FILE 20220531 101817 81150130 NMDUC thesis final file 20th june 2019

Dissertation

Discovery of anti-tuberculosis drug candidates
targeting Mycobacterium tuberculosis ClpC1
through cell-free in-vitro assay

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Graduate School, Myongji University
Department of

Interdisciplinary Program of Bio-modulation

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Minh Duc Nguyen

Thesis Advisor Joo Won Suh

June, 2019

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Discovery of anti-tuberculosis drug candidates
targeting Mycobacterium tuberculosis ClpC1
through cell-free in-vitro assay

Submitted in partial fulfillment of the requirements for the
Ph.D. degree in Biological Science

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”

June, 2019
Graduate School. Myongji University
Department of
Interdisciplinary Program of Bio-modulation
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”

Minh Duc Nguyen

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Discovery of anti-tuberculosis drug candidates
targeting Mycobacterium tuberculosis ClpC1
through cell-free in-vitro assay
Graduate School, Myongji University
Department of Interdisciplinary Programs of Bio-modulation
“

Minh Duc Nguyen
We at this moment recommend that the dissertation by the
above candidate for the Ph.D. degree in Biological science be
accepted.
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Chair, Evaluation Committee
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June, 2019

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Acknowledgments
I would like to express my sincere gratitude to Professor Joo Won Suh and Professor
Lee Hanki, my research supervisors, for their patient guidance, enthusiastic encouragement,
and useful critiques of this research work. I would also like to thank Dr. YongU Kim, for his
advice and assistance in keeping my progress on schedule. My thanks are also extended to
Mr. Johnson Eldin for his help in doing data analysis, to Ms. Jinhua Cheng for her support in
my experiments.
I would also like to extend my thanks to the technicians of the laboratory of the center
for Nutraceutical and Pharmaceutical Materials for their help in offering me the resources in
running the program.
Finally, I wish to thank my family for their support and encouragement throughout my
study.

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Table of Contents

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List of Tables

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iv

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List of Figures

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Abstract

vii

Introduction

01

Tuberculosis

01

MDR-, XDR-TB and new anti-tuberculosis drugs development

5

ATP-dependent protease

10

Clp protease family

14

Clp ATPase and ClpP – the proteolytic component

15

Mycobacterium tuberculosis and its protein degradation system

16

Cell-free protein synthesis system from Escherichia coli cells

18

Molecular Docking

20

N-terminal of ClpC1 protein

22

The aim of this dissertation

24

Chapter 1 - Development of biochemical assay using ClpC1 for screening compounds
targeting ClpC1
1.1 Materials and Methods
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26
26

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1.1.1 Bacterial strains and Medium cultures
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26

1.1.2 Cloning and Purification DNA

27

1.1.3 Expression and Purification recombinant ClpC1 protein

28

1.1.4 SDS-PAGE for protein electrophoresis

29
1

1.1.5 ATPase activity assay

30

BIOMOL® GREEN reagent

30

ADPTM Glo reagent

31

1.1.6 The anti-tuberculosis drugs
1.2 Results

32
33

1.2.1 Cloning, Expression and Purification of ClpC1

33

1.2.2 ClpC1 Displays Basal ATPase activity

37

1.2.3 Comparison of Luminescence ADP Production Assay and Fluorescence Freephosphate Released Assay with Ecumicin and Rufomycin I
1.2.4 Testing the ATPase activity of ClpC1 with 1-, 2- line anti-TB drugs

40
45

Chapter 2 - Development of biochemical assay using ClpC1, ClpP1 and ClpP2 for
screening anti-tuberculosis lead compounds

48

2.1 Materials and Methods

48

2.1.1 Expression and Purification ClpP1 and ClpP2 protein

48

2.1.2 Measurement concentration protein with Bradford method

50

2.1.3 Exchanged buffer with PD-10 Desalting Column

51

2.1.4 Proteolytic activity of ClpC1/ P1/ P2 complex

52

FITC – Casein

52

SsrA – eGFP

53

2.2 Results
2.2.1 Cloning, Expression and Purification of ClpP1 and ClpP2

54
54

2.2.2 Comparison of FITC-Casein degradation and SsrA-eGFP degradation with
Ecumicin and Rufomycin I

57

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2.2.3 Testing the Proteolysis activity of ClpC1/ P1/ P2 complex with 1-, 2- line anti-TB
drugs

