PUMA-BH3结构域短肽的原核表达、纯化及促凋亡活性鉴定

Bcl-2家族蛋白质在线粒体途径凋亡的调控机制中起着重要的作用,p53正向细胞凋亡调控因子（p53 up-regulated modulator of apoptosis protein,PUMA)是该家族的一种只含有BH3同源区域的促凋亡蛋白。为得到PUMA的BH3结构域短肽并检测其生物学活性,将人工合成的编码PUMA-BH3肽的DNA片段克隆到质粒pTYB2上,构建出表达PUMA-BH3-内含肽-几丁质结合域融合蛋白的原核表达载体pTYB2-PUMA-BH3,转化大肠杆菌BL-21(DE3)中IPTG诱导表达。表达的融合蛋白经几丁质亲和层析、二硫苏糖醇（DTT）的柱内还原,直接获得可溶性PUMA-BH3肽。通过研究重组PUMA-BH3肽在体外条件下对线粒体活力、线粒体肿胀度以及细胞色素c释放的影响来鉴定其生物学活性。结果表明,获得的可溶性PUMA-BH3肽能作用于离体线粒体,引起线粒体活力降低,线粒体肿胀并能诱导细胞色素c释放。环孢菌素A对此有一定的抑制作用,提示PUMA-BH3肽对线粒体的上述作用是通过促进通透性转运孔( PTP)开放实现的。经原核表达及纯化,获得了具有促凋亡活性的PUMA-BH3肽,为进一步研制控制凋亡过程的药物奠定了基础。     

COVID-19 receptor and malignant cancers: Association of CTSL expression with susceptibility to SARS-CoV-2

CTSL is expressed by cancerous tissues and encodes a lysosomal cysteine proteinase that regulates cancer progression and SARS-CoV-2 entry. Therefore, it is critical to predict the susceptibility of cancer patients for SARS-CoV-2 and evaluate the correlation between disease outcomes and the expression of CTSL in malignant cancer tissues. In the current study, we analyzed CTSL expression, mutation rate, survival and COVID-19 disease outcomes in cancer and normal tissues, using online databases. We also performed immunohistochemistry (IHC) to test CTSL expression and western blot to monitor its regulation by cordycepin (CD), and N6, N6-dimethyladenosine (m⁶₂A), respectively. We found that CTSL is conserved across different species, and highly expressed in both normal and cancer tissues from human, as compared to ACE2 or other proteinases/proteases. Additionally, the expression of CTSL protein was the highest in the lung tissue. We show that the mRNA expression of CTSL is 66.4-fold higher in normal lungs and 54.8-fold higher in cancer tissues, as compared to ACE2 mRNA expression in the respective tissues. Compared to other proteases/proteinases/convertases such as TMPRSS2 and FURIN, the expression of CTSL was higher in both normal lungs and lung cancer samples. All these data indicate that CTSL might play an important role in COVID-19 pathogenesis in normal and cancer tissues of the lungs. Additionally, the CTSL-002 isoform containing both the inhibitor_I29 and Peptidase_C1 domains was highly prevalent in all cancers, suggesting its potential role in tumor progression and SARS-CoV-2 entry in multiple types of cancers. Further analysis of the expression of CTSL mutant showed a correlation with FURIN and TMPRSS2, suggesting a potential role of CTSL mutations in modulating SARS-CoV-2 entry in cancers. Moreover, high expression of CTSL significantly correlated with a short overall survival (OS) in lung cancer and glioma. Thus, CTSL might play a major role in the susceptibility of lung cancer and glioma patients to SARS-CoV-2 uptake and COVID-19 severity. Furthermore, CD or m⁶₂A inhibited CTSL expression in the cancer cell lines A549, MDA-MB-231, and/or PC3 in a dose dependent manner. In conclusion, we show that CTSL is highly expressed in normal tissues and increased in most cancers, and CD or m⁶₂A could inhibit its expression, suggesting the therapeutic potential of targeting CTSL for cancer and COVID-19 treatment.     

