Important Proteins D103

Thymosin

Actin dynamics - sequesters G-actin-ATP, prevents it from being hydrolyzed

Profilin

Actin dynamics - promotes restoration of G-actin-ADP to G-actin-ATP, necessary for control of leading edge during cell migration

Cofilin

Actin dynamics - Negative regulator of actin polymerization, accelerates depolymerization at the (-) end, necessary for control of leading edge during cell migration

Formin

Actin dynamics - promotes nucleation of linear actin, necessary for formation of stress fibers

WASP + ARP2/3

Actin dynamics - promotes branching of actin filaments

Small GTPases

Proteins regulated by GTP/GDP binding. Activated by GEFs, deactivated by GAFs, anchored to a membrane with a lipid tail but are not embedded in it.

RHO

Small GTPase, its GEF recruits Formin to promote linear actin filament formation. RHO contributes to the formation of stress fibers.

CDC42

Small GTPase, its GEF activates WASP-ARP2/3 and leads to actin branching and filopodia formation (the little barbed ends of the leading edge)

Rac

Small GTPase, its GEF leads to activation of WAVE-ARP2/3 pathway, leading to actin branching and lamellipodia formation (the broad structure of the leading edge).

CapZ, tropomodulin

Cap the negative and positive ends of actin in sarcomeres, respectively

Troponin/tropomyosin/myosin

Tropomyosin - Binds to the binding site for myosin on actin, prevents myosin from binding to it

Troponin - Calcium sensing complex, displaces tropomyosin in response to calcium concentration increase in the cytoplasm, allowing muscle contraction

Myosin - ATPase, walks along actin decreasing the non-overlapping region of actin and myosin bundles.

Nebulin

Stabilizes F-actin in the sarcomeres

Titin

Immense protein, positions the myosin fiber and anchors it in the sarcomere

Tubulin

Made of alpha and beta tubulin heterodimers that assemble into protofilaments which assemble into sheets, which curve to make tubes. Alpha tubulin GTP is unhydrolyzable while Beta tubulin GTP is. G

Gamma-tubulin

Stabilizes the (-) end of tubulin at the origin site and the gamma tubulin ring complex acts as the nucleation site of tubulin.

Kinesin-13

Catalyzes catastrophic (+) end disassembly of tubulin.

EB1

Binds to (+) end of tubulin, allowing it to recognize other binding factors

Stathmin

Destabilizes microtubules

XMAP215

Binds to the positive end of microtubules, stabilizing them and promoting growth. Phosphorylated during mitosis, preventing binding, and allowing for microtubules to be unstable so they can find chromosomes to attach to.

Astral, interpolar, and kinetochore microtubules

Astral - Radiate out from the spindles, not attached to anything

Interpolar: One from each spindle pole attaches to the other, allows for cells to pull apart and spaces the poles from each other

Kinetochore - attaches to the kinetochore, pulls apart chromatids during division

Keratin

Intermediate filament. Starts as a polar monomer (N and C terminus), forms a coiled homodimer, which assembles with another coiled homodimer into a staggered tetramer. Multiple tetramers tightly associate together. Keratin gives epithelial cells their structure

Lamin

Intermediate filament necessary for maintaining the structure of the nuclear envelope. Phosphorylated during mitosis which allows the nuclear envelope to break down.

What are the 5 ECM components and their individual structure/function

Collagen: Trimeric helix that is processed inside the cell and sent outside the cell as a fibril

Elastin: Alanine and lysine rich helices are crosslinked by proline and glycine to form networks that stretch and relax, providing elasticity to cells

Glycosaminoglycans: Long repeating disaccharide chains that help withstand compressive forces

Proteoglycans: Proteins with GAGs where 95% of the weight is the GAG portion.

Multidomain proteins (fibronectin and laminin): Fibronectin (homodimer) provides binding sites and structure for other proteins and laminin (oligomer of heterotrimers) provides binding sites for cells in migration and attaches cells together during embryonic development

ALL OF THESE ARE MADE BY ECM FIBROBLASTS

Cadherins

Mediate cell-cell adhesion. Cadherins stick out from cells and recognize only the same cadherin on another cell, and only in the presence of Ca2+. Cell binding specificity can be created either by cadherin isoform or by having a drastically different number of a cadherin type in a cell vs another.

Desmosomes

Protein complexes that link neighboring cells by attaching cadherin to the intermediate filaments inside the cell

Adherens junctions

Protein complexes that connect neighboring cells via cadherin binding to the actin cytoskeleton - allows actin belts to form between cells

Integrin

Act as a bridge between cytoskeletal elements actin and intermediate filaments to ECM proteins. This helps cells adhere to their basement membrane

What are the steps of protein sorting

1. Sorting signal

2. Sorting signal receptor on target

3. Protein folding state

4. Translocation

5. Driving force

6. Release after translocation

7. Fate of sorting signal

Describe the steps of mitochondrial import

1. Amphiphatic positively charged helix recognized by receptor protein. Cargo proteins use ATP hydrolysis to keep the cargo unfolded

2. Receptor protein and TOM complex allows protein to enter mitochondria, and binding to TIM-receptor complex allows import of the signal sequence into the matrix.

3. Electric gradient of H+ draws the signal sequence into the matrix due to negative charge buildup there.

4. IMM import ATPase uses ATP hydrolysis to bring the rest of the protein inside the mitochondrial matrix.

5. Once inside the matrix, signal peptidase removes the signal sequence

Inner membrane proteins can either have a hydrophobic stop sequence that locks the protein in the IMM, embedding it there, or can be fully brought into the matrix, then brought back to the IMM by a chaperone protein

Intermembrane multipass proteins are released into the intermembrane space by TOM, then accompanied by chaperones which allow them to embed in the inner membrane.

