Ex) Article Title, Author, Keywords
Ex) Article Title, Author, Keywords
DTT 2022; 1(1): 40-44
Published online July 31, 2022
https://doi.org/10.58502/DTT.22.007
Copyright © The Pharmaceutical Society of Korea.
Kang Mu Kwon1,2 , Hyo Jung Kim1,2
Correspondence to:Hyo Jung Kim, hyojungkim@woosuk.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Proteins are constantly synthesized and degraded to maintain protein homeostasis. The ubiquitin-proteasome system is well known as a targeted protein breakdown process in eukaryotic cells. Although the archaea and actinobacterial families have a relevant system, many prokaryotic cells lack proteasomes. HslUV, a bacterial heat shock protein, is known to perform a proteasomal activity. HslU functions as ATPase, HslV is responsible for protease, and both activities are tightly connected in the two-component HslUV system. Here, we identified the characteristics of HslV from Staphylococcus aureus. Although only multimeric HslUV shows its activity, monomer S. aureus HslV remains stable and successfully crystallized. This study will provide a detailed understanding of the bacterial proteasomal system.
KeywordsHslU, HslV, bacterial proteasomal system, ubiquitin-proteasome, Staphylococcus aureus
Cells continuously create and destroy proteins to maintain protein homeostasis; this cycle is referred to as protein turnover. The protein turnover rate can change in response to internal or external stimuli, including hormones, inflammation, or low energy status (Rolfs et al. 2021). Consequently, misfolded, damaged, or aged proteins are removed from the cellular protein pool and replaced with new proteins. Protein degradation occurs in eukaryotes via two types of proteolytic machineries: the lysosome and proteasome (Ciechanover 2005). In lysosomal proteolysis, proteins are engulfed by membrane-enclosed vesicles, such as autophagosomes or endocytic vesicles. Conversely, the ubiquitin-proteasome system uses a diverse collection of E1, E2, and E3 ubiquitin ligases to selectively target proteins for degradation by the proteasome (Park et al. 2020). The proteasome is a 2,500-kDa cylindrical protein complex containing a 20S catalytic core particle capped by either one or two 19S regulatory particles (Tanaka 2009; Choi et al. 2021). The 20S core particle is responsible for the proteasome’s proteolytic activities using its central chamber, while the 19S regulatory particle recognizes polyubiquitnated substrates. All 20S core particles are composed of the outer (α subunits) and inner (β subunits) heptameric rings (Kunjappu and Hochstrasser 2014). A major function of the α subunit ring is to regulate entry into the central proteolytic chamber, while the β subunit cleaves targeted proteins. However, most bacteria do not have proteasomes. Instead, molecular architecture HslUV (originally discovered in
In this research, a preliminary study of HslV from
The
SDS-PAGE was conducted according to the Laemmli method using 15% (w/v) polyacrylamide gel (Laemmli 1970). The samples were treated with 1% (w/v) SDS and 5% (v/v) 2-mercaptoethanol before electrophoresis in a vertical Mini Gel system (Bio-Rad Laboratories, USA). The proteins were stained with Coomassie Brilliant Blue R250 (Thermo Scientific, USA).
Size-exclusion chromatography was employed to determine the oligomeric state of
Crystals of
For data collection under cryogenic conditions, the crystals were transferred to a cryoprotectant solution with 20% (v/v) glycerol under crystallization conditions for several minutes before being flash frozen in a stream of nitrogen gas at 100 K. Data were obtained from ADSC Quantum 270 CCD in beamline 5C of Pohang Accelerator Laboratory, Republic of Korea (Fig. 4). The peaks obtained from consecutive diffraction images were indexed and integrated using the Scalepack package of HKL2000 software (Otwinowski and Minor 1997). The integrated data were processed to refine the electron density by applying a scale factor. The unit cell of
Table 1 Crystallographic data collection
Data collection | |
Beamline | PAL-5C |
Wavelength (Å) | 0.97 |
Rotation range per image (°) | 1 |
Total rotation range (°) | 180 |
Exposure time per image (s) | 1 |
Space group | C2 |
Unit cell parameters (Å) | a = 221.407 |
b = 209.523 | |
c = 6309.842 | |
Resolution (Å) | 40.0−3.5 |
Total no. of reflections | 31556 |
Rmerge | 0.76 (0.80)a) |
CC1/2 | 0.335 |
I/sigma | 14.9 (13.0) |
a)Numbers in parentheses indicate the statistics for the last resolution shell.
The HslUV is a large complex with > 600 kDa molecular weight, 125 Å width, and 150 Å height. Two hexamers of HslV and 1-2 hexamers of HslU consist the HslUV arrangement. The HslV forms an internal cavity to cleave target proteins, and HslU activates HslV by N-terminal cleavage.
No potential conflict of interest relevant to this article was reported.
We thank the beamline staff members at Pohang Light Source (BL-5C), Republic of Korea. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (No. 2022R1F1A1065273) and “Leaders in INdustry-university Cooperation 3.0” Project, supported by the Ministry of Education and National Research Foundation of Korea.
DTT 2022; 1(1): 40-44
Published online July 31, 2022 https://doi.org/10.58502/DTT.22.007
Copyright © The Pharmaceutical Society of Korea.
