BIO568 Exam 2 PDF

BIO568 EXAM 2 ER Analyze results of mRNA translation in the absence & presence of microsomes. Formulate a conclusion based on the data. Analyzing mRNA translation in the presence and absence of microsomes reveals valuable insights into protein targeting and processing. When mRNA encoding a secretory protein is translated in vitro in the absence of microsomes (rough ER vesicles), the resulting protein is larger than the mature protein secreted by cells. This suggests that the protein undergoes processing within the ER. This observation led to the formulation of the Signal Hypothesis, which proposes that a specific amino acid sequence, termed the signal sequence, directs proteins to the ER. Microsomes play a crucial role in this process. When the same mRNA is translated in the presence of microsomes, the synthesized protein is the same size as the mature protein. This indicates that microsomes contain the necessary machinery for signal sequence removal and protein processing. Conclusion: These experimental results support the Signal Hypothesis, confirming that signal sequences are necessary for targeting proteins to the ER and that microsomes contain the components essential for signal sequence cleavage and protein maturation. Describe how the signal sequence, SRP, SR, signal peptidase, translocator/ Sec61 complex, and GTP hydrolysis are involved in cotranslation of proteins into the ER and how mutations would aTect the pathway. The process of co-translational protein import into the ER involves a series of coordinated steps: The signal sequence, a stretch of hydrophobic amino acids at the N-terminus of a newly synthesized polypeptide, emerges from the ribosome during translation. The signal recognition particle (SRP) recognizes and binds to the signal sequence, temporarily halting translation. This pause in translation prevents premature folding of the protein and ensures proper targeting. The SRP-ribosome complex then interacts with the SRP receptor (SR), located on the ER membrane. This interaction guides the ribosome to the translocator, also known as the Sec61 complex, a protein channel embedded within the ER membrane. Upon binding to the translocator, the SRP releases the signal sequence in a step that requires GTP hydrolysis, allowing translation to resume. As translation continues, the growing polypeptide chain is threaded through the translocator channel into the ER lumen. Inside the ER lumen, the signal peptidase, an enzyme residing within the ER membrane, cleaves the signal sequence from the polypeptide chain, releasing the mature protein into the ER lumen. Mutations in any of the components involved in this pathway can disrupt protein targeting and processing. For example, a mutation in the signal sequence could prevent SRP binding, leading to mislocalization of the protein. Similarly, mutations in the SRP, SR, or translocator could impair ribosome docking and protein translocation. A defective signal peptidase would result in the retention of the signal sequence, potentially aNecting protein folding and function. Describe an experiment that would determine the ER localization signal on a protein To experimentally determine the ER localization signal on a protein, you could conduct a series of experiments: 1. Create fusion proteins: Fuse diNerent segments of the protein of interest to a reporter gene that encodes a readily detectable protein, like green ﬂuorescent protein (GFP). 2. Express fusion proteins: Introduce these fusion protein constructs into cells. 3. Observe localization: Observe the localization of the reporter protein using ﬂuorescence microscopy. If the reporter protein is localized to the ER, the segment of the protein fused to it contains the ER localization signal. This approach will help pinpoint the speciﬁc amino acid sequence responsible for ER targeting. Predict how and if a protein will insert into the ER membrane given it's amino acid sequence The amino acid sequence of a protein can provide valuable clues about its potential insertion into the ER membrane. Proteins destined for insertion into the ER membrane typically contain one or more hydrophobic stretches of amino acids called stop-transfer sequences, in addition to their signal sequence. These stop-transfer sequences function as transmembrane domains, embedding the protein within the membrane. By analyzing the hydrophobicity proﬁle of a protein sequence, you can predict the presence and location of potential transmembrane domains, thus indicating whether and how the protein will insert into the ER membrane. Identify the location, function, and downstream action of IRE1, PERK & ATF6 in the Unfolded Protein Response (UPR) The Unfolded Protein Response (UPR) is a cellular stress response triggered by the accumulation of unfolded or misfolded proteins in the ER lumen. Three key sensor proteins, IRE1, PERK, and ATF6, reside in the ER membrane and play[ crucial roles in sensing and responding to ER stress: 1. IRE1 (Inositol-requiring enzyme 1): This enzyme is located in the ER membrane and possesses both kinase and endoribonuclease activity. Upon sensing misfolded proteins in the ER lumen, IRE1 dimerizes and activates its endoribonuclease activity. This leads to the splicing of a speciﬁc mRNA encoding a transcription factor called XBP1. Spliced XBP1 translocates to the nucleus and activates the transcription of genes involved in protein folding, ER chaperones, and ER- associated degradation (ERAD), enhancing the ER's capacity to cope with stress. 