MCL-1ES, a novel variant of MCL-1, associates with MCL-1L and induces mitochondrial cell death

Kim, Jae-Hong,Sim, Se-Hoon,Ha, Hye-Jeong,Ko, Jeong-Jae,Lee, Kangseok,Bae, Jeehyeon Elsevier 2009 FEBS letters Vol.583 No.17

<P><B>Abstract</B></P><P>Myeloid cell leukemia-1 (MCL-1L) is a pro-survival member of the BCL-2 family that promotes cell survival. In this study, we identify a new splicing variant of human MCL-1 that encodes MCL-1ES (extra short). Sequence analysis indicates that this variant results from splicing within the first coding exon of MCL-1 at a non-canonical GC–AG donor–acceptor pair. The deduced sequence of MCL-1ES encodes a protein of 197 amino acids, and the PEST (proline, glutamic acid, serine, and threonine) motifs present in MCL-1L are absent. MCL-1ES interacts with MCL-1L and induces mitochondrial cell death, suggesting that alternative splicing of <I>MCL-1</I> may control the fate of cells.</P><P><B>Structured summary</B></P><P>MINT-7255705, MINT-7255718, MINT-7255731, MINT-7255743:<I>MCL1-ES</I> (uniprotkb:Q07820-2) <I>physically interacts</I> (MI:0914) with <I>MCL1-1L</I> (uniprotkb:Q07820-1) by <I>anti tag coimmunoprecipitation</I> (MI:0007)</P><P>MINT-7255771:<I>MCL1-ES</I> (uniprotkb:Q07820-2) <I>physically interacts</I> (MI:0914) with <I>Beta actin</I> (uniprotkb:P60709) by <I>anti tag coimmunoprecipitation</I> (MI:0007)</P><P>MINT-7255781:<I>MCL1-ES</I> (uniprotkb:Q07820-2) <I>physically interacts</I> (MI:0914) with <I>GAPDH</I> (uniprotkb:P04406) by <I>anti tag coimmunoprecipitation</I> (MI:0007)</P><P>MINT-7255756:<I>MCL1-ES</I> (uniprotkb:Q07820-2) <I>physically interacts</I> (MI:0914) with <I>COX IV</I> (uniprotkb:P13073) by <I>anti tag coimmunoprecipitation</I> (MI:0007)</P>

Mcl-1 is a Binding Partner of hNoxa

박선영,김태형,Park, Sun-Young,Kim, Tae-Hyoung Korean Society of Life Science 2007 생명과학회지 Vol.17 No.8

Bcl-2 family 단백질은 세포사 조절에 매우 중요한 역할을 하며 세포사 촉진 Bcl-2 family 단백질과 세포사 억제 Bcl-2 family 단백질 사이의 균형적인 상호작용이 세포의 운명을 결정하는 주요인자이다. Bcl-2 family 단백질 중 하나인 Noxa 단백질은 p53 에 의한 전사되는 단백질로 처음 발견되었다. Noxa 단백질이 어떻게 세포사를 조절하는지를 이해하기 위해 Yeast two-hybrid 방법을 통해 Noxa 단백질과 결합하는 파트너 단백질을 검색하였고 이를 통해 세포사 억제 단백질 중 하나인 Mcl-1를 발견하였다. 사람 대장암 세포주인 HCT 116에서 Noxa 단백질과 Mcl-1 단백질이 결합하는 것을 면역침전 방법을 통하여 확인하였다. HCT 116 세포주에서 Mcl-1 단백질 과다발현은 Noxa에 의한 세포사 유도를 크게 억제하였다. Noxa 단백질 과다발현에 의한 세포사 과정에서 Mcl-1 단백질이 분해되는 것을 발견하였고 이는 caspase 억제제인 z-VAD-fmk에 의해서 억제되었다. 이는 Mcl-1 단백질이 cas-pase에 의해서 분해되는 것으로 간주된다. 결론적으로, Noxa와 Mcl-1의 결합은 세포사 과정 중 caspase에 의한 Mcl-1 단백질 분해를 유도를 매개할 수 있을 것으로 추측된다. The Bcl-2 family proteins play critical roles in regulation of apoptosis, and the balanced interaction of pro- and anti-death members is a key factor in determining the cell fate. Noxa, a BH3-only Bcl-2-family member, has been originally identified as a target gene of p53. To understand the mechanism by which human Noxa (hNoxa) regulates the cell death, we screened the hNoxa binding partner using the yeast two hybrid screening and found that anti-death protein Mcl-1 binds to hNoxa. The binding of hNoxa to Mcl-1 was confirmed by immunoprecipitation in human colon cancer cell line HCT 116 cells. Mcl-1 significantly inhibited the hNoxa-induced cell death in HCT 116 cells. During the cell death induced by hNoxa, Mcl-1 protein was degraded. Its degradation was inhibited by z-VAD-fmk, a pancaspase inhibitor, suggesting caspase is responsible for Mcl-1 degradation in response to hNoxa. Together, the results indicate that hNoxa binds to Mcl-1 that is degraded by cas-pases during hNoxa-induced cell death.

