<p>The
ubiquitin-proteasome system is responsible for cellular protein recycling, and
it is a crucial system to maintain proper protein balances in cells. Proteasome
is the main component of the system, and the system is tightly related to
multiple cellular processes. Malfunction of the proteasome could lead to
various diseases including cancer, neurodegenerative diseases and autoimmune
diseases. As a result, researchers have been developing small molecules to
target the proteasome to regulate its function. Currently, three small molecules
have been approved by FDA as proteasome inhibitors to treat hematological
cancer multiple myeloma. However, these small molecules inhibit the same
enzymatic subunit on the proteasome and drug resistance has been observed among
patients administrating these proteasome inhibitors. To develop new small molecules
to target the proteasome, we started to investigate the 19S regulatory particle
of the proteasome. In this work, we presented a workflow of discovering a small
molecule selective binder, TXS-8, to 19S regulatory particle subunit Rpn-6. We
also developed a series of assays to investigate the impact of small molecule
on proteasome activity. At last, we introduced the binding site study of TXS-8,
development of TXS-8-based PROTAC and new proteasome probe development.</p>
<p>We first developed a one-bead-one-compound
(OBOC) library to screen with Rpn-6 to discover potential binders to Rpn-6.
After careful evaluation and validation, TXS-8 was discovered as the best hit
from the screening. Our covalent pull-down experiment with cell lysate later confirmed
TXS-8 as a selective binder of Rpn-6 and proteomic analysis of the pulled down
protein also validated Rpn-6 as the major target of TXS-8.</p>
<p>We then investigated the impact of TXS-8 in
Rpn-6 overexpressed cancer cells like Ramos B-cell and multiple myeloma. TXS-8
was four-fold more toxic in these cells comparing to our control HEK-293T
cells. To understand the cause of cell death when dosed with TXS-8, we began to
investigate the impact of TXS-8 on proteasome activity, but some preliminary
results were inconsistent. By the same time, there is also lack of a general
workflow to investigate the impact of small molecules on proteasome activity.
Therefore, we developed a three-step process to illustrate the general workflow
using TXS-8 as an example. We first knocked down Rpn-6 in HEK-293T cells and
monitored proteasome activity changes with a cell permeable probe our lab
developed. We then transfected HEK-293T cells with a full-length foreign
protein and knocked down Rpn-6 in these cells. We later monitored the
degradation of the foreign protein when dosed with TXS-8. In the last step, we
monitored the proteasome activity changes in primary cell lines when dosed with
TXS-8. From these three steps, we successfully demonstrated a general workflow
to investigate if a small molecule can affect proteasome activity. We also
concluded that TXS-8 was unable to affect proteasome activity at non-lethal
concentration.</p>
<p> To
further investigate TXS-8 and provide guidance for future structural
optimization to improve potency, we proposed two methods on investigating the
general binding site of TXS-8 on Rpn-6 using cross-linking techniques that is
currently ongoing. We also modified TXS-8 into proteolysis targeting chimeras
(PROTACs) to investigate if TXS-8-based PROTAC can improve toxicity and
selectively induce Rpn-6 degradation in cells. However, no significant cell
toxicity or Rpn-6 degradation was observed when dosed with TXS-8-based PROTACs.</p>
Finally,
Due to limitation of cell permeable probes, we were unable to
investigate the impact of TXS-8 on the caspase-like β1 and trypsin-like β2
subunit of the proteasome in our previous studies. Although TXS-8 did not alter
the chymotrypsin-like activity at non-lethal concentration, examining the
effect of TXS-8 on the caspase-like and trypsin-like activity could still
benefit our research. Besides, we also desire to expand our proteasome activity
toolbox by developing more sensitive proteasome probes. Therefore, by analyzing
and combing the commercially available proteasome probes and LLVY-Rh probes, we
decided to develop selective proteasome probes for the β1 and β2 subunit to
provide useful tools for future potential small molecule proteasome regulator
characterization.
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/14993049 |
| Date | 16 July 2021 |
| Creators | Wenzhi Tian (11142939) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/DISCOVERY_OF_A_SELECTIVE_BINDER_OF_PROTEASOMAL_SUBUNIT_RPN-6_AND_ITS_EFFECT_ON_PROTEASOME_ACTIVITY/14993049 |
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