62

Chapter 3 - Screening anti-tuberculosis lead compounds through biochemical and
biophysical assay developed

65

3.1 Materials and Method

65

3.1.1 Prestwick Chemical Library

65

3.1.2 Isolation and Purification procedure of Ecumicin analogues

66

3.1.3 Protein and lead compounds structure

68

3.1.4 Modeling Docking

69

3.2 Results
3.2.1 The ATPase activity and Proteolysis activity with Ecumicin analogues

71
71

3.2.2 Screening anti-tuberculosis lead compounds from the Prestwick Chemical
Library
3.2.3 Docking to ClpC1

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84

Conclusion

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References

101

Korean Abstract

109

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List of Tables
Table 1. First- and second-line anti-tuberculosis drugs based on the World Health
Organization classification

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Table 2. ATPase activity of ClpC1 with Ecumicin and Rufomycin I

43

Table 3. The list of 10 anti-tuberculosis drugs with MIC unit and Molecular Weight

46

Table 4. ATPase activity of ClpC1 with Ecumicin analogues

73

Table 5. List lead compounds of the Prestwick Chemical Library

80

List of Figures

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Figure 1. Global tuberculosis (TB) death rate from 1992 to 2017 (WHO)

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Figure 2. Estimated TB incidence rates, 2017

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Figure 3. Current global pipeline of new anti-tuberculosis drugs

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Figure 4. Architecture and mechanism of ATP-dependent proteases

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Figure 5. ClpP1P2 and ClpP1P1 structures

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Figure 6. Structural Comparison of ClpA/C AAA + ATPase Domains

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Figure 7. Electrophoretic analysis of affinity chromatography using Ni-TED resin for
purification of recombinant Mycobacterium tuberculosis ClpC1

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Figure 8. LB Broth medium and SOB medium for expression recombinant ClpC1
protein

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Figure 9. Basal ATPase activity of recombinant ClpC1 protein

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Figure 10. The stability of recombinant ClpC1 protein in ATPase activity

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Figure 11. Structures of Ecumicin and Rufomycin I.

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Figure 12. ClpC1 ATPase activity in response to Ecumicin (ECU) and Rufomycin I
(RUFI) treatment
Figure 13. The ClpC1 ATPase activity in response to drugs treatment

44
47

Figure 14. Electrophoretic analysis of affinity chromatography using Ni - TED resin for
purification of recombinant Mycobacterium tuberculosis ClpP1

55

Figure 15. Electrophoretic analysis of affinity chromatography using Ni-TED resin for
purification of recombinant Mycobacterium tuberculosis ClpP2

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Figure 16. The proteolytic activity of the ClpC1/ P1/ P2 complex in response to
Ecumicin (ECU) and Rufomycin I (RUFI) treatment

60

Figure 17. The Proteolysis activity of ClpC1/ P1/ P2 complex with SsrA – eGFP as a
substrate

61

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Figure 18. The Proteolytic activity of the ClpC1/ P1/ P2 complex in response to drugs
treatment

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Figure 19. Structures of Ecumicin and analogues

67

Figure 20. N-terminal domain of Mycobacterium tuberculosis ClpC1 3WDB

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Figure 21. Basal ATPase activity of ClpC1 with Ecumicin and Ecumicin analogues

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Figure 22. The degradation FITC – Casein of analogues Ecumicin by ClpC1/ P1/ P2
complex

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Figure 23. ClpC1 ATPase activity (A) and proteolytic activity of the ClpC1/ P1/ P2
complex (B) after treated with 19 hit compounds

82

Figure 24. Images of a Docking Result and Structure Evaluations of 19 hit compounds
86

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Discovery of anti-tuberculosis drug candidates targeting
Mycobacterium tuberculosis ClpC1 through cell-free in-vitro assay

Minh Duc Nguyen
Department of Environmental Engineering and Biotechnology
Graduate School, Myongji University
Advisor Joo-Won Suh

