• Korean Journal of Microbiology
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• v.26 no.4
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• pp.291-297
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• 1988

Three alleles of rbsB gene, rbsB, rbsB103, and rbsB106 from the wild type, the mutant and the revertant strain, respectively, were cloned for overproduction of proteins under the control of lambda $P_{L}$ promoter. Five different species of precursor and mature ribose-binding proteins were purified to homogeneity using DEAE-Sephadex column chromatography, osmotic shock pocedure, CM-Sephadex column chromatography, and Chromatofocusing column chromaography. pI of the precursor proteins and mature proteins were determined and found to be pH 8.0 and 7.5, respectively. The purified proteins were subjected to amino acid sequencing. The results confirmed the amino acid changes deduced from the DNA sequencing.

• Korean Journal of Microbiology
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• v.26 no.4
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• pp.283-290
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• 1988

A spontaneous revertant of mutation rbsB103 that is ribose taxis-positive was characterized. This revertant was found to be export-competent in the export of ribose-binding protein shown by the disappearance of accumulated mutant precursor protein and the export of mature ribose-binding protein to the periplasm. The reversional change was shown to be in the region of risB gene that codes for the amino terminal portion of ribose-binding protein. Analysis by high-performance liquid chromatography of peptide patterns of ribose-binding proteins confirmed the relationship between the wild-type and the revertant proteins as shown for the mutant previously (Iida et al., 1985). When the processing rate of presursor proteins from the wild type and the revertant strain in vivo was compared by pulse-chase experiment, it was found that processing is less efficient than normal in the revertant. Purified mature proteins from both wild-type and revertant were subjected to amino acid sequencing. The results confirmed the amino acid changes deduced from the DNA sequencing and showed that processing of the revertant precursor occured in the correct position even though there are two different amino acids present in the signal sequence.

• Korean Journal of Microbiology
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• v.29 no.5
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• pp.270-277
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• 1991

A mutational alteration in the signal sequence of ribose-binding protein (RBP) of Escherichia coli, rbsB103, completely blocks the export of the protein to the periplasm. Intragenic suppressors for this mutation have been selected on minimal medium with ribose as a sole carbon source. Six suppressor mutations were characterized in detail and were found to have single amino acid wubstitution in the mature portion of RBP, which resulted in the mobility shift of the proteins on SDS polyacrylamide gel. Amino acid changes of these suppressors were localized in several peptides which are packed to form the N terminal domain of typical bilobate conformation of RBP. The involvement of SecB, a molecular chaperone, was investigated in the suppression of signal sequence mutation. Translocation efficency was found to be increased by the presence of SecB for all suppressors. It is likely that the folding characteristics of RBP altered by the suppressor mutations affect the affinity of interaction between SecB and RBP.

• Molecules and Cells
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• v.37 no.1
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• pp.9-16
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• 2014

ADP-ribosylation is a type of posttranslational modification catalyzed by members of the poly(ADP-ribose) (PAR) polymerase superfamily. ADP-ribosylation is initiated by PARPs, recognized by PAR binding proteins, and removed by PARG and other ADP-ribose hydrolases. These three groups of proteins work together to regulate the cellular and molecular response of PAR signaling, which is critical for a wide range of cellular and physiological functions.

• Proceedings of the Korean Biophysical Society Conference
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• 1999.06a
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• pp.43-43
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• 1999

Signal peptides of secretary proteins interact with various membranes and non-membrane components during the translocation. We investigated the interaction of signal peptides of ribose binding protein (RBP) with Escherichia coli (E.coli) signal recognition particle (SRP), SecA and lipid bilayer. Previous studies showed that the functional signal peptides inhibit the GTPase activity of E.coli SRP which consisted of F로 and 4.5S RNA.(omitted)

• Proceedings of the Korean Society of Applied Pharmacology
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• 2002.07a
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• pp.113-113
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• 2002