Pneumocytes are distinguished by highly elevated expression of the ER stress biomarker GRP78, a co-receptor for SARS-CoV-2, in COVID-19 autopsies

Vaccinations are widely credited with reducing death rates from COVID-19, but the underlying host-viral mechanisms/interactions for morbidity and mortality of SARS-CoV-2 infection remain poorly understood. Acute respiratory distress syndrome (ARDS) describes the severe lung injury, which is pathologically associated with alveolar damage, inflammation, non-cardiogenic edema, and hyaline membrane formation. Because proteostatic pathways play central roles in cellular protection, immune modulation, protein degradation, and tissue repair, we examined the pathological features for the unfolded protein response (UPR) using the surrogate biomarker glucose-regulated protein 78 (GRP78) and co-receptor for SARS-CoV-2. At autopsy, immunostaining of COVID-19 lungs showed highly elevated expression of GRP78 in both pneumocytes and macrophages compared with that of non-COVID control lungs. GRP78 expression was detected in both SARS-CoV-2-infected and un-infected pneumocytes as determined by multiplexed immunostaining for nucleocapsid protein. In macrophages, immunohistochemical staining for GRP78 from deceased COVID-19 patients was increased but overlapped with GRP78 expression taken from surgical resections of non-COVID-19 controls. In contrast, the robust in situ GRP78 immunostaining of pneumocytes from COVID-19 autopsies exhibited no overlap and was independent of age, race/ethnicity, and gender compared with that from non-COVID-19 controls. Our findings bring new insights for stress-response pathways involving the proteostatic network implicated for host resilience and suggest that targeting of GRP78 expression with existing therapeutics might afford an alternative therapeutic strategy to modulate host-viral interactions during SARS-CoV-2 infections.     

Sulforaphane inhibits the expression of interleukin-6 and interleukin-8 induced in bronchial epithelial IB3-1 cells by exposure to the SARS-CoV-2 Spike protein

BackgroundA key clinical feature of COVID-19 is a deep inflammatory state known as “cytokine storm” and characterized by high expression of several cytokines, chemokines and growth factors, including IL-6 and IL-8. A direct consequence of this inflammatory state in the lungs is the Acute Respiratory Distress Syndrome (ARDS), frequently observed in severe COVID-19 patients. The "cytokine storm" is associated with severe forms of COVID-19 and poor prognosis for COVID-19 patients. Sulforaphane (SFN), one of the main components of Brassica oleraceae L. (Brassicaceae or Cruciferae), is known to possess anti-inflammatory effects in tissues from several organs, among which joints, kidneys and lungs.PurposeThe objective of the present study was to determine whether SFN is able to inhibit IL-6 and IL-8, two key molecules involved in the COVID-19 "cytokine storm".MethodsThe effects of SFN were studied in vitro on bronchial epithelial IB3-1 cells exposed to the SARS-CoV-2 Spike protein (S-protein). The anti-inflammatory activity of SFN on IL-6 and IL-8 expression has been evaluated by RT-qPCR and Bio-Plex analysis.ResultsIn our study SFN inhibits, in cultured IB3-1 bronchial cells, the gene expression of IL-6 and IL-8 induced by the S-protein of SARS-CoV-2. This represents the proof-of-principle that SFN may modulate the release of some key proteins of the COVID-19 "cytokine storm".ConclusionThe control of the cytokine storm is one of the major issues in the management of COVID-19 patients. Our study suggests that SFN can be employed in protocols useful to control hyperinflammatory state associated with SARS-CoV-2 infection.     

Bak but not Bax is essential for Bcl-xS-induced apoptosis

Bcl-x(S), a proapoptotic member of the Bcl-2 protein family, is localized in the mitochondria and induces apoptosis in a caspase- and BH3-dependent manner by a mechanism involving cytochrome c release. The way in which Bcl-x(S) induces caspase activation and cytochrome c release, as well as the relationship between Bcl-x(S) and other proapoptotic members of the Bcl-2 family, is not known. Here we used embryonic fibroblasts derived from mice deficient in the multidomain proapoptotic members of the Bcl-2 family (Bax and Bak) and the apoptotic components of the apoptosome (Apaf-1 and caspase-9) to unravel the cascade of events by which Bcl-x(S) promotes apoptosis. Our results show that Bak but not Bax is essential for Bcl-x(S)-induced apoptosis. Bcl-x(S) induced activation of Bak, which in turn promoted apoptosis by apoptosome-dependent and -independent pathways. These findings provide the first evidence that a proapoptotic Bcl-2 family protein induces apoptosis exclusively via Bak.     