Tuberculosis has been with humans for a long time, a disease that should have only
belonged to the past but is still growing today. Despite all efforts of humans to prevent and
control of tuberculosis, it still affects 8 - 9 million new TB cases and 2 million people died
from the disease annually. The global TB rate continues to increase by 1 % per year with the
widespread of drug-resistant TB. Therefore, the development and research to find new antiTB drugs are becoming an extremely urgent mission. Our studies will focus on research and
development the high throughput anti-tuberculosis drugs screening system, especially for invitro active with specific target protein of Mycobacterium tuberculosis, ClpC1 protein and
ClpCP complex, through Cell-free protein synthesis (CFPS) systems.
To be able to screen lead anti-tuberculosis drugs, currently, researchers have to carry
out directly on the cells of Mycobacterium tuberculosis. Thus, the requirement set out that all
of the experiments must be performed in bio-security facilities level 3 or 4, to prevent
infection from pathogens. These difficulties posed new challenges for the development of
new drugs against Mycobacterium tuberculosis. Based on this reason, the result from our
studies demonstrated that could perform the screening of anti-TB drug candidates in biosecurity facilities level 1 laboratory with the using Cell-free protein synthesis (CFPS) systems
based on Escherichia coli cell extract.

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ClpC1 is a common stress protein which is also involved in the heat-shock protein
HSP100 family. Beside, ClpC1 is also an integral part of the Mycobacterium tuberculosis
genome. We have focused on the ATP hydrolysis activity of ClpC1 and prevented protein
aggregation of ClpC1/ P1/ P2 protein complex to create a specific research direction for the
high-throughput anti-tuberculosis drugs screening system. Thus, ClpC1 protein was
overexpressed, purified and functionally characterized (93.5 kDa). The steady-state growth of
recombinant ClpC1 protein in Luria-Bertani (LB) Broth High Salt medium is maintained and
stabilize after extraction.
In Mycobacterium tuberculosis, ClpC1 is an ATP-dependent molecular chaperone.
Thus, the determination ATPase activity of ClpC1 was carried out by measurement the
released phosphate with BIOMOL® GREEN reagent or ADP generated from the reaction in
ADP GloTM kinases assay. This activity was not changed significantly and remained the
original activity in 10 days after extraction from Escherichia coli Cell-free protein synthesis
systems. Several potential compounds such as Ecumicin and Rufomycin I were chosen
becoming control compounds with expected ATP hydrolysis activities (Vmax = 4983.16 ±
1282.08; 366.96 ± 5.74, Hill coefficient = 1.19 ± 0.217; 1.974 ± 0.810, K d value = 0.52 ±
0.275; 0.02 ± 0.008 of Ecumicin and Rufomycin I, respectively).
Clp proteases are involved in several cellular processes such as degradation of
misfolded proteins, regulation of short-lived proteins and housekeeping removal of
dysfunctional proteins. To gain proteolytic activity, the ClpP multimer associates with one or
two hexameric rings of Clp ATPases, forming the ClpP‐containing proteolytic complex
(designated the ClpP protease). Therefore, in parallel with studies on ClpC1, experiments on
over-expression, purified and functional characteristics of ClpP1 and ClpP2 were carried out
simultaneously, 21.6 kDa and 23.5 kDa, respectively.

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The proteolysis experiments of the ClpC1/ P1/ P2 complex was carried out based on
the degradation of β - Casein or measurement SsrA - eGFP initial degradation. We showed

not only the ability of degradation protein substrate of the ClpCP complex but also the
significant differences under the effects on protease activity of Ecumicin and Rufomycin I
treatment similar to published studies. Under the effect of Ecumicin and Rufomycin I,
ClpC1's ATPase activities were abnormally stimulated up to 2 - 3 times (Hill coefficient =
1.19 ± 0.217; 1.974 ± 0.810, Kd value = 0.52 ± 0.275; 0.02 ± 0.008 of Ecumicin and
Rufomycin I, respectively) and proteolytic activities changed markedly. The biochemical
assay showed that lead compounds stimulate ClpC1’s ATPase activity by two different
mechanisms while inhibiting the proteolysis activity of ClpC1/ P1/ P2 complex.
Finally, we have used this high throughput screening system with three analogues of
Ecumicin and ten anti-TB drugs. Also, a Chemical library with more than 1,000 compounds
was accepted by the FDA, which has also been screened. Notably, we found 19 potential
compounds with a positive effect on ClpC1's ATPase activity and degradation protein
substrate of the ClpCP complex. We also analyzed the docking between ClpC1 N-terminal
and potential compounds in 3D structure. The results indicated that the presence of four
amino acids (ASN26; TYR27, VAL119, and ARG83) have appeared in Hydrogen bonds and
Van der Waal bonds which are weak and popular bonds in the intracellular. Based on these
results, we can build 2D or 3D pharmacopeia models for Pharmacophore-based virtual
screening with high-performance computing in the future. Pharmacophore approaches are
successful subfields of computer-aided drug design (CADD), which have become one of the
essential tools in hit identification, lead optimization, and rational design of novel anti-TB
drugs.