차세대 이동통신 시스템의 주파수 공유분석을 위한 개선된 MCL 방법

정우기,윤현구,조한신,임재우,육종관,박한규,Chung Woo-Ghee,Yoon Hyun-Goo,Jo Han-Shin,Lim Jae-Woo,Yook Jong-Gwan,Park Han-Kyu 한국전자파학회 2006 한국전자파학회논문지 Vol.17 No.3

본 논문에서는 차세대 이동통신 시스템의 간섭을 분석하기 위해 개선된 MCL(Advanced Minimum Coupling Loss) 방법을 제안하고 기존의 MCL 방법과 비교하였다. 제안된 방법은 OFDM(Orthogonal Frequency Division Multiplex) 기반의 차세대 이동통신 시스템의 송신 전력밀도 스펙트럼을 이용하여 간섭전력을 해석적으로 구한다. 또한 개선된 MCL 방법을 이용하여 기존의 MCL에서는 정확히 계산하기가 불가능했던 FSU(Flexible Spectrum Usage)를 사용하는 차세대 이동통신 시스템과 차세대 이동통신 후보 주파수 대역에 존재하는 고정 마이크로파 시스템과의 공유 간섭을 분석하였다. 피간섭원의 수신 안테나에 도착한 간섭 전력을 계산한 결과, 기존 MCL보다 개선된 MCL로 계산한 결과가 보다 정확했으며 그 차이는 최대 4.5 dB였다. 따라서 본 논문에서 제시한 개선된 MCL 방법은 차세대 이동통신 시스템의 공유 분석에 유용하게 사용될 수 있다고 판단된다. In this paper the analytical method, namely advanced minimum coupling loss(A-MCL), was proposed in order to analyze the coexistence of OFDM-based systems beyond 3G(B3G) with point-to point(PP) fixed service(FS) microwave systems. Our proposed method is based on a power spectral density(PSD) analysis. So it can be easily applicable to analyze the coexistence of OFDM-based systems B3G using flexible spectrum usage(FSU) with other systems, where the conventional MCL method cannot allocate transmit power partially to some subcarriers which overlap the band of a victim system. By applying the conventional MCL method and the A-MCL method, interfering power levels at the receiver of a interfered system are respectively calculated. A-MCL can calculate interference power more accurately than MCL by the maximum value of 4.5 dB. Therefore it can be concluded that our prosed method, namely A-MCL, is applicable to a sharing analysis of OFDM-based systems B3G.

