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BAX Gene
Introduction
The mechanisms of apoptosis may help elucidate the pathologies of uncontrolled cell growth or death. Apoptosis is under the mediation of 2 major pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway.[1][2] The intrinsic pathway is controlled and regulated by the Bcl-2 (B-cell lymphoma 2) family of proteins. The proteins in this family can be classified by their anti-apoptotic (Bcl-2, Bcl-x, Bcl-w, Mcl-1, and A1/Bfl-1) or pro-apoptotic (Bax, Bak, and Bok/Mtd) actions.[3] Both pathways result in caspase activation, leading to the termination of cell life.[4] The BAX gene (Bcl-2 Associated X-protein) is a pro-apoptotic member of the Bcl-2 gene family; it encodes a 21-kDa protein named BAX-alpha, whose association with Bcl-2 researchers believe plays a critical role in regulating intrinsic apoptosis.[4]
Development
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Development
Research has shown that the Bax gene is located on chromosome 19 by utilizing fluorescent in-situ hybridization and human-hamster somatic cell hybrid DNA.[4] Using isolated mitochondria treated to block permeability transition (PT) mitochondrial pore opening, scientists determined that Bax protein allows membrane depolarization and release of mitochondrial contents, including cytochrome c, via a Ca+-dependent opening of permeability transition pores.[5] However, PTP-/- knockout mice still exhibit Bax-dependent cell death; therefore, the suggestion has been that Bax is capable of forming pores in the mitochondrial outer membrane (MOM) on its own by inserting its amphipathic alpha-helical structure directly into the MOM.[6]
Cellular
The intrinsic pathway of apoptosis becomes activated via decreased survival signals. The Bax gene encodes BCL2L4 protein that, upon activation, heterodimerizes with Bcl2 family proteins and alters cellular mitochondria to induce cell death.[7] Bax protein is cytosolic in healthy cells with minimal binding and insertion into the outer mitochondrial membrane. Pro-survival signals, such as Bcl-xl and Bcl-2, ensure Bax does not accumulate on the outer mitochondrial membrane of healthy cells.[8] Although the complete mechanism by which Bax permeabilizes the MOM is unclear, it is known that Bax, upon activation, causes membrane depolarization and mitochondrial leaking of contents such as cytochrome c.[3] Morphological changes in the apoptotic cell include pyknosis (nuclear condensation and shrinkage), cell shrinkage, and membrane blebbing, leading to the release of vesicles containing cellular contents phagocytosed by surviving cells.[9]
Biochemical
Due to alternative splicing, the Bax gene produces multiple isoforms, with Bax-alpha being the most predominant.[4] The hydrophobic pocket of the BH3 domain is the active binding site for regulators such as BH3 and Bcl-xl pro- and anti-apoptotic members of the Bcl-2 family.[3] Transcriptional regulation of the Bax gene largely occurs by tumor suppressor P53.[1] Although the conformational changes that occur upon activation are not entirely understood, it is known that the carboxy-terminal end of the protein is responsible for mitochondrial membrane location and binding.[6] Once bound, the BH1 and BH2 domains are suspected to be pore-forming, resulting in increased membrane permeability and leakage of interspace proteins, which induce downstream cellular effects that result in cell death.[6][3]
Molecular Level
Bax and other pro-apoptotic members of the Bcl-2 family contain 4 Bcl-2 homologous (BH) domains. The BH3 domain contains the active binding site, which consists of alpha-helices embedded within a hydrophobic groove.[3] The BH3 domain allows for heterodimerization with Bcl-2 and Bcl-xl, while its hydrophobic c-terminal allows for outer mitochondrial membrane binding. BH1 and BH2 domains are suspected to be the pore-forming domains of the protein.[6] The full extent of the conformational changes that occur during protein binding to both regulators and the MOM has yet to be seen. Still, it is an area of research that may elucidate possible pharmacological interventions in pathological processes that involve apoptotic dysregulation.
