19. Key functional proteins in skeletal muscle Flashcards
What is the structure of a muscle fibre?
Skeletal muscle fibres are composed of bundles of myofibrils which are segmented into functional units called “sarcomeres”.
What is a sarcomere?
basic unit of measure within muscle, defined as the portion between two adjacent Z lines
It is the functional unit of a myofibril.
- contains:
- Nebulin to help align actin
- Titin to provide elasticity and stabilises myosin
- M line in the middle
- Actin & myosin
What is a costamere?
Rib like structures (=”Costal”) on the surface of the sarcolemma
They are specialised sites of transmembrane complexes, occurring over the entire sarcolemma, where the transmission of force is concentrated
Found specifically at the Z disk
The 3 states of transmembrane linkage - force transmission through costameres
A. relaxed
B. Contracted, forces transmitted
C. Contracted, forces not transmitted - TM complexes not linked to ECM
Where is the Vinculin-Talin-Integrin complex usually localised?
usually localised in adherence junctions, involved in cell adhesion, cell migration, also sometimes cell proliferation
integrin acts as a receptor for laminin at the basal lamina, link to proteins at Z line with interaction with vinculin-talin
α7β1 integrin - what are integrins composed of? And which isoforms are the most common?
Integrins are heterodimers composed of an α and β subunit.
22 heterodimers have been identified, composed of 16 α chains and 8 β chains
The α7B and β1D isoforms are the most common integrins found in adult skeletal muscle.
What is the function of the α7β1 integrin?
- Mediate the processes of cell adhesion & migration
- Regulate the intracellular organisation of the actin cytoskeleton
- Important role in many signalling processes
Talin 1 vs talin 2.
Talin 2 is expressed at higher levels in skeletal muscle than talin 1
Both have a talin head and talin rod.
Describe the role of talin 1.
- interacts with the cytoplasmic domain of β1 integrin and with focal adhesion kinase (FAK) and vinculin
- also binds to F-actin to establish a link between β1 integrins and the cytoskeleton (more mechanical function)
What happens when the talin 1 (TIn1) gene is lost in SM?
Leads to progressive myopathy caused by mechanical failure of myotendinous junctions (MTJs).
What happens when the talin 2 gene is lost?
Mice lacking talin 2 are viable and fertile but develop a myopathy with centrally located nuclei that is associated with defects in the maintenance of MTJs.
What happens when you lose both talin 1 and 2?
SEVERE FUNCTIONAL OUTCOME
Severe defects in myoblast fusion and sarcomere assembly.
*talin 1 and 2 are somewhat mutually exclusive but can perform same function
Describe the structure of vinculin.
1066 aa
Has 4 Vh subdomains:
Vh 1 - talin, α-actinin, α-catenin, IpaA
Linker region: signalling intermediate
Arp 2/3, ponsin, vinexin, VASP
Vt region: how it interacts with actin
F-actin, PIP2, paxillin
What is vinculin involved in? And what is the result of mutations in its splice variant?
Vinculin is a membrane-cytoskeletal protein involved in linkage of integrin to the actin cytoskeleton.
Muscle specific splice variant = Metavinculin
*Mutations in metavinculin are assoc. with Idiopathic dilated cardiomyopathy (DCM) in humans
What does the Dystrophin-Glycoprotein Complex do & where is it found?
It is specific to striated muscle fibres and is aka dystrophin associated complex. DGC is localised at costameres.
It is a multimeric protein complex that acts to link the basal lamina to the actin cytoskeleton in SM fibres.
What are the physiological roles of the DGC?
- stabilise the sarcolemma during contraction especially during forced lengthening
- DGC forms rib-like lattices on the cytoplasmic face of the sarcolemma - “costameres”
- mechanical links between the sarcomeres within muscle fibre and laminin in the ECM.
Describe Dystrophin & its brief structure.
Gene = 427kDa
Large rod-like protein enriched at SM sarcomeres.
Key intracellular component of the DGC.
Contains:
- 2 actin-binding domains (ABD)
- 24 Spectrin-like repeats
- 4 Hinge regions (H)
- 1 Cysteine-rich domain (CR)
- 1 C-Terminal domain (CT)
What is the function of dystrophin?
Acts to link the sarcolemma to the actin cytoskeleton by direct interactions with F-actin & βDystroglycan
May also act to modulate signalling pathways through interactions with Syntrophin & Dystrobrevin.
Describe in more depth the structure of the Dystrophin protein.
2 ACTIN BINDING DOMAINS:
- One N terminal, composed of calponin homology domains (CH)
- One centrally located, composed of spectrin-like repeats 11, 12, 13, 15, 17
24 SPECTRIN-LIKE REPEATS:
- Create the rod-like structure of the Dystrophin protein
4 HINGE REGIONS:
- regions of flexibility
1 CYSTEINE-RICH DOMAIN:
- composed of WW domain, two EF hand domains and a ZZ domain
- site of protein-protein interactions
1 C-TERMINAL DOMAIN:
- site of interaction with Syntrophin at SBD & Dystrobrevin at DBBD
What happens when Dystrophin is defective or absent?