BOK and NOXA are essential mediators of p53-dependent apoptosis

Cellular stress leads to DNA damage and activation of the intrinsic apoptotic pathway in which translocation of mitochondrial cytochrome c to the cytosol plays a critical role. Previous studies have suggested alternative mechanisms responsible for this process. We examined initiation mechanisms of the intrinsic apoptotic pathway using human neuroblastoma and breast cancer cells. Results indicated that translocation of cytochrome c does not require prior activation of caspases but rather depends on activation of specific BCL-2 family members, depending upon the type of death signal. Thus, DNA damage-induced apoptosis requires new protein synthesis, accumulation of p53 tumor suppressor protein, and p53-dependent induction of BOK and NOXA genes, while a role for BAX in this pathway is not essential. In contrast, apoptosis induced by staurosporine does not require protein synthesis but is characterized by translocation of BAX. Based on these findings, we propose a model of the intrinsic apoptotic cascade induced by DNA damage where proapoptotic BOK substitutes for a function of BAX.     

B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these “BH3 mimetics” in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.     

Apolipoprotein l6, a novel proapoptotic Bcl-2 homology 3-only protein, induces mitochondria-mediated apoptosis in cancer cells

Cancer cells frequently possess defects in the genetic and biochemical pathways of apoptosis. Members of the Bcl-2 family play pivotal roles in regulating apoptosis and possess at least one of four Bcl-2 homology (BH) domains, designated BH1 to BH4. The BH3 domain is the only one conserved in proapoptotic BH3-only proteins and plays an important role in protein-protein interactions in apoptosis by regulating homodimerization and heterodimerization of the Bcl-2 family members. To date, 10 BH3-only proapoptotic proteins have been identified and characterized in the human genome. The completion of the Human Genome Project and the availability of various public databases and sequence analysis algorithms allowed us to use the bioinformatic database-mining approach to identify one novel BH3-only protein, apolipoprotein L6 (ApoL6). The full-length cDNA of ApoL6 was identified, cloned, and functionally expressed in p53-null colorectal cancer cells (DLD-1). We found that overexpression of wild-type ApoL6 induced mitochondria-mediated apoptosis in DLD-1 cells characterized by release of cytochrome c and Smac/DIABLO from mitochondria and activation of caspase-9, whereas ApoL6 BH3 domain deletion allele did not. In addition, overexpression of ApoL6 also induced activation of caspase-8. Furthermore, we showed that adenovirus harboring the full-length cDNA of ApoL6 induced marked apoptosis in a variety of cancer cell types, and ApoL6 recruited and interacted with lipid/fatty acid components during the induction of apoptosis. To our knowledge, this is the first example that intracellular overproduction of an apolipoprotein induces marked apoptosis.     

Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.     

Vaccinia Virus F1L Interacts with Bak Using Highly Divergent Bcl-2 Homology Domains and Replaces the Function of Mcl-1