Biosynthesis, Modification, and Biodegradation of Bacterial Medium-Chain-Length Polyhydroxyalkanoates

김도영,Hyung Woo Kim,정문규,이영하 한국미생물학회 2007 The journal of microbiology Vol.45 No.2

Medium-chain-length polyhydroxyalkanoates (MCL-PHAs), which have constituents with a typical chainlength of C6-C14, are polyesters that are synthesized and accumulated in a wide variety of Gram-negativebacteria, mainly pseudomonads. These biopolyesters are promising materials for various applications becausethey have useful mechanical properties and are biodegradable and biocompatible. The versatile metaboliccapacity of some Pseudomonas spp. enables them to synthesize MCL-PHAs that contain various functionalsubstituents; these MCL-PHAs are of great interest because these functional groups can improve the physicalproperties of the polymers, allowing the creation of tailor-made products. Moreover, some functional substituentscan be modified by chemical reactions to obtain more useful groups that can extend the potentialapplications of MCL-PHAs as environmentally friendly polymers and functional biomaterials for use in biomedicalfields. Although MCL-PHAs are water-insoluble, hydrophobic polymers, they can be degraded bymicroorganisms that produce extracellular MCL-PHA depolymerase. MCL-PHA-degraders are relativelyuncommon in natural environments and, to date, only a limited number of MCL-PHA depolymerases havebeen investigated at the molecular level. All known MCL-PHA depolymerases share a highly significantsimilarity in amino acid sequences, as well as several enzymatic characteristics. This paper reviews recentadvances in our knowledge of MCL-PHAs, with particular emphasis on the findings by our research group. Medium-chain-length polyhydroxyalkanoates (MCL-PHAs), which have constituents with a typical chain length of C6-C14, are polyesters that are synthesized and accumulated in a wide variety of Gram-negative bacteria, mainly pseudomonads. These biopolyesters are promising materials for various applications because they have useful mechanical properties and are biodegradable and biocompatible. The versatile metabolic capacity of some Pseudomonas spp. enables them to synthesize MCL-PHAs that contain variou s functional substituents; these MCL-PHAs are of great interest because these functional groups can improve the physical properties of the polymers, allowing the creation of tailor-made products. Moreover, some functional substituents can be modified by chemical reactions to obtain more useful groups that can extend the potential applications of MCL-PHAs as environmentally friendly polymers and functional biomaterials for use in biomedical fields. Although MCL-PHAs are water-insoluble, hydrophobic polymers, they can be degraded by microorganisms that produce extracellular MCL-PHA depolymerase. MCL-PHA-degraders are relatively uncommon in natural environments and, to date, only a limited number of MCL-PHA depolymerases have been investigated at the molecular level. All known MCL-PHA depolymerases share a highly significant similarity in amino acid sequences, as well as several enzymatic characteristics. This paper reviews recent advances in our knowledge of MCL-PHAs, with particular emphasis on the findings by our research group.

KRIBB11 accelerates Mcl-1 degradation through an HSF1-independent, Mule-dependent pathway in A549 non-small cell lung cancer cells

Kang, Min-Jung,Yun, Hye Hyeon,Lee, Jeong-Hwa Elsevier 2017 Biochemical and biophysical research communication Vol. No.

<P><B>Abstract</B></P> <P>The Bcl-2 family protein, Mcl-1 is known to have anti-apoptotic functions, and depletion of Mcl-1 by cellular stresses favors the apoptotic process. Moreover, Mcl-1 levels are frequently increased in various cancer cells, including non-small cell lung cancer (NSCLC), and is implicated in resistance to conventional chemotherapy and in cancer metastasis. In this study, we demonstrated that KRIBB11 accelerates the proteasomal degradation of Mcl-1 in the NSCLC cell line, A549. While KRIBB11 is an inhibitor of HSF1, we found that KRIBB11 induced Mcl-1 degradation in an HSF1-independent manner. Furthermore, this process was triggered via increase ubiquitination by the E3 ligase, Mule, rather than via de-ubiquitination by USP9X. Additionally, we found that Mcl-1 levels were only transiently reduced by KRIBB11: Mcl-1 levels were gradually restored as KRIBB11 activity diminished. However, we found that this effect was blocked in BIS (Bcl-2 interacting cell death suppressor, also called BAG3)-depleted cells, and that BIS prevents Mcl-1 from undergoing HSP70-driven proteasomal degradation, through an interaction with HSP70. Taken together, our results suggest that targeting Mcl-1 with KRIBB11 treatment, while simultaneously downregulating BIS, could be a therapeutic strategy in NSCLC.</P> <P><B>Highlights</B></P> <P> <UL> <LI> KRIBB11 accelerates Mcl-1 degradation in HSF1-independent manner. </LI> <LI> KRIBB11 triggers proteasomal degradation of Mcl-1 via E3 ligase Mule. </LI> <LI> Downregulation of BIS expression facilitates the continuous destabilization of Mcl-1 following KRIBB11 treatment. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

