A protein bridge to death between the ER and mitochondria.

Commitment to apoptotic cell death occurs at the mitochondria and is regulated by BCL-2 family proteins localized to this organelle. However, BIK, a resident protein of the endoplasmic reticulum, inhibits mitochondrial BCL-2 proteins to promote apoptosis. In a recent paper in the JBC, Osterlund et al. investigated this conundrum. Surprisingly, they discovered that these endoplasmic reticulum and mitochondrial proteins moved toward each other and met at the contact site between the two organelles, thereby forming a 'bridge to death'.

Design, Synthesis, and Evaluation of BCL-2 Targeting PROTACs.

BCL-2, a member of the BCL-2 protein family, is an antiapoptotic factor that regulates the intrinsic pathway of apoptosis. Due to its aberrant activity, it is frequently implicated in haematopoietic cancers and represents an attractive target for the development of therapeutics that antagonize its activity. A selective BCL-2 inhibitor, venetoclax, was approved for treating chronic lymphocytic leukaemia, acute myeloid leukemia, and other haematologic malignancies, validating BCL-2 as an anticancer target. Since then, alternative therapeutic approaches to modulate the activity of BCL-2 have been explored, such as antibody-drug conjugates and proteolysis-targeting chimeras. Despite numerous research groups focusing on developing degraders of BCL-2 family member proteins, selective BCL-2 PROTACs remain elusive, as disclosed compounds only show dual BCL-xL/BCL-2 degradation. Herein, we report our efforts to develop BCL-2 degraders by incorporating two BCL-2 binding moieties into chimeric compounds that aim to hijack one of three E3 ligases: CRBN, VHL, and IAPs. Even though our project did not result in obtaining a potent and selective BCL-2 PROTAC, our research will aid in understanding the narrow chemical space of BCL-2 degraders.

BCL-2 protein family: attractive targets for cancer therapy.

Acquired resistance to cell death is a hallmark of cancer. The BCL-2 protein family members play important roles in controlling apoptotic cell death. Abnormal over-expression of pro-survival BCL-2 family members or abnormal reduction of pro-apoptotic BCL-2 family proteins, both resulting in the inhibition of apoptosis, are frequently detected in diverse malignancies. The critical role of the pro-survival and pro-apoptotic BCL-2 family proteins in the regulation of apoptosis makes them attractive targets for the development of agents for the treatment of cancer. This review describes the roles of the various pro-survival and pro-apoptotic members of the BCL-2 protein family in normal development and organismal function and how defects in the control of apoptosis promote the development and therapy resistance of cancer. Finally, we discuss the development of inhibitors of pro-survival BCL-2 proteins, termed BH3-mimetic drugs, as novel agents for cancer therapy.

Development of venetoclax performance using its new derivatives on BCL-2 protein inhibition.

Cancer cells are resistant to apoptosis and this is one of the most obvious symptoms of cancer in humans. One of the most exciting strategies for treating cancer is to design regulators that increase cell death and stop cell growth. Members of the BCL-2 family of proteins play an important role in the regulation of apoptosis. In this study, an attempt was made to improve the performance of one of the anticancer drugs by designing new analogs of venetoclax (VNT). For this purpose, molecular docking studies were performed to determine the best binding state of VNT and its newly designed derivatives at the protein-binding site to estimate the binding energy. The best analog in terms of free energy was VNT-12 with the lowest energy (-12.15 kcal/mol). Finally, to investigate the inhibitory effect of the compounds on BCL-2 protein, molecular dynamics simulation was used, and by performing the relevant analyses during the simulation, it was observed that the newly designed ligand had better performance in inhibiting BCL-2 protein compared to VNT.

Correlation of ultrasound features with Bcl-2 protein expression in basal cell carcinomas (BCCs).

