REVIEW+QUESTIONS+EXAM+2

 QUIEN BORRO LAS PREGUNTAS ANTERIORES!? REGRESENLA POR FAVOR! No se quien las borr, pero aqui las voy a poner. Si habian contestaciones, favor de volver a copiarlas! Exam 2 BIOLOGY OF THE CELL Review questions or concepts

VESICULAR TRAFFIC, SECRETION, AND ENDOCYTOSIS 1. Compare and contrast the biosynthetic pathway (of a secretory protein) with the endocytic pathway.

2. Describe the three main vesicle types and compare and contrast their roles in vesicular traffic. Se estudiaron tres tipos de vesículas las cuales se diferencian principalmente por la cubierta de proteínas que tienen y por el trayecto que las mismas recorren.


 * Vesículas cubiertas de clatrina: van del complejo trans Golgi hacia los endosomas y de la membrana plasmática a los endosomas. En otros casos del complejo trans Golgi hacia lisosomas, melanosomas o vesículas “platelet” en un movimiento antero grado.


 * Vesículas cubiertas de COP1: del complejo cis Golgi hacia el RE en un movimiento retrogrado (devuelve al RE proteínas residentes que se van escapando.)


 * Vesículas cubiertas de COP2: del RE hacia el complejo cis Golgi en un movimiento antero grado.

Tres tipos de vesiculas --> se diferencian principalmente por la cubierta de proteinas y por el trayecto que recorren.

a. Vesiculas cubiertas de clatrina: transportan material del trans Golgi complex hacia los endosomas y de la membrana plasmatica a los endosomas. Tambien pueden transportar material del trans Golgi complex a lisosomas, melanosomas o vesiculas platelet en movimiento anterogrado.

b. Vesiculas cubiertas de COP1: transportan material del cis Golgi complex hacia el RE en movimiento retrogrado. Devuelve al RE proteinas residentes que se hayan escapado.

c. Vesiculas cubiertas de COP2: transportan material del RE hacia el cis Golgi complex en movimiento anterogrado.

3. Explain the role of the molecular components that are involved in vesicle coating, cargo recruitment and budding for COPI, COPII and clathrin vesicles.

PIPs (phosphoinositides)--> regulan el ensamblaje de las cubiertas de clatrina en la membrana plasmatica y el aparato de Golgi.

Coat-recruitment GTPases --> controlan el ensamblaje de las cubiertas de clatrina en edosomas y de las cubiertas de COP1 y COP2 en las membranas del Golgi y RE (Arf y Sar1)

Las cubiertas de clatrina consisten de la union de varios triskelions que se ensamblan en forma de canasta para cubrir la parte citosolica de la membrana.

Entre la capa de clatrina y la membrana se encuentra una capa de adaptor proteins que sirven para unir la capa de clatrina con la membrana.

Las adaptor proteins tambien atrapan varias proteinas transmembranales que se enlazan a moleculas cargo y las atrapan dentro de la vesicula.

En el caso de las vesiculas cubiertas de clatrina el "budding" es inducido por la misma secuencia de ensamblaje de las cubiertas.

Para las cubiertas de COP2, el GEF de Sar1 que se encuentra en la membrana del RE se une a Sar1 que se encuentra en el citosol causando el intercambio de GDP por GTP. En este estado activado, la proteina Sar1 se inserta en la membrana del RE y recluta subunidades de proteinas de cubierta a la membrana del RE para que se inicie el proceso de "budding".

Las cubiertas de COP1 se comienzan a ensamblar tan pronto salen las vesiculas cubiertas de COP2.

4. Describe the molecular structure of COPI, COPII and clathrin vesicles and the relationship between its structure and function? 5. How does the monomeric GTPases function as molecular switches? What is the function of GAPs and GEFs? Mention the roles of the following monomeric G proteins: Ras, Ran, Arf/Sar, Rab.

a. Las GTPasas monomericas tienen como funcion controlar el ensamblaje o desensamblaje de las cubiertas de proteinas de los diferentes tipos de vesiculas. Esto es mediado a traves del intercambio de GDP por GTP y/o la hidrolisis de GTP a GDP.

