Effects of a Phosphomimetic Mutant of RAP80 on Linear Polyubiquitin Binding Probed by Calorimetric Analysis

Thach, Thanh Trung,Jee, Jun-Goo,Lee, Sang-Ho Korean Chemical Society 2012 Bulletin of the Korean Chemical Society Vol.33 No.4

RAP80 plays a key role in DNA damage responses by recognizing K63-linked polyubiquitin moieties through its two ubiquitin-interacting motif (UIM) domains. The linker between the two UIMs possesses a phosphorylation site, but the relationship between phosphorylation and polyubiquitin recognition remains elusive. We investigated the interaction between a phosphorylation-mimic RAP80 mutant S101E and linear polyubiquitins, structurally equivalent to the K63-linked ones, using isothermal titration calorimetry (ITC). ITC analysis revealed differential binding affinities for linear tetraubiquitin by otherwise equivalent UIMs in S101E. Mutational analysis supported such differential polyubiquitin recognition by S101E. Our results suggest a potential crosstalk between polyubiquitin recognition and phosphorylation in RAP80.

Molecular Determinants of Polyubiquitin Recognition by Continuous Ubiquitin-Binding Domains of Rad18

Thach, Trung Thanh,Lee, Namsoo,Shin, Donghyuk,Han, Seungsu,Kim, Gyuhee,Kim, Hongtae,Lee, Sangho American Chemical Society 2015 Biochemistry Vol.54 No.12

<P>Rad18 is a key factor in double-strand break DNA damage response (DDR) pathways via its association with K63-linked polyubiquitylated chromatin proteins through its bipartite ubiquitin-binding domains UBZ and LRM with extra residues between them. Rad18 binds K63-linked polyubiquitin chains as well as K48-linked ones and monoubiquitin. However, the detailed molecular basis of polyubiquitin recognition by UBZ and LRM remains unclear. Here, we examined the interaction of Rad18(201–240), including UBZ and LRM, with linear polyubiquitin chains that are structurally similar to the K63-linked ones. Rad18(201–240) binds linear polyubiquitin chains (Ub<SUB>2</SUB>–Ub<SUB>4</SUB>) with affinity similar to that of a K63-linked one for diubiquitin. <I>Ab initio</I> modeling suggests that LRM and the extra residues at the C-terminus of UBZ (residues 227–237) likely form a continuous helix, termed the “extended LR motif” (ELRM). We obtained a molecular envelope for Rad18 UBZ-ELRM:linear Ub<SUB>2</SUB> by small-angle X-ray scattering and derived a structural model for the complex. The Rad18:linear Ub<SUB>2</SUB> model indicates that ELRM enhances the binding of Rad18 with linear polyubiquitin by contacting the proximal ubiquitin moiety. Consistent with the structural analysis, mutational studies showed that residues in ELRM affect binding with linear Ub<SUB>2</SUB>, not monoubiquitin. <I>In cell</I> data support the idea that ELRM is crucial in the localization of Rad18 to DNA damage sites. Specifically, E227 seems to be the most critical in polyubiquitin binding and localization to nuclear foci. Finally, we reveal that the ubiquitin-binding domains of Rad18 bind linear Ub<SUB>2</SUB> more tightly than those of RAP80, providing a quantitative basis for blockage of RAP80 at DSB sites. Taken together, our data demonstrate that Rad18(201–240) forms continuous ubiquitin-binding domains, comprising UBZ and ELRM, and provides a structural framework for polyubiquitin recognition by Rad18 in the DDR pathway at a molecular level.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/2015/bichaw.2015.54.issue-12/bi5012546/production/images/medium/bi-2014-012546_0008.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/bi5012546'>ACS Electronic Supporting Info</A></P>

Effects of a Phosphomimetic Mutant of RAP80 on Linear Polyubiquitin Binding Probed by Calorimetric Analysis

Thanh Trung Thach,지준구,이상호 대한화학회 2012 Bulletin of the Korean Chemical Society Vol.33 No.4

RAP80 plays a key role in DNA damage responses by recognizing K63-linked polyubiquitin moieties through its two ubiquitin-interacting motif (UIM) domains. The linker between the two UIMs possesses a phosphorylation site, but the relationship between phosphorylation and polyubiquitin recognition remains elusive. We investigated the interaction between a phosphorylation-mimic RAP80 mutant S101E and linear polyubiquitins, structurally equivalent to the K63-linked ones, using isothermal titration calorimetry (ITC). ITC analysis revealed differential binding affinities for linear tetraubiquitin by otherwise equivalent UIMs in S101E. Mutational analysis supported such differential polyubiquitin recognition by S101E. Our results suggest a potential crosstalk between polyubiquitin recognition and phosphorylation in RAP80.