BACKGROUND: The expression of the Bcl-2 protein is frequently observed in basal cell carcinomas (BCCs), making it a significant biological marker and potential therapeutic target. Skin ultrasonography offers a noninvasive means of obtaining anatomical information about cutaneous tumors. OBJECTIVES: The purpose of this study was to investigate the correlation between ultrasound features and Bcl-2 expression in BCCs, to provide a reference for developing pharmacological treatment plans. METHODS: According to the Bcl-2 protein expression, 74 BCCs confirmed by surgical pathology were divided into high Bcl-2 expression BCCs (HB-BCCs) and low Bcl-2 expression BCCs (LB-BCCs). Preoperative lesion ultrasound features were analyzed retrospectively based on Liang's criteria, which included the following features: shape, surface, keratinization, base, infiltration level, internal echogenicity, distribution of hyperechoic spots, posterior echogenic changes, internal Doppler signal, and lesion size (maximum diameter and infiltration depth). The differences of two groups were compared using a chi-square test or a paired t-test. RESULTS: Based on ultrasound features, cystic areas were more frequent in LB-BCCs (χ2 = 7.015, P = .008). Furthermore, LB-BCCs exhibited greater infiltration depth than HB-BCCs (4.86 ± 2.12 mm vs. 2.72 ± 1.40 mm, P = .000), had a higher propensity to infiltrate the subcutaneous tissue (χ2 = 12.422, P = .002), and displayed a more abundant internal Doppler signal within the lesions (χ2 = 24.696, P = .000). Conversely, maximum diameter of the lesions, shape, surface, keratinization, base, hyperechoic spots distribution, and posterior echogenic changes of the lesions did not differ significantly between the two groups. CONCLUSIONS: Ultrasound features are correlated with Bcl-2 protein expression level in BCCs. LB-BCCs show greater infiltration depth, subcutaneous infiltration, more cystic changes and more abundant internal Doppler signal than HB-BCCs, which may suggest a potential basis for drug selection in BCC chemotherapy.

Apoptosis Regulation in Osteoarthritis and the Influence of Lipid Interactions.

Osteoarthritis (OA) is one of the most common chronic diseases in human and animal joints. The joints undergo several morphological and histological changes during the development of radiographically visible osteoarthritis. The most discussed changes include synovial inflammation, the massive destruction of articular cartilage and ongoing joint destruction accompanied by massive joint pain in the later stadium. Either the increased apoptosis of chondrocytes or the insufficient apoptosis of inflammatory macrophages and synovial fibroblasts are likely to underly this process. In this review, we discuss the current state of research on the pathogenesis of OA with special regard to the involvement of apoptosis.

In Silico Mining of Natural Products Atlas (NPAtlas) Database for Identifying Effective Bcl-2 Inhibitors: Molecular Docking, Molecular Dynamics, and Pharmacokinetics Characteristics.

The Bcl-2 protein has a vital function in controlling the programmed cell doom of mitochondria. If programmed cell death signals are obstructed, an imbalance between cell survival and death will occur, which is a significant reason for cancer. Therefore, the Bcl-2 protein was identified as a possible therapeutic target for carcinoma treatment. Herein, the Natural Products Atlas (NPAtlas) compounds were virtually screened, seeking potent inhibitors towards the Bcl-2 protein. The performance of AutoDock Vina software to predict the docking score and pose of the investigated compounds was first validated according to the available experimental data. Based on the validated AutoDock Vina parameters, the NPAtlas database was filtered against the Bcl-2 protein. The natural compounds with docking scores less than that of the venetoclax (calc. -10.6 kcal/mol) were submitted to MD simulations, followed by MM-GBSA binding energy calculations. According to MM-GBSA//200 ns MD simulations, saquayamycin F (NPA002200) demonstrated promising binding affinity with a ΔGbinding value of -53.9 kcal/mol towards the Bcl-2 protein when compared to venetoclax (ΔGbinding = -50.6 kcal/mol). The energetical and structural analyses showed a great constancy of the saquayamycin F inside the Bcl-2 protein active site. Moreover, the ADMET and drug-likeness features of the saquayamycin F were anticipated, indicating its good oral bioavailability. According to in silico computations, saquayamycin F is proposed to be used as a therapeutic agent against the wild-type Bcl-2 protein and warrants further experimental assays.

Role of Bcl-2, p53, and Ki-67 expression in basal cell carcinoma and their association with aggressive and non-aggressive histological phenotypes.

