Exploring Interactions of

Beclin-1 Protein with Bcl-2

Proteins

Fidan Aliyeva
English 324
July 29, 2016

Abstract
Beclin-1 is a highly conserved eukaryotic protein that mediates autophagy, an important
cellular process in which contents of the cell are degraded and recycled. Beclin-1 contains
BH3 (Bcl Homology) domain in its intrinsically disordered region. BH3 domain interacts
with cellular and viral Bcl-2 (B-cell Lymphoma 2) proteins. Autophagy is inhibited by this
interaction. Autophagy is a survival mechanism and its disruption can lead to
neurodegenerative disorders, muscular and infectious diseases, cancer, etc. Studying
interactions between BH3 domain of Beclin-1 and Bcl-2 proteins can advance scientific
knowledge and help in designing of therapeutic drugs for related conditions. Proposed
studies would include expression and purification of Beclin-1 and Bcl-2 proteins, Isothermal
Titration Calorimetry (ITC), and Western Blot.

Introduction
Beclin-1 Bcl-2-interacting coiled-coil protein is an important protein that is encoded by
the BECN1 gene, which is important for embryonic survival and normal development, and
also plays role in phagocytosis. (Yue) Beclin-1 protein plays a crucial role in the innate
immune response, tumor suppression, and embryonic development. It also mediates
autophagy, cell survival process where some of the cellular contents like damaged
organelles, long-lived or aggregated proteins, and pathogens, are degraded. Beclin-1 is
important for autophagys vesicle nucleation step. (Sinha) Autophagy is necessary for cells
survival during nutrient deprivation, environmental stress, aging, and infection. During
autophagy those intracellular debris are recycled to generate metabolic precursors like
amino acids and ATP. (Chen) When autophagy is disrupted it could lead to the
development of many conditions, such as neurodegenerative disorders, various types of
cancer, infectious diseases, etc. (Yonekawa) Inhibition of autophagy happens when BH3
(Bcl Homology) domain of Beclin-1 interacts with Bcl-2 (B-cell Lymphoma 2) proteins.
This interaction prevents Beclin-1 from assembling the pre-autophagosomal structure,
thus inhibiting autophagy. (Marquez) Understanding of the mechanism of those
interactions can be very important to determining how to control/affect them and
therefore regulate the process of autophagy. This proposal explains which studies can be
done to further the understanding of those interactions, which could shed light on how to
design therapeutic agents for treatment of diseases/disorders related to inhibition of
autophagy.

Beclin-1 domain architecture

Human Beclin-1 consists of 450 amino acid residues and can share 29-99% identity with
other eukaryotic homologs. The structure of the Beclin-1 was determined by performing
various structural, biophysical and bioinformatics studies, where the sequence
conservation was analyzed and four structurally distinct domains (regions) were
determined to be common to all Beclin-1 homologs. The four regions are an intrinsically
disordered region (IDR), a flexible helical domain (FHD), a coiled-coil domain (CCD), and
a --repeat autophagy-specific domain (BARAD). IDR of Beclin-1 contains BH3 (Bcl

Homology) domain, residues 105-130, but only in the presence of binding to Bcl-2 proteins.
IDRs boundaries or function are not well delineated which leaves room for new and
extensive research. FHD, CCD, and BARAD are highly conserved. From the research done
by several biochemical groups it is presumed that only IDR of Beclin-1 is important for
interactions with Bcl-2 proteins and the extensive research should be focusing on this
particular region of Beclin-1. (Mei)

Intrinsically disordered region (IDR)

