Trametinib potentiates TRAIL‐induced apoptosis via FBW7‐dependent Mcl‐1 degradation in colorectal cancer cells

Trametinib is a MEK1/2 inhibitor and exerts anticancer activity against a variety of cancers. However, the effect of Trametinib on colorectal cancer (CRC) is not well understood. In the current study, our results demonstrate the ability of sub‐toxic doses of Trametinib to enhance TRAIL‐mediated apoptosis in CRC cells. Our findings also indicate that Trametinib and TRAIL activate caspase‐dependent apoptosis in CRC cells. Moreover, Mcl‐1 overexpression can reduce apoptosis in CRC cells treated with Trametinib with or without TRAIL. We further demonstrate that Trametinib degrades Mcl‐1 through the proteasome pathway. In addition, GSK‐3β phosphorylates Mcl‐1 at S159 and promotes Mcl‐1 degradation. The E3 ligase FBW7, known to polyubiquitinate Mcl‐1, is involved in Trametinib‐induced Mcl‐1 degradation. Taken together, these results provide the first evidence that Trametinib enhances TRAIL‐mediated apoptosis through FBW7‐dependent Mcl‐1 ubiquitination and degradation.     

A novel BH3 mimetic efficiently induces apoptosis in melanoma cells through direct binding to anti‐apoptotic Bcl‐2 family proteins,including phosphorylated Mcl‐1

The Bcl‐2 family modulates sensitivity to chemotherapy in many cancers, including melanoma, in which the RAS/BRAF/MEK/ERK pathway is constitutively activated. Mcl‐1, a major anti‐apoptotic protein in the Bcl‐2 family, is extensively expressed in melanoma and contributes to melanoma's well‐documented chemoresistance. Here, we provide the first evidence that Mcl‐1 phosphorylation at T163 by ERK1/2 and JNK is associated with the resistance of melanoma cell lines to the existing BH3 mimetics gossypol, S1 and ABT‐737, and a novel anti‐apoptotic mechanism of phosphorylated Mcl‐1 (pMcl‐1) is revealed. pMcl‐1 antagonized the known BH3 mimetics by sequestering pro‐apoptotic proteins that were released from Bcl‐2/Mcl‐1. Furthermore, an anthraquinone BH3 mimetic, compound 6, was identified to be the first small molecule to that induces endogenous apoptosis in melanoma cells by directly binding Bcl‐2, Mcl‐1, and pMcl‐1 and disrupting the heterodimers of these proteins. Although compound 6 induced upregulation of the pro‐apoptotic protein Noxa, its apoptotic induction was independent of Noxa. These data reveal the promising therapeutic potential of targeting pMcl‐1 to treat melanoma. Compound 6 is therefore a potent drug that targets pMcl‐1 in melanoma.     

Reactive oxygen species‐mediated endoplasmic reticulum stress response induces apoptosis of Mycobacterium avium‐infected macrophages by activating regulated IRE1‐dependent decay pathway

Mycobacterium avium, a slow‐growing nontuberculous mycobacterium, causes fever, diarrhoea, loss of appetite, and weight loss in immunocompromised people. We have proposed that endoplasmic reticulum (ER) stress‐mediated apoptosis plays a critical role in removing intracellular mycobacteria. In the present study, we investigated the role of the regulated IRE1‐dependent decay (RIDD) pathway in macrophages during M. avium infection based on its role in the regulation of gene expression. The inositol‐requiring enzyme 1 (IRE1)/apoptosis signal‐regulating kinase 1 (ASK1)/c‐Jun N‐terminal kinase (JNK) signalling pathway was activated in macrophages after infection with M. avium. The expression of RIDD‐associated genes, such as Bloc1s1 and St3gal5, was decreased in M. avium‐infected macrophages. Interestingly, M. avium‐induced apoptosis was significantly suppressed by pretreatment with irestatin (inhibitor of IRE1α) and 4μ8c (RIDD blocker). Macrophages pretreated with N‐acetyl cysteine (NAC) showed decreased levels of reactive oxygen species (ROS), IRE1α, and apoptosis after M. avium infection. The expression of Bloc1s1 and St3gal5 was increased in NAC‐pretreated macrophages following infection with M. avium. Growth of M. avium was significantly increased in irestatin‐, 4μ8c‐, and NAC‐treated macrophages compared with the control. The data indicate that the ROS‐mediated ER stress response induces apoptosis of M. avium‐infected macrophages by activating IRE1α‐RIDD. Thus, activation of IRE1α suppresses the intracellular survival of M. avium in macrophages.     