Introduction: There is increasing evidence that immunohistochemical expression of p53, Ki-67, and Bcl-2 is associated with aggressive (aBCC) and less aggressive (nBCC) histological subtypes and may have a prognostic role. Aim: To investigate the clinicopathological features and immunohistochemical expressions of p53, Ki-67, and Bcl-2 in cutaneous basal cell carcinoma focusing on histological subtypes. Their roles and possible interactions in the development and progression of BCC are discussed. Material and methods: A total of 50 BCC samples from 50 patients from Western Mexico between June 2018 and June 2019 were included. Paraffin-embedded samples were immunostained with p53, Ki-67, and Bcl-2 antibodies. Semi-quantitative analysis was performed to determine the intensity and positivity of immunostained cells. Parametrical and non-parametrical tests were performed according to the sample's distribution. Results: Samples included 21 nBCC and 29 aBCC. The statistical analysis showed statistical association when grouped as non-aggressive and aggressive subtypes for p53 (p = 0.04) and Bcl-2 (p < 0.01). An inverse negative correlation was found between age and Bcl-2 expression. No statistical association was found between Ki-67 immunoreactivity and any of the other variables. Conclusions: We found that a high expression of Bcl-2 and a low expression of p53 was associated with more indolent histopathological features of BCC and therefore better outcomes. These findings suggest that examination of p53 and Bcl-2 expression in BCC patients may provide valuable prognostic information. These biomarkers may play a role in the development and progression of some cases of BCC.

[Effect of an antioxidant on vascular wall cell apoptosis markers after reconstructive operations]. / Vliianie antioksidanta na markery apoptoza kletok sosudistoi stenki posle rekonstruktivnykh operatsii.

AIM: This study was aimed at determining Bcl-2 and Bax proteins expression before and after reconstructive-repairing operations in patients with atherosclerosis obliterans of lower extremities and at assessing the effect of an antioxidant (vitamin E at a dose of 100 mg once daily for 1 month after surgery) on the dynamics of changes of Bcl-2 and Bax proteins in the postoperative period. PATIENTS AND METHODS: The study included a total of 60 patients with stage III-IV lower limb atherosclerosis obliterans. All patients underwent reconstructive-repairing operations on the arteries of the aortofemoral segment. After surgery the patients were divided into two groups. Group A included 30 patients who during 1 month received additionally to basic therapy vitamin E at a daily dose of 100 mg. Group B was composed of 30 patients receiving basic therapy alone according to the National guidelines of managing patients with peripheral artery disease. All patients before, on POD 1, and 1 month after surgery were subjected to venous blood test aimed at determining Bcl-2 and Bax apoptosis proteins expression by means of enzyme-linked immunosorbent assay. RESULTS: In patients of groups A and B, the baseline level of Bcl-2 protein (4.75 and 4.2 ng/ml, respectively) was comparable with that in apparently healthy volunteers (5.3 ng/ml). The baseline levels of Bax protein in patients of the operated groups (26.9 and 26.0 ng/ml, respectively) were increased compared with the values in healthy volunteers (16.5 ng/ml). On POD 1 there was increased expression of Bax protein in Group A and B patients to 39.4 and 30.2 ng/ml, respectively. One month after surgery, Group B patients demonstrated a decrease in the Bcl-2 values below the baseline level - 1.1 ng/ml (p=0.003), with the Bax level continuing to increase - 36 ng/ml (p=0.004). In turn, Group A patients after 1 month were found to have increased levels of the Bcl-2 protein - 5.75 ng/ml, with the Bax level returning to the baseline values - 27.4 ng/ml. CONCLUSION: In stage III and IV lower limb obliterating atherosclerosis, the level of the Bax proapoptoric protein was higher than that in healthy volunteers. On POD 1, there occurred increased expression of the pro-apoptotic protein Bax and activation of apoptosis markers. On the background of using vitamin E at a dose of 100 mg once daily for 1 month, there was a decrease in level of the Bax propapoptotic protein (p=0.003) and an increase in level of the anti-apoptotic Bcl-2 protein level (p=0.0007).

A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-2.

Programmed cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of harmful cells. Besides an extrinsic pathway, an intrinsic (mitochondrial) apoptotic pathway exists where mitochondria are actively involved in cellular clearance in response to internal stress signals. Pro-apoptotic (death) and anti-apoptotic (survival) members of the B cell CLL/lymphoma-2 (Bcl-2) protein family meet at the mitochondrion's surface where they accurately regulate apoptosis. Overexpression of the anti-apoptotic Bcl-2 protein is a hallmark for many types of cancers and in particular for many treatment resistant tumors. Bcl-2 is a membrane protein residing in the mitochondrial outer membrane. Due to its typical membrane protein features including very limited solubility, it is difficult to express and to purify. Therefore, most biophysical and structural studies have used truncated, soluble versions. However, to understand its membrane-coupled function and structure, access to sufficient amount of full-length human Bcl-2 protein is a necessity. Here, we present a novel, E. coli based approach for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)), solubilized in detergent micelles, which allows for easy exchange of the detergent.