Dissipation of forces through the myofiber become disrupted.
- membranes become permeable
- high rate of muscle fiber breakdown & regeneration
Duchenne’s Muscular Dystrophy: severe complete dysfunction of protein
Becker’s MD: allelic - partially functional protein
In the absence of Dystrophin, other DGC members fail to accumulate on the sarcolemma indicating a role for Dystrophin in stabilising these proteins.
*β-dg, β-sg, α-sg, α-db2, α1-syn, γ-sg
Absence results in:
- muscles containing varying muscle fibre size
- inflammatory cell infiltrate
- accumulating fibrous tissue
- membrane permeability.
Dystrophin can also regulate multiple signalling pathways via interactions via…?
Syntrophin/Dystrobrevin - coordinate the assembly of multiple signalling proteins to the DGC
- Grb2
- calmodulin
- SAPK
- nNOS
nNOS - produces NO during muscle contraction to facilitate vasodilation
What is the Dystroglycan complex composed of?
Laminin
αDystroglycan
βDystroglycan
αDystroglycan & βDystroglycan come from same protein, cleavage produces alpha
What is the function of the Dystroglycan complex?
Acts as a TM linker between the ECM & the plasma membrane/cytoskeleton
Links to the actin cytoskeleton of the muscle fibre by interaction with Dystrophin.
What are laminins and what are they composed of?
Laminins are a family of large, extracellular trimeric proteins.
Composed of α chain, γ chain & β chain
*α chain necessary for interaction w receptors
What is the function of laminin?
Forms direct link b/w ECM & Dystroglycan to maintain skeletal muscle structure
What happens when laminin is defective?
Congenital Muscular Dystrophy (CMD)
- genetic deficiency of laminin α2 chain (Merosin)
- variable muscle fibre size
- presence of immature fibres (central nucleation)
- increased connective tissue (fibrosis)
αDystroglycan
extracellular protein containing multiple sites for glycosylation
requires post-translational modifications for proper function
Function: receptor for laminin in SM
βDystroglycan
TM protein that binds αDystroglycan & Dystrophin
Function: provides link between subsarcolemmal cytoskeleton & ECM
Dystroglycan dysfunction
No known association b/w mutations in dystroglycan & human disease.
H/e, mutations in some glycosyltransferases result in Dystroglycan dysfunction as a result of lack of glycosylation.
Lack of glycosylation doesn’t affect Dystroglycan expression at the sarcolemma but it required for proper ligand binding (laminin).
P’lation of βdystroglycan targets βdystroglycan protein for destruction if not bound to dystrophin.
What is the sarcoglycan complex composed of?
αSarcoglycan, γSarcoglycan, δSarcoglycan, βSarcoglycan and sarcospan
Describe the function of the sarcoglycan complex?
Not fully understood, appears to have both mechanical and non mechanical roles that mediate interactions among ECM, sarcolemma & cytoskeleton.
Forms lateral association with dystroglycan complex.
May function to stabilise the DGC at sarcolemma.
What are the consequences of defective sarcoglycans?
Absence of sarcoglycans lead to alterations in membrane permeability, ultimately resulting in cell death.
Genetic deletion of sarcoglycan subunits results in a subset of Limb-girdle Muscular Dystrophy (LGMD), referred to as “sarcoglycanopathies”.
Desmin: what is it & what is its function?
Muscle specific type III intermediate filament protein expressed in skeletal, cardiac & smooth muscle
Function: links adjacent myofibers at level of the Z disc and binds myofibrils to the sarcolemma at level of costameres.
Desmin is not required for normal muscle development and is essential for the maintenance of myofibril integrity following stress
Desminopathy
- very rare diseases (only 60 patients reported to date)
- desmin-related myofibrillar myopathy
- mutation in desmin gene prevents formation of desmin filaments
- results in misalignment of sarcomeres, disorganisation of muscle fibres, and muscle cell death
What is Titin?
- Largest known protein in nature
- Anchored at Z & M lines of sarcomere
- Maintains rigid contact with the thick filament along the A band
- flexible along the I band
- “molecular bungee cord” of skeletal muscle - functions to protect muscle fibres from damage due to over stretching
What are Titin’s 4 main roles in skeletal muscle?
- Provide a structural framework through association with sarcomeric proteins
- Centering the thick filament within the sarcomere during contraction
- Act as a molecular spring
- Signalling mediator (has kinase domain)
Titinopathy
aka Tibial Muscular Dystrophy
(Limb Girdle muscular dystrophy 2J)
- Detected in the Finnish population (1/10000 occurrence)
- Alter the structure & function of Titin
- May disrupt Titin’s interactions with other sarcomeric proteins
- Disrupts normal muscle contraction, resulting in muscle weakness and wasting over time
- Not known why disease is confined to lower leg muscles.
