrowth in Aplysia neurons has not been examined. In the present study, we cloned Aplysia ARF6 (ApARF6) and revealed that an overexpression of enhanced green fluorescent protein (EGFP)‐fused constitutively active ApARF6 (ApARF6‐Q67L‐EGFP) could induce neurite outgrowth in Aplysia sensory neurons. Further, we observed that ApARF6‐induced neurite outgrowth was inhibited by the co‐expression of a Sec7 activity‐deficient mutant of ApSec7 (ApSec7‐E159K). The pleckstrin homology domain of ApSec7 may bind to active ApARF6 at the plasma membrane and prevent active ApARF6‐induced functions, including intracellular vacuole formation in HEK293T cells. The results of the present study suggest that activation of ARF6 signaling could induce neurite outgrowth in Aplysia neurons and may be involved in downstream signaling of ApSec7‐induced neurite outgrowth in Aplysia neurons.

Analysis of the effects of δ-Aminolevulinic acid on the proliferation and apoptosis of mammalian cells

δ-Aminolevulinic acid (ALA) is a compound which is widely present in the biosphere and plays an important role in the living body as an intermediate of the tetrapyrrole compound biosynthesis pathway that leads to heme in mammals and chlorophyll in plants. ALA is of interest as a biodegradable mediator, a growth regulator, a precursor of heme proteins, and an effective agent used in therapy of cancer. It has been recently reported that ALA is commonly used in dermatology, due to good effects of skin therapy. Although for the last few decades a substantial amount of research has been focused on the elucidation of the mechanism of ALA and the improvement of its therapeutic activity, it’s effect on the cell functions and growth was not cleared. Here, we identified that ALA treatment could attenuate cell proliferation of HEK293T and HaCaT cells. In addition, ALA treatement could induce apoptosis of HeLa cells. These results suggest that apoptosis induced by ALA treatment might be responsible for inhibition of cell proliferation. These results propose the possibility of the improved therapeutic strategy making ALA one of the effective drugs used in human cancers. 요 약: δ-Aminolevulinic acid (ALA)는 생물권내에 폭넓게 존재하는 화합물이고, 포유류에서 헴(heme)과 식물에서 엽록소 생성을 하게하는 경로에서 만들어진 테트라피롤의 중간물질로써 생체내 중요한 역할을 한다. ALA는 생분해성 매개자, 성장 조절자, 헴단백질의 전구체 그리고 암 치료에서 사용되는 효과적인 물질로써 관심이 있다. 최근에는 ALA가 피부 치료의 좋은 효과를 가지고 있어 피부과학에서 빈번히 사 ★ Corresponding author Phone : +82-(0)54-530-1213; +82-(0)42-629-8785 Fax : +82-(0)82-54-530-1218; +82-(0)42-629-8789 E-mail : jangdj@knu.ac.kr; leeja@hnu.kr 이 저자들은 본 논문에 공동으로 기여했음 224 Yong-Woo Jun, Kun-Hyung Kim, Su-Yeon Jo, Jin-A Lee and Deok-Jin Jang Analytical Science & Technology 용된다고 보고되어 있는데, 하지만 지난 몇 십 년 동안, 많은 연구가 ALA 메커니즘의 설명과 치료적 활 성 개선에 초점을 맞춰왔지만, 세포 기능과 세포생장에 대한 ALA의 효과는 아직 불분명하다. 본 연구에 서는 ALA의 약물학적 효과가 HEK293T 뿐만 아니라, HaCaT와 HeLa 세포에서 세포분열을 억제하는 것 으로 확인을 하였다. 또한, ALA가 처리된 세포에서는 세포예정사가 유도되는 것을 확인하였다. 이러한 결과들은 특정 세포에 ALA가 처리되면 세포증식을 억제하며, 특히 인간의 암세포의 죽임을 유발하는 효 과적인 약물 중 하나로써 ALA를 개선된 치료 전략으로 가능성을 제시한다.

Identification of N-terminal amino acids of ApPDE4 involved in targeting to plasma membrane and cellular morphological change by expression of N-terminal peptide

PDE plays an important role in cAMP-mediated cellular signaling within the cells. The proper targeting of each PDE is mediated by unique N-terminal of each PDE isoform. It has been recently reported that supershort-, short- and long-forms of PDE4 in Aplysia were cloned in Aplysia. Long-form of ApPDE4 was localized at plasma membrane and presynaptic terminal in Aplysia sensory neurons. However, it remains elusive which part of ApPDE4 is minimal region for the proper targeting and what are the effects on the cell functions. Here, we identified that N-terminal 13 amino acids of ApPDE4 long-form is minimal regions for the plasma membrane targeting. In addition, overexpression of ApPDE4(N20)-mRFP could induce morphological changes in HEK293T cells. Interestingly, mRFP-(PH), which selectively binds to PI4,, could induce morphological changes in similar with that by ApPDE4(N20)-mRFP. These results suggested that binding of ApPDE4(N20) to lipids including PI4, might be responsible for targeting of ApPDE4 to plasma membrane and morphological changes in HEK293T cells.