THE CHAPERONE UNC-45 HAS A CRUCIAL ROLE IN MAINTAINING THE STRUCTURE AND FUNCTION OF THE MUSCLE CONTRACTILE APPARATUS DURING AGING Restricted; Files Only
Matheny, Courtney (Summer 2022)
Published
Abstract
As longevity increases, age-related diseases will become a greater public health concern. Sarcopenia is the age-related decline in muscle mass and function without any underlying disease. The molecular mechanisms responsible for this pathology remain unknown. Muscle function is dependent on having properly organized and functioning thick filaments, which are primarily composed of myosin. UNC-45 is required for the folding of the myosin head initially after translation and likely re-folds the myosin head to regain functionality after thermal or chemical stress causes unfolding. UNC-45 was first discovered using C. elegans, which is an excellent model organism for studying muscle biology and aging. We observe an early onset of sarcopenia when UNC-45 is perturbed during adulthood. We observe that during adult aging, there is a sequential decline of HSP-90, UNC-45, and then myosin. Myosin and UNC-45 protein decline are independent of steady state mRNA levels. Loss of UNC-45 is correlated with an increase in phosphorylation of the protein. By mass spectrometry S111 has been identified as being phosphorylated and this modification may affect binding to HSP-90. A longevity mutant with delayed onset of sarcopenia also shows a delay in the loss of HSP-90, UNC-45, and myosin. We also see a decrease in UNC-45 protein, but not transcript, in an hsp-90 loss of function mutant, suggesting a role for HSP-90 in stabilizing UNC-45. These results lead us to propose the model that during aging, a loss of HSP-90 leads to UNC-45 being post translationally modified, such as phosphorylation, and degraded, which then leads to a loss of myosin, and thus reduction in muscle mass and function. A better understanding of how myosin and its chaperone proteins are regulated and affected by aging will lead to better preventative care and treatment of sarcopenia and, possibly, the age-related decline of heart muscle function.
Table of Contents
Table of Contents
Chapter 1: Introduction ……………………………………………………………………………1
Muscle Anatomy…………………………………………………………………………………….1
Overview ……………………………………………………………………………………..1
Myosin structure and mechanism…………………………………………………………..3
The myosin head chaperone UNC-45……………………………………………………………..5
Discovery and importance ……………………………………………………………………5
Structure and mechanism …………………………………………………………………….7
Interacting proteins …………………………………………………………………………….9
Sarcopenia …………………………………………………………………………………………….11
Overview ………………………………………………………………………………………..11
Molecular discoveries made in models other than C. elegans ……………………………12
Therapeutic discoveries made in models other than C. elegans …………………………15
Caenorhabditis elegans as a model organism to study muscle assembly, maintenance, and aging ………………………………………………………………………………….17
Overview of C. elegans as a model to study muscle ……………………………..17
Muscle aging discoveries made using C. elegans ………………………………...19
Scope of Dissertation ………………………………………………………………………………..25
Figures ………………………………………………………………………………………………….28
Figure 1.1………………………………………………………………………………………..28
Figure 1.2………………………………………………………………………………………..29
Figure 1.3………………………………………………………………………………………..30
Figure 1.4………………………………………………………………………………………..31
Figure 1.5………………………………………………………………………………………..32
Figure 1.6………………………………………………………………………………………..33
Literature cited…………………………………………………………………………………………35
Chapter 2: In vivo mutational analysis of conserved residues of UNC-45 using C. elegans
Introduction ……………………………………………………………………………………………44
Results…………………………………………………………………………………………………..49
Dominant-negative inhibition of thick filament assembly by expression of mutant UCS proteins in C. elegans …………………………………………………………………………49
Effects on unc-45 temperature sensitive mutants on sarcomere organization, MHC B and UNC-45 protein levels…………………………………………………………………….51
Discussion ……………………………………………………………………………………………..53
Materials and methods ……………………………………………………………………………….60
Figures and Tables…………………………………………………………………………………….63
Figure 2.1………………………………………………………………………………………..63
Figure 2.2………………………………………………………………………………………..64
Figure 2.3………………………………………………………………………………………..65
Figure 2.4………………………………………………………………………………………..66
Figure 2.5………………………………………………………………………………………..67
Figure 2.6………………………………………………………………………………………..68
Figure 2.7……………………………………………………………………………………….69
Table 2.1………………………………………………………………………………………..70
Table 2.2………………………………………………………………………………………..70
Acknowledgements ………………………………………………………………………………….70
Literature cited ……………………………………………………………………………………….71
Chapter 3: UNC-45 has a crucial role in maintaining muscle sarcomeres during aging in C. elegans
Introduction ……………………………………………………………………………………………75