A small hairpin RNA targeting myeloid cell leukemia-1 enhances apoptosis in host macrophages infected with Mycobacterium tuberculosis

Fei-yu Wang,Yu-qing Zhang,Xin-min Wang,Chan Wang,Xiao-fang Wang,Jiang-dong Wu,Fang Wu,Wan-jiang Zhang,Le Zhang 한국미생물학회 2016 The journal of microbiology Vol.54 No.4

Myeloid cell leukemia-1 (Mcl-1) plays an important role in various cell survival pathways. Some studies indicated that the expression of Mcl-1 was upregulated in host cells during infection with the virulent Mycobacterium tuberculosis strain, H37Rv. The present study was designed to investigate the effect of inhibiting Mcl-1 expression both in vivo and in vitro on apoptosis of host macrophages infected with M. tuberculosis using a small hairpin (sh)RNA. Mcl-1 expression was detected by the real time-polymerase chain reaction, western blotting, and immunohistochemistry. Flow cytometry and transmission electron microscopy were used to measure host macrophage apoptosis. We found elevated Mcl-1 levels in host macrophages infected with M. tuberculosis H37Rv. The expression of Mcl-1 was downregulated efficiently in H37Rv-infected host macrophages using shRNA. Knockdown of Mcl-1 enhanced the extent of apoptosis in H37Rv-infected host macrophages significantly. The increased apoptosis correlated with a decrease in M. tuberculosis colony forming units recovered from H37Rv-infected cells that were treated with Mcl-1-shRNA. Reducing Mcl-1 accumulation by shRNA also reduced accumulation of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax, in H37Rv-infected host macrophages. Our results showed that specific knockdown of Mcl-1 expression increased apoptosis of host macrophages significantly and decreased the intracellular survival of a virulent strain of M. tuberculosis. These data indicate that interference with Mcl-1 expression may provide a new avenue for tuberculosis therapy.

세포사멸을 유도하는 새로운 단백질인 MCL-1ES BH3M의 클로닝 및 기능연구

김재홍,박미라,하혜정,이강석,배지현 한국발생생물학회 2008 발생과 생식 Vol.12 No.3

BCL-2 family members are essential protein for the regulation of cell death and survival consisting both anti- apoptotic and pro-apoptotic proteins. In the present study, we designed and cloned a new apoptotic molecule MCL-1ES BH3M coding a modified protein of MCL-1L. Compared to MCL-1L protein, MCL-1ES BH3M lacks the PEST motifs known to be involved in MCL-1L protein degradation and has seven mutated residues in BH3 domain critical for dimerization with BCL-2 family members. Overexpression of Mcl-1ES BH3M induced death of different cells, and its cell killing effect was not blocked by forced expression of the pro-survival protein MCL-1L. Expression of MCL-1ES BH3M protein led to the activation of caspase 9 and caspase3, suggesting apoptotic cell death, and confocal fluorescent microscopic analyses showed that MCL-1ES BH3M was partially localized in mitochondria. In conclusion, we reported a new apoptotic molecule and determined its cell death activity in cells

Biosynthesis, Modification, and Biodegradation of Bacterial Medium-Chain-Length Polyhydroxyalkanoates

Kim, Do-Young,Kim, Hyung-Woo,Chung, Moon-Gyu,Rhee, Young-Ha The Microbiological Society of Korea 2007 The journal of microbiology Vol.45 No.2