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Introduction
Tuberculosis
Tuberculosis is known to be one of the most dangerous diseases in human history.
Tuberculosis is most common in the lungs but can also affect the central nervous system
(meningitis), lymphatic system, circulatory system, genitourinary system, bones, and joints 61.

Currently, TB is the most common of bacterial infection, affecting 2 billion people or 1/3 of
the population, with 9 million new cases annually, causing 1.5 million deaths (estimated
2016), most of them in developing countries. Most (90 %) cases of TB infection are
asymptomatic. 10 % of those in their lifetime will develop symptoms of tuberculosis, and if
left untreated, it will kill 50 % of the victims. In 2016, about 4,100 people died every day,
compared with 3,300 cases in a few years ago, making it the world's deadliest infectious
disease. In 2017, 10 million people fell ill with TB, and 1.6 million died from the disease
(including 0.3 million among people with HIV), an estimated 1 million children became sick
with TB and 230,000 children died of TB (including children with HIV associated TB). The
death toll from the disease has plummeted, according to the WHO 62. Globally, TB incidence
is falling at about 2 % per year, with an estimated 54 million lives were saved through TB
diagnosis and treatment between 2000 and 2017. This expectation of accelerating to a 4 – 5 %
annual decline to reach the 2020 milestones of the End TB Strategy. However, the distraction
in TB control programs, the outbreak of the HIV/AIDS epidemic, and migration have
prompted the rise of tuberculosis. Multidrug-resistant TB (MDR-TB) remains a public health
crisis and a health security threat. WHO estimates that there were 558,000 new cases with
resistance to rifampicin – the most effective first-line drug, of which – 82 % had MDR-TB.
Multidrug-Resistant Tuberculosis (MDR) vaccines are on the rise. Thus, ending the TB
epidemic by 2030 is among the health targets of the Sustainable Development Goals61.

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Figure 1. Global tuberculosis (TB) death rate has fallen by almost half since 1990, but more
than 4,000 people a day are still dying from this preventable disease, says the World Health
Organization’s (WHO) Global tuberculosis report61.

2

Figure 2. Estimated TB incidence rates, 201762.

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The leading causative agent of most cases of tuberculosis is Mycobacterium
tuberculosis, a disease-causing bacterium in the Mycobacterium genus. First discovered in
1882 by Robert Koch, M. tuberculosis has an abnormal waxy coating on the surface of the
cell (mainly mycolic acid), due to its cellular water repellency that is easily accessible 47.
Easily detect acid substitutes used based on gram staining techniques. Under the microscope,
M. tuberculosis retains the dye after being treated with an acid solution, so it was classified as
"acid-fast bacillus" (AFB)46. This species is not classified gram positive or gram negative
because they do not have this chemical characteristic, although the cell walls contain
peptidoglycan. These are aerobic bacteria and require high levels of oxygen. Tuberculosis has
the appearance of a small rodent, which can tolerate weak disinfectants and survive in the dry
state for weeks, but in natural conditions only grows in the host. M. tuberculosis breaks down
every 15 - 20 hours, very slowly compared to other bacteria with a time-division in minutes
(Escherichia coli can divide around every 20 minutes). Also, due to the abnormal cell wall,
rich in fat (e.g., mycolic acid), M. tuberculosis is highly resistant, and this is also an
important virulence factor.

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MDR-, XDR-TB and new anti-tuberculosis drugs development
Prolonged treatment and poor compliance are the leading cause of drug-resistant TB
and treatment failure. The number of multi-drug resistant TB cases (MDR-TB, at least
Isoniazid (INH) and Rifampicin (RMP), the two most potent anti-TB drugs) reached nearly
480,000 worldwide, with around 3.5 % of new cases and 20.5 % of cases were previously
treated with MDR-TB21. Notably, extensively drug-resistant TB (XDR-TB) is a more severe