Al estar en su estado desactivado (enlazada a GDP) se comienza el proceso de ensamblaje de la cubierta y en estado activado (enlazada a GTP) la cola de la proteina se inserta en la membrana blanco para comenzar el desensamblaje de la cubierta de proteinas.

b. GAPs --> GTPase-activating proteins: catalizan la hidrolisis de GTP a GDP para desactivar las GTPasas monomericas.

GEFs --> guanine-nucleotide-exchange factors: activan las GTPasas monomericas catalizando el intercambio de GDP por GTP.

c. Arf/Sar --> familia de GTPasas monomericas que controlan el ensamblaje, desensamblaje y "budding" de cubiertas COP y clatrina.

Ras --> regula los procesos de transduccion de señales relacionadas con el crecimiento y la diferenciacion celular.

familia Rab --> participan en la fusion de vesiculas y en la especificidad del transporte vesicular. Son las que le dan direccion a la vesicula.

Ran --> transporte de peptidos y proteinas a traves de la membrana nuclear.

6. What determines the specific targeting, tethering and fusion of a transport vesicle and the target membrane compartment? Describe the molecular components. What is the role of derivatives of phosphatidyl inositol phospholipids? How are the SNARE proteins involved in the process of membrane fusion? 7. How do retrieval signals ensure that proteins are kept residents of a particular membrane compartment? 8. Mention the main functions of the Golgi apparatus. What types of protein modification can only occur in the Golgi apparatus? 9. How can scientist track proteins as they move from the ER to the Golgi? 10. Compare and contrast the vesicular transport with the cisternal maturation models that explain transport through the Golgi apparatus.

11. Describe the roles of lysosomes. Lysosomes are large and spherical organelles that contain acid hydrolases. This enzymes control intracellular digestion of macromolecules from phagocytosis (ingestion of other dying cells or larger extracellular material), endocytosis (where recepetor proteins are recycled from the cell surface), and autophagy (organelles unnedeed are delivery to lysosomes). The lysosomes need to be activated by proteolytic cleveage and require an acid environment.

12. Describe the steps that ensure that a lysosomal enzyme will be targeted to a lysosome rather that a secretory vesicle. They carry a unique marker in the form of mannose 6-phosphate (M6P) groups, which are added exclusively to the N-linked oligosaccharides of these soluble lysosomal enzymes as they pass through the lumen of the cis Golgi network. The M6P groups are recognized by transmembrane M6P receptor proteins, which are present in the trans Golgi network. The receptor proteins bind to lysosomal hydrolases on the lumenal side of the membrane and to adaptins in assembling clathrin coats on the cytosolic side. In this way, they help package the hydrolases into clathrin-coated vesicles that bud from the trans Golgi network. The vesicles subsequently deliver their contents to a late endosome.

13. Describe the steps that occur between the binding of an LDL particle to the plasma membrane of a cell and the entry of cholesterol molecules to the cytosol. Most cholesterol is transported in the blood as cholesteryl esters in the form of lipid-proteins = low-density lipoproteins (LDLs). When a cell needs cholesterol for membrane synthesis:
 * It makes transmemnbrane receptor proteins for LDL and inserts them to theplasma membrane.
 * Once there, the LDL-receptors diffuse until they associate with clathrin-coated pits.
 * Clathrin-coated pits are constantly pinching off to form vesicles, therefore any LDL particles bound to LDL receptors in the pits are raidly internalized in coated vesicles.
 * After shedding the clathrin coat, vesicles deliver their contents to early endosomes.
 * Once LDL and LDL receptors encounter low pH in endosomes, LDL is released from its receptor and is delivered via late endosomes to lysosomes.
 * In the lysosomes LDL is hydrolyzed to free cholesterol.
 * Now cholesterol is available to the cell.

14. How does the role of phagocytosis in the life of a unicellular organism compare to its role in a multicellular organism? What are the major steps that occur during phagocytosis?

