Structural insights into the oligomerization of FtsH periplasmic domain from <i>Thermotoga maritima</i>

An, Jun Yop,Sharif, Humayun,Kang, Gil Bu,Park, Kyung Jin,Lee, Jung-Gyu,Lee, Sukyeong,Jin, Mi Sun,Song, Ji-Joon,Wang, Jimin,Eom, Soo Hyun Elsevier 2018 Biochemical and biophysical research communication Vol.495 No.1

<P><B>Abstract</B></P> <P>Prompt removal of misfolded membrane proteins and misassembled membrane protein complexes is essential for membrane homeostasis. However, the elimination of these toxic proteins from the hydrophobic membrane environment has high energetic barriers. The transmembrane protein, FtsH, is the only known ATP-dependent protease responsible for this task. The mechanisms by which FtsH recognizes, unfolds, translocates, and proteolyzes its substrates remain unclear. The structure and function of the ATPase and protease domains of FtsH have been previously characterized while the role of the FtsH periplasmic domain has not clearly identified. Here, we report the 1.5–1.95 Å resolution crystal structures of the <I>Thermotoga maritima</I> FtsH periplasmic domain (<I>tm</I>PD) and describe the dynamic features of <I>tm</I>PD oligomerization.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We report the 1.5–1.95A resolution crystal structures of the <I>Thermotoga maritima</I> FtsH periplasmic domain (<I>tm</I>PD). </LI> <LI> Acidic residues at the pore region of <I>tm</I>PD are highly conserved among only the thermophilic, FtsH homologs. </LI> <LI> The negative charge repulsion of <I>tm</I>PD destabilizes its hexamerization. </LI> <LI> Dynamical features of the PD in hexameric full-length FtsH may be relevant for the substrate recognition. </LI> </UL> </P>

Inactivation of the PtdIns(4)P phosphatase Sac1 at the Golgi by H₂O₂ produced via Ca²⁺-dependent Duox in EGF-stimulated cells

Park, Sujin,Lim, Jung Mi,Park, Seon Hwa,Kim, Suree,Heo, Sukyeong,Balla, Tamas,Jeong, Woojin,Rhee, Sue Goo,Kang, Dongmin Elsevier 2019 FREE RADICAL BIOLOGY AND MEDICINE Vol.131 No.-

<P><B>Abstract</B></P> <P>Binding of epidermal growth factor (EGF) to its cell surface receptor induces production of H<SUB>2</SUB>O<SUB>2</SUB>, which serves as an intracellular messenger. We have shown that exogenous H<SUB>2</SUB>O<SUB>2</SUB> reversibly inactivates the phosphatidylinositol 4-phosphate [PtdIns(4)P] phosphatase Sac1 (suppressor of actin 1) at the Golgi complex of mammalian cells by oxidizing its catalytic cysteine residue and thereby increases both the amount of Golgi PtdIns(4)P and the rate of protein secretion. Here we investigated the effects of EGF on Sac1 oxidation and PtdIns(4)P abundance at the Golgi in A431 cells. EGF induced a transient increase in Golgi PtdIns(4)P as well as a transient oxidation of Sac1 in a manner dependent on elevation of the intracellular Ca<SUP>2+</SUP> concentration and on H<SUB>2</SUB>O<SUB>2</SUB>. Oxidation of Sac1 occurred at the Golgi, as revealed with the use of the Golgi-confined Sac1-K2A mutant. Knockdown of Duox enzymes implicated these Ca<SUP>2+</SUP>-dependent members of the NADPH oxidase family as the major source of H<SUB>2</SUB>O<SUB>2</SUB> for Sac1 oxidation. Expression of a Golgi-targeted H<SUB>2</SUB>O<SUB>2</SUB> probe revealed transient EGF-induced H<SUB>2</SUB>O<SUB>2</SUB> production at this organelle. Our findings have thus uncovered a previously unrecognized EGF signaling pathway that links intracellular Ca<SUP>2+</SUP> mobilization to events at the Golgi including Duox activation, H<SUB>2</SUB>O<SUB>2</SUB> production, Sac1 oxidation, and PtdIns(4)P accumulation.</P> <P><B>Highlights</B></P> <P> <UL> <LI> EGF elicits transient H<SUB>2</SUB>O<SUB>2</SUB> production at the Golgi by Ca<SUP>2+</SUP>-dependent Duox. </LI> <LI> This H<SUB>2</SUB>O<SUB>2</SUB> inactivates Sac1 at the Golgi by transiently oxidizing its catalytic Cys. </LI> <LI> Transient inactivation of Sac1 results in a transient increase in Golgi PtdIns(4)P. </LI> <LI> This new EGF signaling pathway links Ca<SUP>2+</SUP> to Golgi PtdIns(4)P via Duox-H<SUB>2</SUB>O<SUB>2</SUB>-Sac1. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Heterogeneous Exhaustion Status of Tumor-Infiltrating CD8 T Cells Determines Distinct Subgroups of Hepatocellular Carcinoma Patients