form of MDR-TB caused by bacteria that do not respond to the most effective second-line
anti-TB drugs, often leaving patients without any further treatment options. In 2017, MDRTB remained a public health crisis and a health security threat. About 8.5 % of MDR-TB
cases had extensively drug-resistant TB (XDR-TB) in 2017. Worldwide, only 55 % of MDRTB patients are currently successfully treated. In 2016, WHO approved the use of a short,
standardized regimen for MDR-TB patients who do not have strains that are resistant to
second-line TB medicines. This regimen takes 9 – 12 months and is much less expensive than
the conventional treatment for MDR-TB, which can take up to 2 years. Patients with XDRTB or resistance to second-line anti-TB drugs cannot use this regimen, however, and need to
be put on more extended MDR-TB regimens to which 1 of the new drugs (bedquiline and
delamanid) may be added62. MDR-TB treatment regimens are currently recommended by the
WHO for 20 months of treatment with a second drug for most patients and are associated
with multiple (and sometimes severe) side-effects and treatment rates, lower value.
Widespread drug-resistant TB (XDR-TB, MDR-TB plus fluoroquinolone resistance and at
least one of the other three injections) have been reported in 84 countries and accounts for 9
% of all drug-resistant tuberculosis cases21. Although treatable, anti-tuberculous drugs require
extensive chemotherapy with second line anti-tuberculosis drugs, are expensive and produce
severe unfavorable reactions. Drug-resistant TB may stem from genetic mutations that lead to
loss of sensitivity to antibiotics. Such modifications may occur at the target or drug trigger41.
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To summarize the most common variations can arise in the first or second line antituberculosis drugs, Table 1. The emergence of widespread TB drug resistance, in which
bacilli are resistant to Rifampicin, Isoniazid, Fluoroquinolone within at least one of the three
injections (e.g., Amikacin, Kanamycin, or Capreomycin) causing a severe threat to the
tuberculosis control41. The remaining patients with less effective treatment options. Therefore,
there is a high demand for new drugs to fight tuberculosis.
The Global Alliance for Drug Development () recommends
that an ideal new TB drug meet requirements: shorten the duration of treatment and improve
treatment for approximately 2 billion people MDR's infected Mycobacterium tuberculosis.
Moreover, which must be effective against latent TB, this is necessary to provide a
comprehensive assessment of recent and ongoing efforts to produce new anti-tuberculosis
drugs.

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Antibiotic

Mechanism and target
Inhibits mycolic acid
synthesis;

Isoniazid

Rifampicin

The primary target is InhA
and secondary objectives
are KasA and DfrA
Inhibits transcription;
RNA polymerase β-subunit

The mutation associated
with resistance
katG (required for drug
activation);
inhA (promoter mutations)

rpoB

First-line
Inhibits arabinogalactan

synthesis; possibly EmbB

embB

Pyrazinamide

Unknown (possible
inhibits FAS-I or alters
membrane energetics)

pncA (required for drug
activation)

Streptomycin

Inhibits protein synthesis
30S ribosomal subunit

rpsL and rrs

Fluoroquinolones

Inhibits DNA gyrase

gyrB

Ethionamide

Inhibits mycolic acid
synthesis; InhA

Ethambutol

Cycloserine
Second-line
p-aminosalicylic
acid

Inhibits peptidoglycan
synthesis by blocking the
synthesis

ethA (required for drug
activation)
inhA (promoter mutation)
alr (overproduction)
ddl (overproduction)

Inhibits folate metabolism;
Possibly dihydropteroate
synthase

thyA

Capreomycin

Inhibits protein synthesis

tlyA and rrs

Kanamycin

Inhibits protein synthesis

rrs

Amikacin

Inhibits protein synthesis

rrs
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Table 1. First- and second-line anti-tuberculosis drugs based on the World Health
Organization classification19.
However, during the 1960s, the available therapies developed relatively slowly.
Treatments with available anti-tuberculosis drugs are complicated not only the long duration
of treatment but also the various side effects and not to mention the interactions of the drug. It
is the urgent needs mentioned above that motivated the search for a new anti-tuberculosis
medicine with specific targets in tuberculosis. One of the popular emerging new destination
to inhibit Mycobacterium tuberculosis is a bacterial protease. Interactions among bacterial
protease and microbial pathogenesis have been established by various research, and the
results show that they are potential targets for the development of new antibacterial drugs 41.
The current criteria for developing new drug candidates and regimens for the treatment of TB
are more stringent:
1) Fully authenticated safety document;
2) Stronger than existing drugs to reduce treatment time;
3) Inhibit new targets for the treatment of tuberculosis and MDR-TB;
4) Compatible with antiretroviral therapy, as many patients co-infected with HIV;

5) No antagonism with the current tuberculosis drugs or other drug candidates to be
able to create a regimen that includes at least three active drugs;
6) Kill M. tuberculosis in its various physiological states.