15. Describe the roles of early and late endosomes. -Early endosomes mature into late endosomes before fusing with lysosomes. Endocytosed molecules are initially delivered in vesicules to early endosomes, and here is where the endocytosed molecules first meet the lysosomal hydrolases. Late endosomes contain endocytosed molecules were not recycled to the plasma membrane. Mature lysosomes form by a maturation process from late endosomes.

INTERCELLULAR SIGNALING

1. Describe the 5 types of signals that mediate intercellular communication. A. Contact-dependent signaling: Requires cells to be in direct membrane-membrane contact. Signal molecules remain bound to the surface of the signaling cell and influence only cells that contact it. B. Paracrine signaling: Short range cell-cell communication via secreted signal molecules that act on neighboring cells. The secreted molecules may act as local mediators. C. Autocrine signaling: A cell secretes signal molecules that act back on itself. D. Synaptic signaling: Communicating cell-cell junction that allows signals to pass from a nerve cell to another cell via small signal molecules known as neurotransmitters. E. Endocrine signaling: Signaling via hormone release by endocrine glands into the blood stream and carried to distant target cells that have specific hormone receptors.

2. What is meant by signal transduction? Signal Transduction is the conversion of signal from one physical or chemical form to another. Conversion of an extracellular signal to an intracellular signal.

3. What are the general steps by which signal transduction can occur? First, the extracellular signal molecule binds to a receptor protein that is embedded in the plasma membrane of the target cell and activates one or more intracellular signaling pathways mediated by a series of signaling proteins. Finally, one or more of the intracellular signaling proteins alters the activity of effector proteins and thereby the behavior of the cell, such as altering its metabolism, gene expression or, cell shape or movement.

4. Compare and contrast the components of signaling between ligands with intracellular receptors, GPCRs or RTKs. 5. What is a second messenger? Why do you suppose it is called this? A second messenger is a method of cellular signaling whereby a diffusable signaling molecule is rapidly produced/secreted, which can then go on to activate effector proteins within the cell to exert a cellular response. Secondary messengers are a component of signal transduction cascades. Secondary messenger systems can be activated by diverse means, either by activation of enzymes that synthesize them, as is the case with the activation of cyclases that synthesize cyclic nucleotides, or by opening of ion channels to allow influx of metal ions, such as in Ca2+ signaling. Since they are activated by these so called first messengers they are called second messengers. These small molecules may then go on to exert their effect by binding to and activating effector molecules such as protein kinases, ion channels, and a variety of other proteins, thus continuing the signaling cascade.

6. What is meant by the term amplification in regard to signal transduction? How does the use of a reaction cascade result in amplification of the signal? How does it increase the possibilities for regulation?

7. Describe the steps in the signaling pathway by which nitric oxide mediates dilation of blood vessels. Nitric Oxide is a signaling molecule that activates intracellular receptors. Nitric Oxide plays a mayor role in smooth muscle relaxation in mammalian cells via blood vessel dilatation. Acetylcholine released by nerve terminals in the blood vessel wall activates nitric oxide synthases in endothelial cells lining the blood vessel, causing the endothelial cells to produce nitric oxide from the degradation of the aminoacid argenine. The nictric acid diffuses out of the endothelial cells and into the neighboring smooth muscle cells, where it binds to and activates guanylyl cyclase to produce the small intracellular signaling molecule cyclic GMP. The cyclic GMP triggers a response that causes the smooth muscle to relax, enhancing blood flow through the vessel dilatating them.

8. Describe the steps in the signaling pathway of a steroid hormone (estrogen). [|http://www.youtube.com/watch?v=tK4lzxQuxTM&NR=1]

In the absence of hormone, estrogen receptors are largely located in the cytosol. Hormone binding to the receptor triggers a number of events starting with migration of the receptor from the cytosol into the nucleus, dimerization of the receptor, and subsequently binding of the receptor dimer to specific sequences of DNA known as hormone response elements. The DNA/receptor complex then recruits other proteins which are responsible for the transcription of downstream DNA into mRNA and finally protein which results in a change in cell function. Estrogen receptors also occur within the cell nucleus and both estrogen receptor subtypes have a DNA-binding domain and can function as transcription factors to regulate the production of proteins. The receptor also interacts with activator protein 1 and Sp-1 to promote transcription, via several coactivators such as PELP-1.