The Bcl-2 family regulates induction of apoptosis at the mitochondria. Essential to this regulation are the interactions between Bcl-2 family members, which are mediated by Bcl-2 homology (BH) domains. Vaccinia virus F1L is a unique inhibitor of apoptosis that lacks significant sequence similarity with the Bcl-2 family and does not contain obvious BH domains. Despite this, F1L inhibits cytochrome c release from mitochondria by preventing Bak and Bax activation. Although F1L constitutively interacts with Bak to prevent Bak activation, the precise mechanism of this interaction remains elusive. We have identified highly divergent BH domains in F1L that were verified by the recent crystal structure of F1L (Kvansakul, M., Yang, H., Fairlie, W. D., Czabotar, P. E., Fischer, S. F., Perugini, M. A., Huang, D. C., and Colman, P. M. (2008) Cell Death Differ. 15, 1564–1571). Here we show that F1L required these BH domains to interact with ectopically expressed and endogenous Bak. The interaction between F1L and Bak was conserved across species, and both F1L and the cellular antiapoptotic protein Mcl-1 required the Bak BH3 domain for interaction. Moreover, F1L replaced Mcl-1 during infection, as the Bak·Mcl-1 complex was disrupted during vaccinia virus infection. In contrast to UV irradiation, vaccinia virus infection did not result in rapid degradation of Mcl-1, consistent with our observation that vaccinia virus did not initiate a DNA damage response. Additionally, Mcl-1 expression prevented Bak activation and apoptosis during infection with a proapoptotic vaccinia virus devoid of F1L. Our data suggest that F1L replaces the antiapoptotic activity of Mcl-1 during vaccinia virus infection by interacting with Bak using highly divergent BH domains.     

MUC1-C influences cell survival in lung adenocarcinoma Calu-3 cells after SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces coronavirus disease 2019 (COVID-19) and may increase the risk of adverse outcomes in lung cancer patients. In this study, we investigated the expression and function of mucin 1 (MUC1) after SARS-CoV-2 infection in the lung epithelial cancer cell line Calu-3. MUC1 is a major constituent of the mucus layer in the respiratory tract and contributes to pathogen defense. SARS-CoV-2 infection induced MUC1 C-terminal subunit (MUC1-C) expression in a STAT3 activation-dependent manner. Inhibition of MUC1-C signaling increased apoptosis-related protein levels and reduced proliferation-related protein levels; however, SARS-CoV-2 replication was not affected. Together, these results suggest that increased MUC1-C expression in response to SARS-CoV-2 infection may trigger the growth of lung cancer cells, and COVID-19 may be a risk factor for lung cancer patients.     

Induction of BIM, a proapoptotic BH3-only BCL-2 family member, is critical for neuronal apoptosis

Sympathetic neuronal death induced by nerve growth factor (NGF) deprivation requires the macromolecular synthesis-dependent translocation of BAX from the cytosol to mitochondria and its subsequent integration into the mitochondrial outer membrane, followed by BAX-mediated cytochrome c (cyt c) release. The gene products triggering this process remain unknown. Here, we report that BIM, a member of the BH3-only proapoptotic subfamily of the BCL-2 protein family, is one such molecule. NGF withdrawal induced expression of BIM(EL), an integral mitochondrial membrane protein that functions upstream of (or in parallel with) the BAX/BCL-2 and caspase checkpoints. Bim deletion conferred protection against developmental and induced neuronal apoptosis in both central and peripheral populations, but only transiently, suggesting that BIM--and perhaps other BH3-only proteins--serve partially redundant functions upstream of BAX-mediated cyt c release.     

Utilizing mitochondrial events as biomarkers for imaging apoptosis

Cells undergoing apoptosis show a plethora of time-dependent changes. The available tools for imaging apoptosis in live cells rely either on the detection of the activity of caspases, or on the visualization of exposure of phosphatidyl serine in the outer leaflet of the cell membrane. We report here a novel method for the detection of mitochondrial events during apoptosis, namely translocation of Bax to mitochondria and release of cytochrome c (Cyt c) using bimolecular fluorescence complementation. Expression of split yellow fluorescent protein (YFP) fragments fused to Bax and Cyt c, resulted in robust induction of YFP fluorescence at the mitochondria of apoptotic cells with very low background. In vivo expression of split YFP protein fragments in liver hepatocytes and intra-vital imaging of subcutaneous tumor showed elevated YFP fluorescence upon apoptosis induction. Thus, YFP complementation could be applied for high-throughput screening and in vivo molecular imaging of mitochondrial events during apoptosis.     

BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations

Background

Epidermal growth factor receptor (EGFR) mutations are present in the majority of patients with non-small cell lung cancer (NSCLC) responsive to the EGFR tyrosine kinase inhibitors (TKIs) gefitinib or erlotinib. These EGFR-dependent tumors eventually become TKI resistant, and the common secondary T790M mutation accounts for half the tumors with acquired resistance to gefitinib. However, the key proapoptotic proteins involved in TKI-induced cell death and other secondary mutations involved in resistance remain unclear. The objective of this study was to identify the mechanism of EGFR TKI-induced apoptosis and secondary resistant mutations that affect this process.

Methods and Findings

To study TKI-induced cell death and mechanisms of resistance, we used lung cancer cell lines (with or without EGFR mutations), Ba/F3 cells stably transfected with EGFR mutation constructs, and tumor samples from a gefitinib-resistant patient. Here we show that up-regulation of the BH3-only polypeptide BIM (also known as BCL2-like 11) correlated with gefitinib-induced apoptosis in gefitinib-sensitive EGFR-mutant lung cancer cells. The T790M mutation blocked gefitinib-induced up-regulation of BIM and apoptosis. This blockade was overcome by the irreversible TKI CL-387,785. Knockdown of BIM by small interfering RNA was able to attenuate apoptosis induced by EGFR TKIs. Furthermore, from a gefitinib-resistant patient carrying the activating L858R mutation, we identified a novel secondary resistant mutation, L747S in cis to the activating mutation, which attenuated the up-regulation of BIM and reduced apoptosis.

Conclusions

Our results provide evidence that BIM is involved in TKI-induced apoptosis in sensitive EGFR-mutant cells and that both attenuation of the up-regulation of BIM and resistance to gefitinib-induced apoptosis are seen in models that contain the common EGFR T790M and the novel L747S secondary resistance mutations. These findings also suggest that induction of BIM may have a role in the treatment of TKI-resistant tumors.     

Reconstitution of interactions of Murine gammaherpesvirus 68 M11 with Bcl-2 family proteins in yeast

One of the mechanisms of defense against viral infection is induction of apoptosis in infected cells. To escape this line of protection, genomes of many viruses encode for proteins that inhibit apoptosis. Murid herpesvirus 4 gene M11 encodes for homologue of cellular Bcl-2 proteins that inhibits apoptosis and autophagy in infected cell. To study a role of M11 in regulation of apoptosis we have established a yeast model system in which the action of M11 together with proapoptotic proteins Bax, Bak and Bid can be studied. When expressed in yeast, M11 did not inhibit autophagic pathway, so only effects of expression of M11 on activity of coexpressed proapoptotic proteins could be observed. In this experimental setting M11 potently inhibited both proapoptotic multidomain proteins Bax and Bak. The antiapoptotic activity of M11 was suppressed by coexpression of proapoptotic BH3-only protein tBid, indicating that M11 inhibits apoptosis likely by the same mechanism as cellular antiapoptotic proteins Bcl-2 or Bcl-XL.     

GdCl3 Induced Hep G2 Cell Death through Mitochondrial and External Death Pathways without Significant Elevation of ROS Generation

Gadolinium (Gd) compounds have important applications as MRI contrast and potential anticancer agents. The present study investigated the mechanisms of the proapoptotic effect of gadolinium chloride (GdCl₃) on hepatoblastoma cell line (Hep G2) tumor cells. The experimental results indicated that GdCl₃ induced apoptosis of Hep G2 at high concentration and with long time incubation; however, unlike the actions on normal cell lines, GdCl₃ did not cause any oxidative stress on tumor cells. Cytochrome c (Cyt c) and apoptosis inducing factor release, Bax translocation, collapse of mitochondria membrane potential, caspase 3 and 8 activation, and Bid cleavage were observed along with a sustained activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2 terminal kinase (JNK). Addition of ERK and JNK inhibitor attenuated the effect of GdCl₃ induced apoptosis and Cyt c release. All the results suggested a novel mechanism that GdCl₃ induced Hep G2 cell death through intrinsic and external death pathways without significant elevation of reactive oxygen species generation. The present work provided new insight to understand the mechanisms of the biological effects of GdCl₃ and implications for the development of anticancer Gd agents.