Function
The Bax gene encodes BCL2L4 protein that, upon activation, heterodimerizes with Bcl2 family proteins and alters cellular mitochondria to induce cell death.[7] When Bax protein is activated, it binds and induces MOM permeabilization. Such permeabilization results in mitochondrial swelling and rupture with subsequent leakage of intermembrane space proteins, specifically cytochrome c and endonuclease G.[10] Every mechanism in which Bax increases mitochondrial membrane permeability is poorly understood, but scientists know that binding to permeability transition pore (PTP) causes conformational changes that increase permeability.[11] However, PTP-knockout mice still exhibit Bax-dependent cell death; therefore, there have been suggestions that Bax can form pores in the MOM by inserting its amphipathic alpha-helical structure directly into the MOM.[6]
Mechanism
Bax protein exists constitutively in the cytoplasm until activated. It binds to multiple anti-apoptotic proteins, such as Bcl-xl, that inhibit Bax translocation to the MOM; therefore, Bax-mediated apoptosis depends on the concentration of both pro- and anti-apoptotic proteins.[6] Bax becomes activated by binding upregulated death signals such as BH3-only proteins or t-Bid at its BH3 hydrophobic pocket domain. Activation causes conformational changes that lead to translocation to the MOM, where Bax induces mitochondrial outer membrane permeabilization (MOMP) by 2 possible mechanisms described above.[6][2] MOMP leads to mitochondrial swelling and rupture, causing the release of periplasmic proteins such as cytochrome C.[2] Cytochrome C binds and activates cytosolic caspases, which are known to be the effector proteases of cell death.[2][10]
Testing
Researchers originally discovered Bax using immunoprecipitation studies with Bcl-2.[6] They discovered its genetic location using FISH and parsed out its function using various methods.[5] Most recent studies aim to identify Bax's role in pathological diseases using genetic knockout models or virally infected over-expression of Bax.[2][12]
Pathophysiology
Because of the Bax gene's role in cell apoptosis, dysfunction carries links to various pathologies related to cell accumulation (i.e., cancer) or cell loss (ie, heart failure, Alzheimer disease).[2] Tumorigenesis largely depends on the cell's ability to overcome cell cycle regulators and apoptosis. Research has shown that both loss of function and gain of function mutations in Bcl-2 family proteins, including Bax, increase the likelihood of human tumorigenesis.[13] Research demonstrates that Bax deletion increases the oncogenic gene activation and the progression of certain cancers, such as Philadelphia-positive leukemia.[12]
Clinical Significance
Bax dysregulation can lead to apoptotic dysfunction, increasing the likelihood of multiple pathologies. As a tumor suppressor gene, Bax has a critical role in preventing tumorigenesis, and loss of function often occurs in human tumors. Loss of function mutations can also lead to incomplete negative selection and peripheral tolerance dysfunction, thus causing autoimmune complications. Chronic neurodegenerative conditions, such as Huntington disease, Parkinson disease, or Alzheimer disease, often depend on aberrant apoptotic cell death. Lastly, although necrosis is the primary mechanism of injury during myocardial infarction, it has been seen that reperfusion injury is an apoptosis-driven event.[13]
References
Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, Green DR. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science (New York, N.Y.). 2004 Feb 13:303(5660):1010-4 [PubMed PMID: 14963330]
Level 3 (low-level) evidenceReed JC. Mechanisms of apoptosis. The American journal of pathology. 2000 Nov:157(5):1415-30 [PubMed PMID: 11073801]
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Level 3 (low-level) evidenceApte SS, Mattei MG, Olsen BR. Mapping of the human BAX gene to chromosome 19q13.3-q13.4 and isolation of a novel alternatively spliced transcript, BAX delta. Genomics. 1995 Apr 10:26(3):592-4 [PubMed PMID: 7607685]
Narita M, Shimizu S, Ito T, Chittenden T, Lutz RJ, Matsuda H, Tsujimoto Y. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proceedings of the National Academy of Sciences of the United States of America. 1998 Dec 8:95(25):14681-6 [PubMed PMID: 9843949]
Level 3 (low-level) evidenceShamas-Din A, Kale J, Leber B, Andrews DW. Mechanisms of action of Bcl-2 family proteins. Cold Spring Harbor perspectives in biology. 2013 Apr 1:5(4):a008714. doi: 10.1101/cshperspect.a008714. Epub 2013 Apr 1 [PubMed PMID: 23545417]
Level 3 (low-level) evidenceOltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993 Aug 27:74(4):609-19 [PubMed PMID: 8358790]
Level 3 (low-level) evidenceEdlich F,Banerjee S,Suzuki M,Cleland MM,Arnoult D,Wang C,Neutzner A,Tjandra N,Youle RJ, Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol. Cell. 2011 Apr 1; [PubMed PMID: 21458670]
Zhang Y, Chen X, Gueydan C, Han J. Plasma membrane changes during programmed cell deaths. Cell research. 2018 Jan:28(1):9-21. doi: 10.1038/cr.2017.133. Epub 2017 Oct 27 [PubMed PMID: 29076500]
Kuwana T, Newmeyer DD. Bcl-2-family proteins and the role of mitochondria in apoptosis. Current opinion in cell biology. 2003 Dec:15(6):691-9 [PubMed PMID: 14644193]
Level 3 (low-level) evidenceSchwarz M, Andrade-Navarro MA, Gross A. Mitochondrial carriers and pores: key regulators of the mitochondrial apoptotic program? Apoptosis : an international journal on programmed cell death. 2007 May:12(5):869-76 [PubMed PMID: 17453157]
Shi L, Long YY, Sha MQ, Luo X, Huang P, Chen Y. [BAX Deletion Accelerates Progression of BCR-ABL-Induced B-ALL in Mice]. Zhongguo shi yan xue ye xue za zhi. 2020 Feb:28(1):29-33. doi: 10.19746/j.cnki.issn.1009-2137.2020.01.006. Epub [PubMed PMID: 32027249]
Kelly PN, Strasser A. The role of Bcl-2 and its pro-survival relatives in tumourigenesis and cancer therapy. Cell death and differentiation. 2011 Sep:18(9):1414-24. doi: 10.1038/cdd.2011.17. Epub 2011 Mar 18 [PubMed PMID: 21415859]
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