9. Describe the types of nuclear receptors, their structure and their main function. 10. Describe the importance of GPCRs. 11. Compare and contrast the structure of GPCRs with the structure of nuclear receptors, and RTKs. 12. What is the role of trimeric G proteins in a signaling pathway? How it compares with monomeric G proteins. 13. Describe the structure of trimeric G proteins. Consists of three subunits (α, β and γ). The α and the γ subunits have covalently attached lipid molecules that help to bind them to the plasma membrane, and the α subunit has GDP bound. The three-dimensional structure of an inactive G protein, based on transducin, the G protein in visual transduction. The α subunit contains the GTPase domain and binds to one side of the β subunit, which locks the GTPase domain in an inactive conformation that binds GDP. The γ subunit binds to the opposite side of the β subunit.

14. Describe the types of G a, its associated effectors, second messenger involved, and receptor examples. 15. How it is possible that the same first messenger, such acetylcholine, can evoke different responses in different target cells? That the same second messenger, such as cAMP evoke different responses in target cells? That the same response, such as cardiac muscle contraction or glycogen breakdown, can be initiated by different stimuli?

16. Describe the steps between the binding of a ligand such as epinephrine or glucagon to its receptor and the activation of the adenylyl cyclase. Once cAMP is synthesized, describe the steps that lead to the release of glucose into the bloodstream (liver cell).

====**• En respuesta a una crisis (stress o peligro) las glándulas adrenales secretan la hormona epinefrina, estas al igual que la hormona glucagón aumentan la concentración de glucosa en la sangre(hepatocitos).**====

**• El AMPc en el citosol activa el efector PKA(cinasa de proteína A), ésta fosforila enzimas metabólicas relacionadas a aumentar la concentración de glucosa en la sangre.**
17. Once PKA is activated, what is its function in the cytoplasm and/or in the nucleus?

====**PKA inactivo tiene 4 subunidades: 2 reguladoras y 2cataliticas.AMPc se liga y separa las subunidades reguladoras,las subunidades cataliticas acyuan como cinasas fosforilando proteínas en el citosol produciendose una respuesta celular.**==== ====**En el núcleo de la celula PKA activa factores de transcripción; CREB (cAMP response elements binding),estos se enlazan a una secuencia de DNA conocidos como CRE(cAMP responses elements), los cuales regulan la expresion de genes especificos.**====

 PKA contiene cuatro subunidades: dos reguladoras y dos cataliticas. A bajas concentraciones se encuentra inactivado, pero a altas concentraciones de cAMP, este se enlaza a la holoenzima causando un cambio en conformacion de las subunidades reguladoras y con esto, el desprendimiento de las subunidades.

En celulas de mamiferos se encuentran al menos dos tipos de PKA. El tipo 1 se encuentra principalmente en el citosol mientras que el tipo 2 se encuentra en la membrana plasmatica, en la membrana externa de la mitocondria, la membrana nuclear y microtubulos. Una vez activado, se liberan las subunidades cataliticas y estas se mueven hacia el nucleo donde pueden fosforilar proteinas reguladoras de genes. A la vez, activa la fosfodiesterasa adyacente, que baja los niveles de cAMP para que se mantenga rapida la respuesta de PKA. 

 18. Describe the processes that mediate “turning off” the cascade. Include the role of phosphatases, phosphodiesterases, and desensitization, sequestration and downregulation of receptors. Emphasize the role of receptor phosphorylation and arresting binding in the latter three.