Atypical APC/C‐dependent degradation of Mcl‐1 provides an apoptotic timer during mitotic arrest

The initiation of apoptosis in response to the disruption of mitosis provides surveillance against chromosome instability. Here, we show that proteolytic destruction of the key regulator Mcl‐1 during an extended mitosis requires the anaphase‐promoting complex or cyclosome (APC/C) and is independent of another ubiquitin E3 ligase, SCF^Fbw7. Using live‐cell imaging, we show that the loss of Mcl‐1 during mitosis is dependent on a D box motif found in other APC/C substrates, while an isoleucine‐arginine (IR) C‐terminal tail regulates the manner in which Mcl‐1 engages with the APC/C, converting Mcl‐1 from a Cdc20‐dependent and checkpoint‐controlled substrate to one that is degraded independently of checkpoint strength. This mechanism ensures a relatively slow but steady rate of Mcl‐1 degradation during mitosis and avoids its catastrophic destruction when the mitotic checkpoint is satisfied, providing an apoptotic timer that can distinguish a prolonged mitotic delay from normal mitosis. Importantly, we also show that inhibition of Cdc20 promotes mitotic cell death more effectively than loss of APC/C activity through differential effects on Mcl‐1 degradation, providing an improved strategy to kill cancer cells.     

Phosphorylation of Mcl‐1 by CDK1–cyclin B1 initiates its Cdc20‐dependent destruction during mitotic arrest

The balance between cell cycle progression and apoptosis is important for both surveillance against genomic defects and responses to drugs that arrest the cell cycle. In this report, we show that the level of the human anti‐apoptotic protein Mcl‐1 is regulated during the cell cycle and peaks at mitosis. Mcl‐1 is phosphorylated at two sites in mitosis, Ser64 and Thr92. Phosphorylation of Thr92 by cyclin‐dependent kinase 1 (CDK1)–cyclin B1 initiates degradation of Mcl‐1 in cells arrested in mitosis by microtubule poisons. Mcl‐1 destruction during mitotic arrest requires proteasome activity and is dependent on Cdc20/Fizzy, which mediates recognition of mitotic substrates by the anaphase‐promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Stabilisation of Mcl‐1 during mitotic arrest by mutation of either Thr92 or a D‐box destruction motif inhibits the induction of apoptosis by microtubule poisons. Thus, phosphorylation of Mcl‐1 by CDK1–cyclin B1 and its APC/C^Cdc20‐mediated destruction initiates apoptosis if a cell fails to resolve mitosis. Regulation of apoptosis, therefore, is linked intrinsically to progression through mitosis and is governed by a temporal mechanism that distinguishes between normal mitosis and prolonged mitotic arrest.     

Mcl‐1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cells

Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl‐2 family proteins such as Bcl‐2 and Bcl‐x_L potently inhibit apoptosis, no studies have examined the use of the Bcl‐2 family protein, Mcl‐1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl‐1 protein and a Mcl‐1 mutant protein that is not degraded by the proteasome in a serum‐free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl‐1 led to increased viabilities in fed‐batch culture, with cell lines expressing the Mcl‐1 mutant maintaining ～90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl‐1‐expressing cell lines were isolated that consistently showed increases in antibody production of 20–35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl‐1‐expressing cell lines, and Mcl‐1‐expressing cells exhibited 3‐fold lower caspase‐3 activation when compared with the control cell lines. Altogether, the expression of Mcl‐1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