Kang Mu Kwon1,2 , Hyo Jung Kim1,2
1College of Pharmacy, Woosuk University, Wanju, Korea
2Research Institute of Pharmaceutical Sciences, Woosuk University, Wanju, Korea
Correspondence to:Hyo Jung Kim, hyojungkim@woosuk.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Proteins are constantly synthesized and degraded to maintain protein homeostasis. The ubiquitin-proteasome system is well known as a targeted protein breakdown process in eukaryotic cells. Although the archaea and actinobacterial families have a relevant system, many prokaryotic cells lack proteasomes. HslUV, a bacterial heat shock protein, is known to perform a proteasomal activity. HslU functions as ATPase, HslV is responsible for protease, and both activities are tightly connected in the two-component HslUV system. Here, we identified the characteristics of HslV from Staphylococcus aureus. Although only multimeric HslUV shows its activity, monomer S. aureus HslV remains stable and successfully crystallized. This study will provide a detailed understanding of the bacterial proteasomal system.
Keywords: HslU, HslV, bacterial proteasomal system, ubiquitin-proteasome, Staphylococcus aureus
Cells continuously create and destroy proteins to maintain protein homeostasis; this cycle is referred to as protein turnover. The protein turnover rate can change in response to internal or external stimuli, including hormones, inflammation, or low energy status (Rolfs et al. 2021). Consequently, misfolded, damaged, or aged proteins are removed from the cellular protein pool and replaced with new proteins. Protein degradation occurs in eukaryotes via two types of proteolytic machineries: the lysosome and proteasome (Ciechanover 2005). In lysosomal proteolysis, proteins are engulfed by membrane-enclosed vesicles, such as autophagosomes or endocytic vesicles. Conversely, the ubiquitin-proteasome system uses a diverse collection of E1, E2, and E3 ubiquitin ligases to selectively target proteins for degradation by the proteasome (Park et al. 2020). The proteasome is a 2,500-kDa cylindrical protein complex containing a 20S catalytic core particle capped by either one or two 19S regulatory particles (Tanaka 2009; Choi et al. 2021). The 20S core particle is responsible for the proteasome’s proteolytic activities using its central chamber, while the 19S regulatory particle recognizes polyubiquitnated substrates. All 20S core particles are composed of the outer (α subunits) and inner (β subunits) heptameric rings (Kunjappu and Hochstrasser 2014). A major function of the α subunit ring is to regulate entry into the central proteolytic chamber, while the β subunit cleaves targeted proteins. However, most bacteria do not have proteasomes. Instead, molecular architecture HslUV (originally discovered in
In this research, a preliminary study of HslV from
The
SDS-PAGE was conducted according to the Laemmli method using 15% (w/v) polyacrylamide gel (Laemmli 1970). The samples were treated with 1% (w/v) SDS and 5% (v/v) 2-mercaptoethanol before electrophoresis in a vertical Mini Gel system (Bio-Rad Laboratories, USA). The proteins were stained with Coomassie Brilliant Blue R250 (Thermo Scientific, USA).
Size-exclusion chromatography was employed to determine the oligomeric state of
Crystals of
For data collection under cryogenic conditions, the crystals were transferred to a cryoprotectant solution with 20% (v/v) glycerol under crystallization conditions for several minutes before being flash frozen in a stream of nitrogen gas at 100 K. Data were obtained from ADSC Quantum 270 CCD in beamline 5C of Pohang Accelerator Laboratory, Republic of Korea (Fig. 4). The peaks obtained from consecutive diffraction images were indexed and integrated using the Scalepack package of HKL2000 software (Otwinowski and Minor 1997). The integrated data were processed to refine the electron density by applying a scale factor. The unit cell of
Table 1 . Crystallographic data collection.
Data collection | |
Beamline | PAL-5C |
Wavelength (Å) | 0.97 |
Rotation range per image (°) | 1 |
Total rotation range (°) | 180 |
Exposure time per image (s) | 1 |
Space group | C2 |
Unit cell parameters (Å) | a = 221.407 |
b = 209.523 | |
c = 6309.842 | |
Resolution (Å) | 40.0−3.5 |
Total no. of reflections | 31556 |
Rmerge | 0.76 (0.80)a) |
CC1/2 | 0.335 |
I/sigma | 14.9 (13.0) |
a)Numbers in parentheses indicate the statistics for the last resolution shell..
The HslUV is a large complex with > 600 kDa molecular weight, 125 Å width, and 150 Å height. Two hexamers of HslV and 1-2 hexamers of HslU consist the HslUV arrangement. The HslV forms an internal cavity to cleave target proteins, and HslU activates HslV by N-terminal cleavage.
No potential conflict of interest relevant to this article was reported.
We thank the beamline staff members at Pohang Light Source (BL-5C), Republic of Korea. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST) (No. 2022R1F1A1065273) and “Leaders in INdustry-university Cooperation 3.0” Project, supported by the Ministry of Education and National Research Foundation of Korea.
Table 1 Crystallographic data collection
Data collection | |
Beamline | PAL-5C |
Wavelength (Å) | 0.97 |
Rotation range per image (°) | 1 |
Total rotation range (°) | 180 |
Exposure time per image (s) | 1 |
Space group | C2 |
Unit cell parameters (Å) | a = 221.407 |
b = 209.523 | |
c = 6309.842 | |
Resolution (Å) | 40.0−3.5 |
Total no. of reflections | 31556 |
Rmerge | 0.76 (0.80)a) |
CC1/2 | 0.335 |
I/sigma | 14.9 (13.0) |
a)Numbers in parentheses indicate the statistics for the last resolution shell.