El turning off puede ocurrir de varias maneras. Puede ser por la desactivación de receptores, a los cuales se les liga una molécula mensajera que induce la endocitosis del receptor y su secuestración temporera en endosomas. En algunos casos, esta secuestración puede llevar a la degradación del receptor en lisosomas, esto se conoce como receptor down regulation. Otros receptores pueden ser desactivados aun estando en la membrana plasmática por una fosforilación o metilación. La desensitivacion de los receptores también puede ocurrir en algún paso downstream del receptor. Puede ser por un cambio en proteínas intracelulares que permiten la transducción de la señal, o por la producción de alguna proteína inhibidora que bloquee su proceso. Las GPCRs se inactivan por su fosforilación mediante PKA, PKC o alguna GPCR kinases(GRKs). Las GRKs fosforilan serinas y treoninas en un GPCR. Esto sucede luego de que un ligando haya activado el receptor, ya que es esto lo que activa a su vez a la GRK. Una vez el receptor esta fosforilado, aumenta su afinidad por arrestina, lo que evita que el receptor active a su proteina G correspondiente y en entonces, es endocitado. Arrestina también funciona como adaptor protein para formar el coating de clatrinas para el receptor a endocitar.


 * fosfatasa- defosforila, es parte de la respuesta de "negative feedback" que causa MAPK

fosfodiesterasa- hidroliza cAMP y cGMP por lo tanto disminuye la señal intracelular**



18. Describe the processes that mediate “turning off” the cascade. Include the role of phosphatases, phosphodiesterases, and desensitization, sequestration and downregulation of receptors. Emphasize the role of receptor phosphorylation and arresting binding in the latter three.

19. What is the mechanism of formation of IP3? What is the relationship between IP3 and an elevation of intracellular calcium?


 * IP3 es sintetizado por la proteína efectora fosfolipasa C (PLC). PLC rompe PIP2 y forma diacilglicerol (se queda en la memb.) y IP3. IP3 funciona como ligando en canales de Ca2+ en el Reticulo endoplásmico, IP3 hace que se abran los canales y aumenta la concentración de Ca2+ en el citoplasma.**

 20. How is the [Ca2+] of the cytosol maintained a such low level? How does the concentration change in response to stimuli? 21. What is the role of calcium-binding proteins such as calmodulin in eliciting a response? 22. Describe the relationship between phosphatidylinositol, diacylglycerol, calcium ions, and protein kinase C. 23. Explain the process of phototransduction of rod cells. 24. Explain the role of epinephrine and acetylcholine in the fight or flight response through adrenergic receptors and muscarinic receptors. What would be the effect of antagonists and agonists in this system? 25. Compare and contrast muscular nicotinic receptors with muscarinic receptors. What is the first messenger, receptor structure, signal transduction mechanism? 26. Describe the structure of receptor tyrosine kinases. 27. Describe the steps between the binding of an insulin molecule at the surface of a target cell and the activation of PI3K. How does the action of insulin differ from NGF as ligand of its RTK? 28. What is the role of Ras in signaling pathways? How is this affected by the activity of a Ras-GAP? How does Ras differ from a heterotrimeric G protein? 29. What are the SH2 and SH3 domains, and what roles do they play in signaling pathways? Dominios SH2 en proteinas adaptadoras tiene afinidad por tirosinas fosforiladas en el donino citosolico de los RTK, los dominos SH3 tienen afinidad por regiones ricas en prolina de los cuales un ejemplo es Sos, que está dentro de la cascada. 30. Describe how the development of Drosophila eyes help discover the relationship between Ras and RTKs. 31. How does MAP kinase cascade alter the transcriptional activity of a cell?

32. Why FRET can be used as a tool to study protein interactions?

====**FRET (fluorescence energy transfer), es una técnica utilizada para medir la interacción entre dos proteínas mediante la emisión de fluorescencia a un determinado largo de onda. Se ha demostrado la disociación del complejo Gα-Gβγ en segundos luego de la adición del ligando, de esta manera se ha evidenciado el ciclo de la proteína G. Puede ser utilizado para seguir la formación y disociación de otros complejos proteínas -proteínas en células vivas.**//====