结核分枝杆菌对宿主巨噬细胞死亡方式的调控

结核分枝杆菌（Mycobacterium tuberculosis,Mtb）感染后能抑制宿主巨噬细胞（M西）的免疫反应,并在其中生存、复制。研究表明Mtb减毒株感染主要诱导宿主Mφ凋亡,凋亡能抑制胞内Mtb的活力;而Mtb毒力株感染能抑制凋亡的完成,诱导Mφ坏死,最终导致Mtb扩散、感染临近细胞。通过对Mtb感染诱导宿主Mφ不同死亡方式的讨论,进一步认识Mtb的致病机制。     

ESX‐1 and phthiocerol dimycocerosates of Mycobacterium tuberculosis act in concert to cause phagosomal rupture and host cell apoptosis

Although phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, little is known about their mechanism of action. Localized in the outer membrane of mycobacterial pathogens, DIM are predicted to interact with host cell membranes. Interaction with eukaryotic membranes is a property shared with another virulence factor of Mtb, the early secretory antigenic target EsxA (also known as ESAT‐6). This small protein, which is secreted by the type VII secretion system ESX‐1 (T7SS/ESX‐1), is involved in phagosomal rupture and cell death induced by virulent mycobacteria inside host phagocytes. In this work, by the use of several knock‐out or knock‐in mutants of Mtb or Mycobacterium bovis BCG strains and different cell biological assays, we present conclusive evidence that ESX‐1 and DIM act in concert to induce phagosomal membrane damage and rupture in infected macrophages, ultimately leading to host cell apoptosis. These results identify an as yet unknown function for DIM in the infection process and open up a new research field for the study of the interaction of lipid and protein virulence factors of Mtb.     

结核分枝杆菌RD1区研究进展

RD1区是结核分枝杆菌(MTB)在长期传代过程中丢失的重要保护性抗原,RD1区仅存在于致病性分枝杆菌中,而在卡介苗(BCG)及环境分枝杆菌中缺失。RD1区基因全长9.5kb,共有9个开放读码框,分别编码9个蛋白。RD1区是MTB毒力的关键因素之一,同时RD1区存在一种新分泌表达体系,能保证ESAT6和CFP10蛋白分泌表达。RD1区蛋白有较强的免疫原性,在MTB的预防和诊断中将可能发挥巨大作用,并有可能成为筛选抗MTB药物的理想靶抗原。     

Intersection of mitochondrial fission and fusion machinery with apoptotic pathways: Role of Mcl‐1

Mitochondria actively contribute to apoptotic cell death through mechanisms including the loss of integrity of the outer mitochondrial membrane, the release of intermembrane space proteins, such as cytochrome c, in the cytosol and the caspase cascade activation. This process is the result of careful cooperation not only among members of the Bcl‐2 family but also dynamin‐related proteins. These events are often accompanied by fission of the organelle, thus linking mitochondrial dynamics to apoptosis. Emerging evidences are suggesting a fine regulation of mitochondrial morphology by Bcl‐2 family members and active participation of fission–fusion proteins in apoptosis. The debate whether in mitochondrial morphogenesis the role of Bcl‐2 family members is functionally distinct from their role in apoptosis is still open and, above all, which morphological changes are associated with cell death sensitisation. This review will cover the findings on how the mitochondrial fission and fusion machinery may intersect apoptotic pathways focusing on recent advances on the key role played by Mcl‐1.     

The structure of glucose‐1‐phosphate thymidylyltransferase from Mycobacterium tuberculosis reveals the location of an essential magnesium ion in the RmlA‐type enzymes