( Hyung-don Kim ),( Gi-won Song ),( Seongyeol Park ),( Min Kyung Jung ),( Kijong Yi ),( Min Hwan Kim ),( Hyo Jeong Kang ),( Chang-hoon Yoo ),( Kyung Hwan Kim ),( Sukyeong Eo ),( Deok-bog Moon ),( Seun 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: T-cell exhaustion, or an impaired capacity to secrete cytokines and proliferate with overexpression of immune checkpoint receptors, occurs during chronic viral infections but has also been observed in tumors, including hepatocellular carcinomas (HCCs). We investigated features of exhaustion in CD8⁺ T cells isolated from HCC specimens. Methods: We obtained HCC specimens, along with adjacent non-tumor tissues and blood samples, from 79 patients who underwent surgical resection at Asan Medical Center (in Seoul, Korea) from April 2016 through December 2017. Intrahepatic lymphocytes and tumor-infiltrating T cells were analyzed by flow cytometry. Tumor-infiltrating CD8⁺ T cells were sorted by flow cytometry into populations based on expression level of programmed cell death 1 (PDCD1 or PD1): PD1-high, PD1-intermediate, and PD1-negative. Sorted cells were analyzed by RNA-seq. Proliferation and production of interferon gamma (IFNG) and tumor necrosis factor (TNF) by CD8⁺ T cells were measured in response to anti-CD3 and antibodies against immune checkpoint receptors including PD1, hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3), lymphocyte activating 3 (LAG3), or isotype control. Tumor-associated antigen-specific CD8⁺ T cells were identified using HLA-A^*0201 dextramers. Results: PD1-high, PD1-intermediate, and PD1-negative CD8+ T cells from HCCs had distinct gene expression profiles. PD1-high cells expressed higher levels of genes that regulate T-cell exhaustion than PD1-intermediate cells. PD1-high cells expressed TIM3 and LAG3, and a low proportion was TCF1⁺, TBET^high/ eomesodermin^low, and CD127+. PD1-high cells produced the lowest amounts of IFNG and TNF upon anti-CD3 stimulation. Differences in proportions of PD1-high CD8⁺ T cells led to the identification of 2 subgroups of HCCs; HCCs with a larger proportion of PD1-high cells were more aggressive than HCCs with a smaller proportion. Incubation of CD8⁺ T cells from HCCs with high proportions of PD1-high cells with antibodies against PD1 and TIM3 or LAG3 further restored proliferation and production of IFNG and TNF in response to anti-CD3. Conclusions: We found HCC specimens to contain CD8⁺ T cells that express different levels of PD1. HCCs with high proportions of PD1-high CD8⁺ T cells express TIM3 and/or LAG3 and produce low levels of IFNG and TNF in response to anti-CD3. Incubation of these cells with antibodies against PD1 and TIM3 or LAG3 further restore proliferation and production of cytokines; HCCs with high proportions of PD1-high CD8⁺ T cells might be more susceptible to combined immune checkpoint blockade-based therapies.

Association Between Expression Level of PD1 by Tumor-Infiltrating CD8⁺ T Cells and Features of Hepatocellular Carcinoma

Kim, Hyung-Don,Song, Gi-Won,Park, Seongyeol,Jung, Min Kyung,Kim, Min Hwan,Kang, Hyo Jeong,Yoo, Changhoon,Yi, Kijong,Kim, Kyung Hwan,Eo, Sukyeong,Moon, Deok-Bog,Hong, Seung-Mo,Ju, Young Seok,Shin, Eui- Elsevier 2018 Gastroenterology Vol.155 No.6