Beclin-1 contains a long, almost one-third of the whole protein, intrinsically disordered
region at its C-terminus, residues 1-140. Disorder of the IDR of Beclin-1 has been confirmed
by multiple structural and biophysical studies. When a full-length Beclin-1 is within
phosphatidylinositol 3-kinase complexes a BH3 domain is recognized as an unconnected
to any polypeptide chain four-turn helix. However, no identifiable side-chain electron
density has been shown to be visible and other proteins within the complex are also
missing many residues in the same region. (Mei)
IDR of Beclin-1 lacks a well-packed hydrophobic core. It contains 35% polar and charged
residues, which are disorder-promoting residues. 6% of those residues are glycines and 6%
are prolines. IDRs are very poorly conserved among Beclin-1 homologs with the exception
of the short regions around the invariant (never changing) cysteine containing motifs
(CxxC), residues 18-21 and 137-140. (Mei)
IDR of Beclin-1 contains several binding motifs, which include Anchor regions, which are
sequences flanking or overlapping IDRs that are predicted by the program ANCHOR to
nucleate binding-associated folding, and Eukaryotic Linear Motifs (ELMs), which are
short, evolutionary plastic, linear sequence motifs shown to be key for various proteinprotein interactions that were identified using the ELM server17. IDR of Beclin-1 contains
three complete Anchor regions, residues 13-49, 79-103, 116-127, and a fourth Anchor region
consists of residues 137-145 that extends into the FHD (residues 141-171). BH3 domain (10513) includes the third Anchor region (116-127). (Mei)

BH3 domain was the first Beclin-1 -MoRF (-molecular recognition feature), region that
undergoes disorder-to-helix transitions upon binding to partners, to be identified. BH3
domain is disordered, but it folds into a helix upon binding to various Bcl-2 proteins or in
the presence of 2,2,2-trifluoroethanol (TFE), a chemical that includes helicity in -MoRFs
even in the absence of their binding partners. Residues 76-105 of IDR of Beclin-1, which
includes the second Anchor region, is likely also an -MoRF, whereas residues 50-78,
which do not contain an Anchor region, do not form an -MoRF. (Mei)

Bcl-2 proteins
Bcl2 (B-cell lymphoma 2) is a family of evolutionary related proteins that govern
mitochondrial outer membrane permeabilization, which is responsible for engaging the
apoptotic cascade in numerous cell death pathways. (Erlich) All proteins in Bcl2 family
share at least one of four homologous regions which are called Bcl homology (BH)
domains including BH1, BH2, BH3, BH4. Those BH domains control Bcl2 protein
interactions and contribute to the function of these proteins in apoptosis and cell survival.
There have been 25 members of Bcl2 family identified and they can be divided into three
subgroups, pro-apoptotic, anti-apoptotic, also known as pro-survival, and BH3-only
proteins that are also pro-apoptotic in nature. Pro-apoptotic Bax-like proteins include Bax,
Bak, Bok, and Bik which promote cell death. Anti-apoptotic Bcl2-like proteins include
Bcl2, Bcl-xL, Mcl-1, Bcl-w and A-1 which suppress cell death and promote cell survival. Proapoptotic BH3-only proteins like Bad, Bid, Bim, Noxa, and Puma can interact with either
of the other two subgroups of the Bcl2 family members. (Elmore)
BH3 domains were originally discovered in the context of apoptosis regulators and they
mediate binding of pro-apoptotic Bcl-2 family members to anti-apoptotic Bcl-2 family
members. BH3 domains also function in the regulation of autophagy, pathway involved in
cellular and tissue homeostasis. Anti-apoptotic Bcl-2 homologs downregulate autophagy
through interactions with the essential autophagy effector Beclin-1. BH3 domain of Beclin1 is necessary and sufficient for binding to anti-apoptotic Bcl-2 homologs and required for
Bcl-2-mediated inhibition of autophagy. BH3 domain of Beclin-1 serves as a key structural
motif that enables Bcl-2 to function not only as an anti-apoptotic protein, but also as an

anti-autophagy protein. (Sinha) Beclin-1 is a key determining factor as to whether cells

undergo autophagy or apoptosis. (Marquez)

Objective
By performing various studies and experiments and then analyzing collected data the
mechanism by which BH3 domain of Beclin-1 in its IDR binds to Bcl-2 proteins can be
better understood. Understanding of those interactions can lead to the advancement in
scientific and medical knowledge, which can benefit many people who suffer from
conditions related to inhibition of autophagy by interactions of BH3 domain of Beclin-1
with Bcl-2 proteins.

