THE CHAPERONE UNC-45 HAS A CRUCIAL ROLE IN MAINTAINING THE STRUCTURE AND FUNCTION OF THE MUSCLE CONTRACTILE APPARATUS DURING AGING Open Access

Matheny, Courtney (Summer 2022)

Permanent URL: https://etd.library.emory.edu/concern/etds/wp988k979?locale=en
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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

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