Results…………………………………………………………………………………………………..77
C. elegans develop sarcopenia and show decreased numbers of assembled thick filaments during aging………………………………………………………………………….77
UNC-45 has a role in maintaining assembled thick filaments and nematode motility during adulthood………………………………………………………………………………..78
UNC-45 degradation may be increased in aging muscles…………………………………79
UNC-45 phosphorylation increases with aging……………………………………………...80
A delayed onset of Sarcopenia is associated with increased UNC-45 and HSP-90……82
HSP-90 may play a pivotal role in UNC-45 protein regulation…………………………….83
Discussion………………………………………………………………………………………………84
Materials and Methods………………………………………………………………………………..88
Figures..…………………………………………………………………………………………………94
Figure 3.1………………………………………………………………………………………..94
Figure 3.2………………………………………………………………………………………..95
Figure 3.3………………………………………………………………………………………..96
Figure 3.4………………………………………………………………………………………..96
Figure 3.5………………………………………………………………………………………..97
Figure 3.6………………………………………………………………………………………..98
Figure 3.7………………………………………………………………………………………..99
Figure 3.8………………………………………………………………………………………100
Figure 3.9………………………………………………………………………………………101
Supplemental Figures and Tables………………………………………………………………102
Table S3.1……………………………………………………………………………………..102
Figure S3.2…………………………………………………………………………………….103
Figure S3.3…………………………………………………………………………………….103
Figure S3.4…………………………………………………………………………………….104
Figure S3.5…………………………………………………………………………………….105
Figure S3.6…………………………………………………………………………………….106
Figure S3.7…………………………………………………………………………………….106
Graphical Abstract…………………………………………………………………………….107
Acknowledgments……………………………………………………………………………………107
Literature cited………………………………………………………………………………………..108
Chapter 4: Indole improves aging muscle mass and function and increases Myosin in aged nematodes in an AHR-1/HSP-90/UNC-45 dependent manner
Introduction …………………………………………………………………………………………..113
Results………………………………………………………………………………………………115
Indole protects against loss of muscle mass and function with age………………….115
Indole increases myosin MHC B and its chaperone UNC-45…………………………116
Indole’s protective qualities are AHR-1, HSP-90, and UNC-45 dependent…………..118
Indole causes more HSP-90 to bind to UNC-45…………………………………………119
Discussion…………………………………………………………………………………………….120
Materials and Methods………………………………………………………………………………122
Figures ………………………………………………………………………………………………...126
Figure 4.1………………………………………………………………………………………126
Figure 4.2………………………………………………………………………………………127
Figure 4.3………………………………………………………………………………………128
Figure 4.4………………………………………………………………………………………129
Supplemental Figures ………………………………………………………………………………130
Figure S4.1…………………………………………………………………………………….130
Figure S4.2…………………………………………………………………………………….131
Figure S4.3…………………………………………………………………………………….132
Acknowledgments …………………………………………………………………………………..133
Literature cited………………………………………………………………………………………..134
Chapter 5: Conclusions and future directions
Overview of findings and significance……………………………………………………...……137
Conserved regions of the UCS domain of UNC-45 are essential for thick filament assembly and organization…………………………………………………………………..137
UNC-45 has a crucial role in maintaining muscle sarcomeres during aging in C. elegans…………………………………………………………………………………………138
Indole improves aging muscle mass and function and increases Myosin in aged nematodes in an AHR-1/HSP-90/UNC-45 dependent manner………………………….141
Additional data – UNC-45 is increased during stress ……………………………………142
Pitfalls and technical difficulties…………………………………………………………………..143
Future directions …………………………………………………………………………………….144
Identification of new clients of UNC-45, as well as new PTMs of UNC-45……………144
Increasing UNC-45 in older animals to alleviate sarcopenia…………………………….145
Knocking down HSP-90 or UNC-45 in young adults……………………………………..146
Analysis of TPR domain unc-45 mutants from the million-mutation project……………146
High throughput drug screening…………………………………………………………….147
Analysis of UNC-45 Phosphorylation………………………………………………………150
Figures …………………………………………………….………………………………………….151
Figure 5.1……………………………………………………………………………………...151
Figure 5.2……………………………………………………………………………………...152
Figure 5.3……………………………………………………………………………………...153
Figure 5.4……………………………………………………………………………………...153
Figure 5.5……………………………………………………………………………………...154
Acknowledgments……………………………………………………………………………………155
Literature Cited ………………………………………………………………………………………155
Abbreviations…………………………………………………………………………………………157
About this Dissertation
| School | |
|---|---|
| Department | |
| Subfield / Discipline | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| Keyword | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
File download under embargo until 19 August 2024 | 2022-06-24 10:12:45 -0400 | File download under embargo until 19 August 2024 |
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|