Phylogenetically conserved Bcl-2 family proteins play a pivotal role in the regulation of apoptosis from virus to human. Members of the Bcl-2 family consist of antiapoptotic proteins such as Bcl-2, Bcl-xL, and Bcl-w, and proapoptotic proteins such as BAD, Bax, BOD, and Bok. It has been proposed that anti- and proapoptotic Bcl-2 proteins regulate cell death by binding to each other and forming heterodimers. A delicate balance between anti- and proapoptotic Bcl-2 family members exists in each cell and the relative concentration of these two groups of proteins determines whether the cell survives or undergoes apoptosis. Mcl-1 (Myeloid cell :leukemia-1) is a member of the Bcl-2 family proteins and was originally cloned as a differentiation-induced early gene that was activated in the human myeloblastic leukemia cell line, ML-1 . Mcl-1 is expressed in a wide variety of tissues and cells including neoplastic ones. We recently identified a short splicing variant of Mcl-1 short (Mcl-IS) and designated the known Mcl-1 as Mcl-1 long (Mcl-lL). Mcl-lL protein exhibits antiapoptotic activity and possesses the BH (Bcl-2 homology) 1, BH2, BH3, and transmembrane (TM) domains found in related Bcl-2 proteins. In contrast, Mcl-1 S is a BH3 domain-only proapoptotic protein that heterodimerizes with Mcl-lL. Although both Mc1-lL and Mcl-lS proteins contain BH domains fecund in other Bcl-2 family proteins, they are distinguished by their unusually long N-terminal sequences containing PEST (proline, glutamic acid, serine, and threonine) motifs, four pairs of arginine residues, and alanine- and glycine-rich regions. In addition, the expression pattern of Mcl-1 protein is different from that of Bcl-2 suggesting a unique role (or Mcl-1 in apoptosis regulation. Tankyrasel (TRF1-interacting, ankyrin-related ADP-related polymerasel) was originally isolated based on its binding to TRF 1 (telomeric repeat binding factor-1) and contains the sterile alpha motif (SAM) module, 24 ankyrin (ANK) repeats, and the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP). Previous studies showed that tankyrasel promotes telomere elongation in human cells presumably by inhibiting TRFI though its poly(ADP-ribosyl)action by tankyrasel . In addition, tankyrasel poly(ADP-ribosyl)ates Insulin-responsive amino peptidase (IRAP), a resident protein of GLUT4 vesicles, and insulin stimulates the PARP activity of tankyrase1 through its phosphorylation by mitogen-activated protein kinase (MAPK). ADP-ribosylation is a posttranslational modification that usually results in a loss of protein activity presumably by enhancing protein turnover. However, little information is available regarding the physiological function(s) of tankyrase1 other than as a PARP enzyme. In the present study, we found tankyrasel as a specific-binding protein of Mcl-1 Overexpression of tankyrasel led to the inhibition of both the apoptotic activity of Mel-lS and the survival action of Mcl-lL in mammalian cells. Unlike other known tankyrasel-interacting proteins, tankyrasel did not poly(ADP-ribosyl)ate either of the Mcl-1 proteins despite its ability to decrease Mcl-1 proteins expression following coexpression. Therefore, this study provides a novel mechanism to regulate Mcl-1-modulated apoptosis in which tankyrasel downregulates the expression of Mcl-1 proteins without the involvement of its ADP-ribosylation activity.

• BMB Reports
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• v.39 no.3
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• pp.319-328
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• 2006

SecA protein, a cytoplasmic ATPase, plays a central role in the secretion of signal peptide-containing proteins. Here, we examined effects of signal peptide and ATP on the oligomerization, conformational change, and membrane binding of SecA. The wild-type (WT) signal peptide from the ribose-binding protein inhibited ATP binding to soluble SecA and stimulated release of ATP already bound to the protein. The signal peptide enhanced the oligomerization of soluble SecA, while ATP induced dissociation of SecA oligomer. Analysis of SecA unfolding with urea or heat revealed that the WT signal peptide induces an open conformation of soluble SecA, while ATP increased the compactness of SecA. We further obtained evidences that the signal peptide-induced oligomerization and the formation of open structure enhance the membrane binding of SecA, whereas ATP inhibits the interaction of soluble SecA with membranes. On the other hand, the complex of membrane-bound SecA and signal peptide was shown to resume nucleotide-binding activity. From these results, we propose that the translocation components affect the degree of oligomerization of soluble SecA, thereby modulating the membrane binding of SecA in early translocation pathway. A possible sequential interaction of SecA with signal peptide, ATP, and cytoplasmic membrane is discussed.