<P><B>Background & Aims</B></P> <P>T-cell exhaustion, or an impaired capacity to secrete cytokines and proliferate with overexpression of immune checkpoint receptors, occurs during chronic viral infections but has also been observed in tumors, including hepatocellular carcinomas (HCCs). We investigated features of exhaustion in CD8<SUP>+</SUP> T cells isolated from HCC specimens.</P> <P><B>Methods</B></P> <P>We obtained HCC specimens, along with adjacent nontumor tissues and blood samples, from 90 patients who underwent surgical resection at Asan Medical Center (Seoul, Korea) from April 2016 through April 2018. Intrahepatic lymphocytes and tumor-infiltrating T cells were analyzed by flow cytometry. Tumor-infiltrating CD8<SUP>+</SUP> T cells were sorted by flow cytometry into populations based on expression level of programmed cell death 1 (PDCD1 or PD1): PD1-high, PD1-intermediate, and PD1-negative. Sorted cells were analyzed by RNA sequencing. Proliferation and production of interferon gamma (IFNG) and tumor necrosis factor (TNF) by CD8<SUP>+</SUP> T cells were measured in response to anti-CD3 and antibodies against immune checkpoint receptors including PD1, hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3), lymphocyte activating 3 (LAG3), or isotype control. Tumor-associated antigen-specific CD8<SUP>+</SUP> T cells were identified using HLA-A*0201 dextramers. PDL1 expression on tumor tissue was assessed by immunohistochemistry.</P> <P><B>Results</B></P> <P>PD1-high, PD1-intermediate, and PD1-negative CD8<SUP>+</SUP> T cells from HCCs had distinct gene expression profiles. PD1-high cells expressed higher levels of genes that regulate T-cell exhaustion than PD1-intermediate cells. PD1-high cells expressed TIM3 and LAG3, and low proportions of TCF1<SUP>+</SUP>, TBET<SUP>high</SUP>/eomesodermin<SUP>low</SUP>, and CD127<SUP>+</SUP>. PD1-high cells produced the lowest amounts of IFNG and TNF upon anti-CD3 stimulation. Differences in the PD1 expression patterns of CD8<SUP>+</SUP> T cells led to the identification of 2 subgroups of HCCs: HCCs with a discrete population of PD1-high cells were more aggressive than HCCs without a discrete population of PD1-high cells. HCCs with a discrete population of PD1-high cells had higher levels of predictive biomarkers of response to anti-PD1 therapy. Incubation of CD8<SUP>+</SUP> T cells from HCCs with a discrete population of PD1-high cells with antibodies against PD1 and TIM3 or LAG3 further restored proliferation and production of IFNG and TNF in response to anti-CD3.</P> <P><B>Conclusions</B></P> <P>We found HCC specimens to contain CD8<SUP>+</SUP> T cells that express different levels of PD1. HCCs with a discrete population of PD1-high CD8<SUP>+</SUP> T cells express TIM3 and/or LAG3 and produce low levels of IFNG and TNF in response to anti-CD3. Incubation of these cells with antibodies against PD1 and TIM3 or LAG3 further restore proliferation and production of cytokines; HCCs with a discrete population of PD1-high CD8<SUP>+</SUP> T cells might be more susceptible to combined immune checkpoint blockade–based therapies.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Transcriptome-based deep learning analysis identifies drug candidates targeting protein synthesis and autophagy for the treatment of muscle wasting disorder

Lee Min Hak,Lee Bada,Park Se Eun,Yang Ga Eul,Cheon Seungwoo,Lee Dae Hoon,Kang Sukyeong,Sun Ye Ji,Kim Yongjin,Jung Dong-sub,Kim Wonwoo,Kang Jihoon,Kim Yi Rang,Choi Jin Woo 생화학분자생물학회 2024 Experimental and molecular medicine Vol.56 No.-

Sarcopenia, the progressive decline in skeletal muscle mass and function, is observed in various conditions, including cancer and aging. The complex molecular biology of sarcopenia has posed challenges for the development of FDA-approved medications, which have mainly focused on dietary supplementation. Targeting a single gene may not be sufficient to address the broad range of processes involved in muscle loss. This study analyzed the gene expression signatures associated with cancer formation and 5-FU chemotherapy-induced muscle wasting. Our findings suggest that dimenhydrinate, a combination of 8-chlorotheophylline and diphenhydramine, is a potential therapeutic for sarcopenia. In vitro experiments demonstrated that dimenhydrinate promotes muscle progenitor cell proliferation through the phosphorylation of Nrf2 by 8-chlorotheophylline and promotes myotube formation through diphenhydramine-induced autophagy. Furthermore, in various in vivo sarcopenia models, dimenhydrinate induced rapid muscle tissue regeneration. It improved muscle regeneration in animals with Duchenne muscular dystrophy (DMD) and facilitated muscle and fat recovery in animals with chemotherapy-induced sarcopenia. As an FDA-approved drug, dimenhydrinate could be applied for sarcopenia treatment after a relatively short development period, providing hope for individuals suffering from this debilitating condition.