Methods
To study the interactions of BH3 domain of Beclin-1 with Bcl-2 proteins both of the
proteins have to be expressed and purified. First of all, bacterial transformation, a process
by which foreign DNA (e.g. plasmid) is introduced into a bacterial cell, would be
performed to clone in a plasmid, circular and double-stranded DNA molecule distinct
from a cell's chromosomal DNA, which contains desired tag and the protein of interest
(Beclin-1 or Bcl-2). Bacterial cells now containing a protein of interest would undergo
protein expression, a process of protein synthesis, modification, and regulation in living
cells (bacteria), at certain conditions (temperature of incubation, amount of antibiotic,
speed of shaker during incubation, etc.) that would have to be determined by trial and
error. IPTG (Isopropyl -D-1-thiogalactopyranoside), a molecular mimic of allolactose, a
lactose metabolite that triggers transcription of the lac operon, would be used to induce
protein expression. Cells would be then harvested and stored at very low freezing
temperature (~ -80oC) until needed for the next step, protein purification.
Protein of interest would be purified using affinity chromatography which makes use of
specific binding interactions between molecules. Protein would be lysed (breaking down
of the membrane) and the cell lysate would be incubated with the affinity support to allow
the protein of interest in the cell lysate to bind to the immobilized ligand. Then the non-

bound sample components would be washed away from the support using appropriate
buffers, solutions that would maintain the binding interaction between target and ligand.
Protein of interest would then be eluted from the immobilized ligand by altering the
buffer conditions so that the binding interaction no longer occurs. Each specific affinity
system requires its own set of conditions which would be determined by trial and error.
After affinity chromatography protein of interest would be concentrating to obtain higher
yields. During protein concentration the concentrators with filters (based on molecular
weight) would be used to allow excess buffer and impurities to be separated from the
protein of interest. Then the protein of interest would be further purified using size
exclusion chromatography, where molecules in solution are separated by their size.
After purification of both proteins, Beclin-1 and Bcl-2, the ITC (Isothermal Titration
Calorimetry) would be used to determine binding affinity of those proteins toward each
other. ITC is a label-free measurement of the binding affinity and thermodynamics of
biomolecular interactions. It works by directly measuring the heat that is either released
or absorbed during a biomolecular binding event.
Western Blot, an analytical technique used to identify specific amino-acid sequences in
proteins, would also be performed to verify expression levels of Beclin-1 and Bcl-2 proteins
in bacterial cells. First, protein of interest would be separated using gel electrophoresis.
Then the separated molecule would be transferred/blotted onto a second matrix (e.g.
nitrocellulose membrane). Next, the membrane would be blocked to prevent any
nonspecific binding of antibodies to the surface of the membrane. Transferred protein
would be complexed with an enzyme-labeled antibody as a probe. An appropriate
substrate would then be added to the enzyme and together they would produce a
detectable product such as a chromogenic precipitate on the membrane for colorimetric
detection. The most sensitive detection methods use a chemiluminescent substrate that,
when combined with the enzyme, produces light as a byproduct. The light output would
be captured using film that is designed for chemiluminescent detection and the intensity
of the signal should correlate with the abundance of the antigen on the membrane.

Methodological Reasoning
Prosed methods were selected based on the simplicity, cost, efficiency, accuracy, and
availability. Bacterial transformation is a relatively simple and cost efficient procedure of
introducing foreign DNA into a cell. Protein expression is a widely used method of
synthesizing substantial amount of protein of interest in living cells. Induction with IPTG
is also a widely accepted method used during protein expression. Protein purifications
using affinity chromatography and size exclusion chromatography methods are very
efficient and commonly used among researchers. The size exclusion chromatography uses
a machine, although expensive, is necessary and can be found at most advanced
laboratories. Isothermal Titration Calorimetry (ITC) is the only technique that can
simultaneously determine all binding parameters in a single experiment, which is very
efficient and it doesnt require modifications of binding partners, either with fluorescent
tags or through immobilization, which is simple and less expensive than other techniques.
Western Blot is an efficient method of determining if the protein of interest is present and
the molecular weight of the protein of interest can also be determined.

Ethical and Technical Concerns

At this point of the study there should be no ethical concerns since the proposed work
doesnt include tests/experiments that would involve humans or animals. If approved, the
research team would include highly qualified and trained individuals who will perform
experiments using all necessary safety precautions and standards.

Timeline
Task

Due Date

Rough draft of the Proposal

7/22/2016

Final draft of the Proposal

7/29/2016

Start date of the Project

9/1/2016 (pending approval)

End date of the Project

5/15/2017 (presumed date)

Publication of the Project

9/1/2017 (presumed date)

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