IMM Space proteins can embed in the mitochondrial membrane then be cleaved to be released into the intermembrane space.

OMM proteins, once released into the intermembrane space from TOM, are accompanied by chaperone proteins that mediate insertion into the outer membrane.

Describe the process of nuclear import/export

1. Nuclear localization signal (positively charged amino acid stretch at N-terminus) binds to cytosolic importin

2. Importin-cargo complex (with protein fully folded) enters the nucleus through nuclear pores

3. The cargo dissociates from the importin, and Ran-GTP attaches to the importin (Ran GEF concentration high in the nucleus)

4. Ran-GTP is transported outside of the nuclear pore using GTP hydrolysis by Ran GAP.

5. Nuclear localization signal is left intact because back and forth import and export cylces may occur for a protein

Export follows the same steps in reverse, except for the importin exits the nucleus with the Cargo and Ran-GTP bound.

Describe import of proteins to the ER

1. N-terminus Hydrophobic import signal sequence is recognizes as soon as it is translated by signal recognition particle.

2. Signal recognition particle arrests translation, and while still bound to the nascent polypeptide and ribosome, it attaches to the signal recognition particle receptor on the ER

3. SRP and SRP-R are still GTP bound, but they act as GAPs for each other, hydrolyzing GTP and causing each to dissociate from the complex, and the translocon recognizes the signal sequence. Translation continues.

4. Signal peptidase cleaves the signal sequence and the protein uses translation as the driving force to enter the ER lumen, where it is folded.

If there is a hydrophobic domain, the domain will stay embedded in the membrane, and signal peptidase will cleave the signal sequence but the translocon will release the protein with the hydrophobic domain embedded in the membrane

The topology of the protein is determined by positively charged amino acids before or after the first hydrophobic domain. If before, the N terminus stays in the ER lumen, but if after, the N terminus stays in the cytosol.

Proteins can also be posttranslationally imported into the ER if they have a signal sequence. Chaperone proteins keep them unfolded and they can localize to the ER.

Multipass proteins embed uniquely to the mitochondria, each hydrophobic domain is stopped and released into the plasma membrane through the side of the translocon as the protein is translated.

BiP

Carries out chaperone-mediated protein folding inside the ER. High rate of misfolded protein increases sequestered BiP, which leads to ER stress. Additionally, BiP uses ATP to draw proteins through the translocon into the ER posttranslationally.

Disulfide isomerase

Forms sulfide bridges

Glucosidase

Removes one glucose from oligosaccharyl tree

Glucosyltransferase

Adds a glucose to an oligosaccharyl tree

Calnexin

A type of lectin (sugar binding protein) that monitors whether a glycoprotein is being correctly folded

Describe the misfolded protein response in the ER

If proteins are being misfolded, there are ER membrane proteins that sense misfolded proteins and initiate a transcriptional response to increase the folding capacity of the ER. This includes decreasing translation of new proteins and activating transcription factors to activate folding factors in the ER.

Misfolded proteins that are sequestering folding factors are translocated back into the cytosol, where they are polyubiquitinated and marked for degradation.

TANGO1

Senses the size of cargo and adjusts budding vesicle size accordingly

Describe the mechanism of anterograde transport from the ER to the Golgi

1. Sar1-GDP is recognized by a receptor in the ER, which acts as a GEF on it, causing it to become Sar1-GTP and sink its hydrophobic tail in the ER membrane

2. Sar1-GTP recruits COPII Sec proteins that have receptors which bind different types of soluble cargo. The membrane begins to curve and bud off, taking transmembrane and soluble cargo with it.

3. As the vesicle buds off the ER, Sar1-GTP is hydrolyzed, causing the COPII coat to dissociate from the vesicle.

4. Rab, a small GTPase that is present on the outside of the vesicle, is recognized by a tether protein of the Golgi. As the tether brings the vesicle closer, the t-SNARE proteins of the Golgi membrane recognize the v-SNARE proteins of the vesicle, and they bind and crosslink, being helically shaped, and this is what causes the vesicle to fuse with the Golgi. To return the v-SNARES back to the ER, they are sent back in COP1 coated vesicles. The COP1 mechanisms mimics that of COP2, but with a different GTPase.

How do missorted proteins return to the ER from the Golgi?

Missorted proteins, such as BiP, if they are in the ER, will have their KDEL motif recognized by a KDEL receptor, which packages it into a COP1 vesicle and ships it back to the ER.

Describe the process of endocytosis

1. Arf1-GDP recruited by Arf1 receptor, exchanges its GDP to GTP

2. Arf1-GTP anchors into membrane, changing the membrane composition in a certain area

3. AP2 adaptor protein binds to the cargo, and clathrin coated vesicle begins to bud off. Dynamin cuts the vesicle off from the membrane and GTP hydrolysis of Arf1-GTP causes the clathrin coat to dissociate

4. The vesicle fuses with the endosome, where the low pH causes receptor dissociation.

5. The fate of the receptor that was endocytosed depends on its sorting signal, but the cargo fuses with the lysosome when the endosome matures into a late endosome.

How does EGFR enter the lysosome

Monoubiquitination of the consensus site causes ESCRT complex to translocate the EGFR to a budding vesicle that does not have a clathrin coat. The monoubiquitination signal brings the protein to the lysosome. Note that polyubiquitination brings a protein to the proteasome.