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, continues to be a major threat to populations worldwide. Whereas the disease is treatable, the drug regimen is arduous at best with the use of four antimicrobials over a six‐month period. There is clearly a pressing need for the development of new therapeutics. One potential target for structure‐based drug design is the enzyme RmlA, a glucose‐1‐phosphate thymidylyltransferase. This enzyme catalyzes the first step in the biosynthesis of l ‐rhamnose, which is a deoxysugar critical for the integrity of the bacterium's cell wall. Here, we report the X‐ray structures of M. tuberculosis RmlA in complex with either dTTP or dTDP‐glucose to 1.6 Å and 1.85 Å resolution, respectively. In the RmlA/dTTP complex, two magnesium ions were observed binding to the nucleotide, both ligated in octahedral coordination spheres. In the RmlA/dTDP‐glucose complex, only a single magnesium ion was observed. Importantly, for RmlA‐type enzymes with known three‐dimensional structures, not one model shows the position of the magnesium ion bound to the nucleotide‐linked sugar. As such, this investigation represents the first direct observation of the manner in which a magnesium ion is coordinated to the RmlA product and thus has important ramifications for structure‐based drug design. In the past, molecular modeling procedures have been employed to derive a three‐dimensional model of the M. tuberculosis RmlA for drug design. The X‐ray structures presented herein provide a superior molecular scaffold for such endeavors in the treatment of one of the world's deadliest diseases.     

结核分枝杆菌与巨噬细胞相互作用的研究进展

结核分枝杆菌（Mycobacterium tuberculosis,MTB）是一种典型的胞内致病菌,巨噬细胞是MTB在体内的主要宿主细胞。巨噬细胞具有强大的吞噬功能,在机体固有免疫和适应性免疫中均发挥着重要作用,可有效保护宿主免受结核分枝杆菌的感染。MTB在与宿主巨噬细胞的长期相互作用过程中,逐渐形成多种逃避杀灭的有效策略,得以在宿主体内存活并增殖。该文从巨噬细胞抗MTB感染及MTB逃避巨噬细胞杀灭两个方面综述国内外的研究进展。     

Differential recruitment of CD63 and Rab7‐interacting‐lysosomal‐protein to phagosomes containing Mycobacterium tuberculosis in macrophages

M.tb is an intracellular pathogen which survives within the phagosomes of host macrophages by inhibiting their fusion with lysosomes. Here, it has been demonstrated that a lysosomal glycoprotein, CD63, is recruited to the majority of M.tb phagosomes, while RILP shows limited localization. This is consistent with the author's findings that CD63, but not RILP, is recruited to the phagosomes in macrophages expressing the dominant negative form of Rab7. These results suggest that M.tb phagosomes selectively fuse with endosomes and lysosomes to escape killing activity while acquiring nutrients.     

PE_PGRS62 promotes the survival of Mycobacterium smegmatis within macrophages via disrupting ER stress-mediated apoptosis

Mycobacterium tuberculosis, the leading causative agent of tuberculosis, remains one of the most deadly infectious pathogens. PE_PGRS proteins become a new focus as their species specificity in mycobacteria, especially in pathogenic mycobacteria. Despite intensive research, PE_PGRS proteins are still a mysterious aspect of mycobacterial pathogenesis with unknown mechanism. Herein, we focused on a PE_PGRS member from M. tuberculosis, PE_PGRS62, characterized by a surface-exposed protein function in disrupting phagolysosome maturation. Expression of PE_PGRS62 in Mycobacterium smegmatis, a nonpathogenic species naturally deficient in PE_PGRS genes, resulted in enhanced resistance to various in vitro stresses and cellular survival in macrophage. As a consequence, the cytokine profiles of macrophage were disturbed and cell apoptosis were inhibited via decreasing endoplasmic reticulum stress response.     

Identification of small peptides mimicking the R2 C‐terminus of Mycobacterium tuberculosis ribonucleotide reductase

Ribonucleotide reductase (RNR) is a viable target for new drugs against the causative agent of tuberculosis, Mycobacterium tuberculosis. Previous work has shown that an N‐acetylated heptapeptide based on the C‐terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. Here the synthesis and binding affinity, evaluated by competitive fluorescence polarization, of several truncated and N‐protected peptides are described. The protected single‐amino acid Fmoc‐Trp shows binding affinity comparable to the N‐acetylated heptapeptide, making it an attractive candidate for further development of non‐peptidic RNR inhibitors. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.