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Figure 3. Current global pipeline of new anti-tuberculosis drugs. Agents currently in
discovery or development for the treatment of tuberculosis (TB) were show3.

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ATP-dependent protease
In bacterial protease, the role of intracellular protein level to adapt to the changing of
both external and internal conditions was determined not only by rates of synthesis but also
by rates of degradation. The half-lives for different proteins in the cell may range from
minutes to days. Moreover, the differential rates of protein degradation are an essential aspect
of protein quality control by eliminating short-lived regulatory proteins, misfolded and
damaged proteins that are arising from folding or assembly with slow speed, chemical or
thermal stress, internal structural instability, and biosynthetic errors41.
Researches have shown that the protease process in both prokaryotic and eukaryotic is
energy-dependent, with inhibition of ATP production in-vivo significantly inhibits protein
degradation. Not only Proteasome family of eukaryotic cells is an ATP-dependent protease,
but also different families have been characterized in bacteria, such as Clp, HslUV, FtsH, and
Lon. All of these proteases have several standard features. First, the access to active sites of
proteolysis was prevented to globular proteins by a restricted pore entrance to the protease
chamber; the protease is ‘self-compartmentalized’. It was assumed that this accessibility
prohibits many degradations of proteins by these proteases. Thus, a result of this process,
proteins must be unfolded and threaded into the protease chamber for degradation.

Moreover, the second common characteristic of these proteases is the requirement of
ATP hydrolysis, by ATPases of the AAA or AAA+ superfamily. The ATPase chaperones or
chaperonins will unfold and transport substrates into the proteolytic chamber. Final of
processive protein degradation, generating peptides of 9 – 13 amino acids, after releasing
from the chamber, that is further degraded by cellular peptidases. Notably, the feature of these
10


proteases has been indicated with the nonpathogenic model organisms Escherichia coli and
Bacillus subtilis41.

Figure 4. Architecture and mechanism of ATP-dependent proteases12.
(A) AAA+ proteases, adaptor proteins and accessory domains.
(B) Protein degradation by ATP-dependent proteases.

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The Lon protease family is a family of proteases. They were found in archaea,
bacteria, and eukaryotes. Escherichia coli Lon protease is the first ATP-dependent protease to
be identified. It is known to represent the ATP-dependent serine peptidases with associates to
form homooligomer consisting of four to eight copies of a single 784 amino acid polypeptide
chain, each subunit carry out both of the protease active site and the ATP binding site. The Nterminal domain (NTD) is mainly interdependent in the recognition and binding of target
proteins17. The central part of the chain (A domain) is the ATPase, while the proteolytic
domain (P domain) was located at its C terminus. In addition to proteolysis, Lon also proved
to be showing chaperone activity as well as promote membrane protein assembly.
Similar to Lon protease, the FtsH/AAA family is another ATP-dependent protease
exhibits chaperone activities. This protease is conserved in eubacteria, mitochondria, and
chloroplasts that are essential for the survival of Escherichia coli. Furthermore, FtsH is the
only protease as it is anchored to the inner membrane while other proteases are cytoplasm.

FtsH has N-terminally located transmembrane segments and a central cytosolic region
consisting of AAA-ATPase and Zn 2+-metalloprotease domains. It forms a homo-oligomer that
active simultaneous with ATPase and protease in a polypeptide33.
Meanwhile, the heat shock proteins HslV and HslU (call as ClpQ and ClpY,
respectively) also are expressed in Escherichia coli in response to cell stress. The HslV
protein is a protease, and the HslU protein is an ATPase, they form a symmetrical complex of
four stacked rings, a double-ring-shaped homohexamer of HslV is capped on each side by a
ring-shaped HslU homohexamer with, and a central hole in which the protease and ATPase
active sites reside4. The mechanism of HslVU degradation is reviewed by Groll et al. 200531.
Besides that, this complex is thought to resemble the hypothetical ancestor of the proteasome,
12