Medium-chain-length polyhydroxyalkanoates (MCL-PHAs), which have constituents with a typical chain length of $C_{6}-C_{14}$, are polyesters that are synthesized and accumulated in a wide variety of Gram-negative bacteria, mainly pseudomonads. These biopolyesters are promising materials for various applications because they have useful mechanical properties and are biodegradable and biocompatible. The versatile metabolic capacity of some Pseudomonas spp. enables them to synthesize MCL-PHAs that contain various functional substituents; these MCL-PHAs are of great interest because these functional groups can improve the physical properties of the polymers, allowing the creation of tailor-made products. Moreover, some functional substituents can be modified by chemical reactions to obtain more useful groups that can extend the potential applications of MCL-PHAs as environmentally friendly polymers and functional biomaterials for use in biomedical fields. Although MCL-PHAs are water-insoluble, hydrophobic polymers, they can be degraded by microorganisms that produce extracellular MCL-PHA depolymerase. MCL-PHA-degraders are relatively uncommon in natural environments and, to date, only a limited number of MCL-PHA depolymerases have been investigated at the molecular level. All known MCL-PHA depolymerases share a highly significant similarity in amino acid sequences, as well as several enzymatic characteristics. This paper reviews recent advances in our knowledge of MCL-PHAs, with particular emphasis on the findings by our research group.

Down-Regulation of Mcl-1 by Small Interference RNA Induces Apoptosis and Sensitizes HL-60 Leukemia Cells to Etoposide

Karami, Hadi,Baradaran, Behzad,Esfehani, Ali,Sakhinia, Masoud,Sakhinia, Ebrahim Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.2

Background: Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs. Myeloid cell leukemia-1 (Mcl-1), a death-inhibiting protein that regulates apoptosis, has been shown to be overexpressed in numerous malignancies. In addition, it has been demonstrated that the expression level of the Mcl-1 gene increases at the time of leukemic relapse following chemotherapy. The aim of this study was to target Mcl-1 by small interference RNA (siRNA) and analyze its effects on survival and chemosensitivity of acute myeloid leukemia cell line HL-60. Materials and Methods: siRNA transfection was performed with a liposome approach. The expression levels of mRNA and protein were measured by real-time quantitative PCR and Western blot analysis, respectively. Trypan blue assays were performed to evaluate tumor cell growth after siRNA transfection. The cytotoxic effects of Mcl-1 siRNA (siMcl-1) and etoposide were determined using MTT assay on their own and in combination. Apoptosis was quantified using a DNA-histone ELISA assay. Results: Transfection with siMcl-1 significantly suppressed the expression of Mcl-1 mRNA and protein in a time-dependent manner, resulting in strong growth inhibition and spontaneous apoptosis. Surprisingly, pretreatment with siMcl-1 synergistically enhanced the cytotoxic effect of etoposide. Furthermore, Mcl-1 down-regulation significantly increased apoptosis sensitivity to etoposide. No significant biological effects were observed with negative control siRNA treatment. Conclusions: Our results suggest that specific suppression of Mcl-1 by siRNA can effectively induce apoptosis and overcome chemoresistance of leukemic cells. Therefore, siMcl-1 may be a potent adjuvant in leukemia chemotherapy.

난소암 세포에서 IEX-1과 MCL-1 단백질들의 세포사멸 기능에 관한 상호작용

윤성민,나순영,이강석,배지현,김홍만 한국발생생물학회 2010 발생과 생식 Vol.14 No.2

Apoptosis is a crucial mechanism for the proper regulation of homeostasis. BCL-2 family proteins are key molecules which control cellular survival and apoptosis. MCL-1(myeloid cell leukemia-1) is a pro-survival member of BCL- 2 family that promotes the survival of cells, and is highly expressed in diverse cancers including ovarian cancer, leukemia, and cervical cancer. Previously we identified IEX-1(immediate early response gene X-1) as a binding partner of MCL- 1. In the present study, we demonstrated that overexpression of IEX-1 induces apoptosis of ovarian cancer cells. Moreover, IEX-1 significantly attenuated the pro-survival function of MCL-1 in these cells. Also, IEX-1-induced cell death activity was able to be modulated by changes in the expression level of MCL-1. Thus, these results suggest that both IEX-1 and MCL-1 modulate each other’s function controlling cellular survival and death and the inhibitory activity of IEX-1 toward MCL-1 may be applied for the development of chemotherapeutics.