Betaine reduces cellular melanin content via suppression of microphthalmia-associated transcription factor in B16-F1 murine melanocytes

조보람,전희진,Trung Thanh Thach,Chunyan Wu,이성준 한국식품과학회 2017 Food Science and Biotechnology Vol.26 No.5

Long-term topical skin care by traditional antimelanogenic agents can raise several safety concerns. An understanding of the molecular mechanisms of active compounds on melanogenesis is, therefore, necessary to address pigmentation issues. Here we revealed that stimulation with 1 mM betaine, an abundant component in rice bran, significantly reduced 21% of intracellular melanin content by suppressing tyrosinase activity and microphthalmia- associated transcription factor (MITF) expression in B16-F1 murine melanocytes. The expression of MITF was suppressed at both mRNA and protein levels by 43 and 44%, respectively. Subsequently, the betaine-stimulated melanocytes showed inhibition of PKA-CREB signaling axis but activation of extracellular-signal-regulated kinase and AKT-GSK3b signaling pathways. This inhibition and activation led to downregulation of MITF expression at both the transcriptional and post-translational levels to suppress melanin synthesis. These findings collectively suggested that betaine is a potential anti-melanogenic compound for functional foods and cosmetics.

Adenylate kinase potentiates the capsular polysaccharide by modulating Cps2D in Streptococcus pneumoniae D39

Prachetash Ghosh,Truc Thanh Luong,Masaud Shah,Trung Thanh Thach,최상돈,이상호,이동권 생화학분자생물학회 2018 Experimental and molecular medicine Vol.50 No.-

Streptococcus pneumoniae is a polysaccharide-encapsulated bacterium. The capsule thickens during blood invasion compared with the thinner capsules observed in asymptomatic nasopharyngeal colonization. However, the underlying mechanism regulating differential CPS expression remains unclear. CPS synthesis requires energy that is supplied by ATP. Previously, we demonstrated a correlation between ATP levels and adenylate kinase in S. pneumoniae (SpAdK). A dose-dependent induction of SpAdK in serum was also reported. To meet medical needs, this study aimed to elucidate the role of SpAdK in the regulation of CPS production. CPS levels in S. pneumoniae type 2 (D39) increased proportionally with SpAdK levels, but they were not related to pneumococcal autolysis. Moreover, increased SpAdK levels resulted in increased total tyrosine kinase Cps2D levels and phosphorylated Cps2D, which is a regulator of CPS synthesis in the D39 strain. Our results also indicated that the SpAdK and Cps2D proteins interact in the presence of Mg-ATP. In addition, in silico analysis uncovered the mechanism behind this protein–protein interaction, suggesting that SpAdK binds with the Cps2D dimer. This established the importance of the ATP-binding domain of Cps2D. Taken together, the biophysical interaction between SpAdK and Cps2D plays an important role in enhancing Cps2D phosphorylation, which results in increased CPS synthesis.

Peptide-Programmable Nanoparticle Superstructures with Tailored Electrocatalytic Activity

Kang, Eun Sung,Kim, Yong-Tae,Ko, Young-Seon,Kim, Nam Hyeong,Cho, Geonhee,Huh, Yang Hoon,Kim, Ji-Hun,Nam, Jiyoung,Thach, Trung Thanh,Youn, David,Kim, Young Dok,Yun, Wan Soo,DeGrado, William F.,Kim, Sun American Chemical Society 2018 ACS NANO Vol.12 No.7

<P>Biomaterials derived <I>via</I> programmable supramolecular protein assembly provide a viable means of constructing precisely defined structures. Here, we present programmed superstructures of AuPt nanoparticles (NPs) on carbon nanotubes (CNTs) that exhibit distinct electrocatalytic activities with respect to the nanoparticle positions <I>via</I> rationally modulated peptide-mediated assembly. <I>De novo</I> designed peptides assemble into six-helix bundles along the CNT axis to form a suprahelical structure. Surface cysteine residues of the peptides create AuPt-specific nucleation site, which allow for precise positioning of NPs onto helical geometries, as confirmed by 3-D reconstruction using electron tomography. The electrocatalytic model system, i.e., AuPt for oxygen reduction, yields electrochemical response signals that reflect the controlled arrangement of NPs in the intended assemblies. Our design approach can be expanded to versatile fields to build sophisticated functional assemblies.</P> [FIG OMISSION]</BR>