• Molecules and Cells
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• v.37 no.7
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• pp.526-531
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• 2014

Human PARP family consists of 17 members of which PARP-1 is a prominent member and plays a key role in DNA repair pathways. It has an N-terminal DNA-binding domain (DBD) encompassing the nuclear localisation signal (NLS), central automodification domain and C-terminal catalytic domain. PARP-1 accounts for majority of poly-(ADP-ribose) polymer synthesis that upon binding to numerous proteins including PARP itself modulates their activity. Reduced PARP-1 activity in ageing human samples and its deficiency leading to telomere shortening has been reported. Hence for cell survival, maintenance of genomic integrity and longevity presence of intact PARP-1 in the nucleus is paramount. Although localisation of full-length and truncated PARP-1 in PARP-1 proficient cells is well documented, subcellular distribution of PARP-1 fragments in the absence of endogenous PARP-1 is not known. Here we report the differential localisation of PARP-1 Nterminal fragment encompassing NLS in PARP-$1^{+/+}$ and PARP-$1^{-/-}$ mouse embryo fibroblasts by live imaging of cells transiently expressing EGFP tagged fragment. In PARP-$1^{+/+}$ cells the fragment localises to the nuclei presenting a granular pattern. Furthermore, it is densely packaged in the midsections of the nucleus. In contrast, the fragment localises exclusively to the cytoplasm in PARP-$1^{-/-}$ cells. Flourescence intensity analysis further confirmed this observation indicating that the N-terminal fragment requires endogenous PARP-1 for its nuclear transport. Our study illustrates the trafficking role of PARP-1 independently of its enzymatic activity and highlights the possibility that full-length PARP-1 may play a key role in the nuclear transport of its siblings and other molecules.

• Development and Reproduction
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• v.2 no.2
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• pp.165-171
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• 1998

p62 is a novel cytoplsmic protein that binds to SH2 domain of p56$^{lck}$, lymphocyte-specific protein tyrosine kinase, and the expression of p62 was observed in most tissues. In addition p62 interacts with various proteins including ubiquitin and atypical PKC isoform, indicating its diverse biological role in different tissues. However, little is known about functional connection between p62 and its binding proteins. In the present study, a novel cellular protein, p62 has been shown to bind to 14-3-3 $\tau$ isoform that is specific for T cells. Moreover, overexpression of p62 in T cells caused to delay onset of UV-induced apoptosis characterized by DNA fragmentation and breakdown of poly (ADP-ribose) polymerase (PARP). Lately, 14-3-3 proteins have been shown to mediate survival signal via interacting proapoptotic Bad protein in the Iymphocyte. These results suggested the presence of p62-mediated regulatory mechanism during apoptosis in T cells, in which activation-induced apoptotic signal could be interfered by p62 and 14-3-3 protein.n.

• Natural Product Sciences
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• v.26 no.3
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• pp.191-199
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• 2020

Fucoidan, a natural component of brown seaweed, has various biological activities such as anti-cancer activity, anti-oxidant, and anti-inflammatory against various cancer cells. However, the fucoidan has been implicated in melanoma cells via apoptosis signaling pathway. Therefore, we investigated apoptosis with fucoidan in A2058 human melanoma cells with dose- and time-dependent manners. In our results, A2058 cells viability decreased at relatively short-time and low-concentration through fucoidan. This effects of fucoidan on A2058 cells appeared to be mediated by the induction of apoptosis, as manifested by morphological changes through DNA-binding dye Hoechst 33342 staining. When a dose of 80 ㎍/mL fucoidan was treated, the cells were observed: crescent or ring-like structure, chromatin condensation, and nuclear fragmentation. With the increase at 100 ㎍/mL fucoidan, the cell membrane is intact throughout the total process, including membrane blebbing and loss of membrane integrity as well as increase of sub-G1 DNA. Furthermore, to understand the exact mechanism of fucoidan-treated in A2058 cells, western blotting was performed to detect apoptosis-related protein expression. In this study, Bcl-2 family proteins can be regulated by fucoidan, suggesting that fucoidan-induced apoptosis is modulated by intrinsic pathway. Therefore, expression of Bcl-2 and Bax may result in altered permeability, activating caspase-3 and caspase-9. And the cleaved form of poly ADP-ribose polymerase was detected in fucoidan-treated A2058 cells. These results suggest that A2058 cells are highly sensitive to growth inhibition by fucoidan via apoptosis, as evidenced by activation of extracellular signal-regulated kinases/p38/Bcl-2 family signaling, as well as alteration in caspase-9 and caspase-3.