2. PERK (Protein kinase RNA-like ER kinase): Also located in the ER membrane, PERK is a kinase that gets activated upon ER stress. Once activated, PERK phosphorylates eIF2α (eukaryotic initiation factor 2α), a key regulator of protein synthesis. This phosphorylation event attenuates general protein translation, reducing the inﬂux of newly synthesized proteins into the ER and alleviating the protein folding load. 3. ATF6 (Activating transcription factor 6): This protein is initially synthesized as an ER membrane-bound precursor. Under ER stress conditions, ATF6 translocates to the Golgi apparatus, where it is cleaved by proteases. This cleavage releases the active form of ATF6, a transcription factor that migrates to the nucleus and activates the expression of genes involved in protein folding, ER chaperones, and ERAD, similar to XBP1. The UPR, through the actions of IRE1, PERK, and ATF6, aims to restore ER homeostasis by either enhancing the ER's protein folding capacity or reducing the protein load through translational attenuation. However, if the stress is too severe or prolonged, the UPR can also trigger apoptosis, a programmed cell death pathway, to eliminate the damaged cell. Justify how a mutation in calnexin or calreticulin will eTect ERAD Calnexin and calreticulin are ER-resident chaperone proteins that assist in the proper folding of newly synthesized glycoproteins. They recognize and bind to specific sugar residues (N-linked glycans) attached to proteins in the ER, providing a platform for proper folding and preventing aggregation. ERAD (ER-associated degradation) is a quality control pathway in the ER that targets misfolded or unfolded proteins for degradation by the proteasome, a cellular machinery responsible for protein breakdown. Mutations in calnexin or calreticulin can disrupt their chaperone function, leading to the accumulation of misfolded proteins in the ER. This accumulation can overwhelm the ERAD machinery, reducing its efficiency in clearing misfolded proteins. As a result, misfolded proteins may accumulate, potentially leading to ER stress and triggering the UPR. GOLGI Predict the eTects of mutation in adaptors, clathrin, COPI, COP II and dynamin on vesicle budding. Adaptor proteins connect cargo receptors to clathrin proteins during the formation of coated pits in receptor-mediated endocytosis. Mutations in adaptor proteins would prevent the recruitment of specific cargo proteins, inhibiting the formation of coated pits and subsequent vesicle budding. Clathrin is a protein that coats the invaginated coated pit and plays a crucial role in vesicle formation. Mutations in clathrin would disrupt the structure of the coated pit, preventing the membrane from curving and forming a vesicle. COPI and COPII are coat proteins involved in vesicle transport between the ER and the Golgi apparatus. COPII facilitates the budding of vesicles from the ER and their transport to the Golgi, while COPI mediates the budding of vesicles from the Golgi, some of which return to the ER. Mutations in COPII would hinder vesicle budding from the ER, affecting protein transport to the Golgi. Conversely, mutations in COPI would disrupt vesicle budding from the Golgi, impacting both protein transport within the Golgi and retrieval of ER-resident proteins. Dynamin is a GTPase that assists in pinching off coated vesicles from the membrane during endocytosis. Mutations in dynamin would prevent the final separation of the vesicle from the membrane, leading to an accumulation of invaginated coated pits unable to release their contents. Explain the role of Sar in vesicular transport and its regulation by GEF & GAP Sar1 is a small GTPase essential for COPII vesicle formation, moving cargo from the ER to the Golgi. It is regulated by GEF and GAP proteins. GEF (Sec12) activates Sar1 by exchanging GDP for GTP, enabling Sar1 to anchor to the ER membrane and recruit COPII coat proteins that form the vesicle. After budding, GAP (Sec23) stimulates GTP hydrolysis on Sar1, returning it to an inactive form, which causes COPII coat disassembly and prepares the vesicle for fusion with the Golgi. This precise GEF- GAP regulation is crucial for eNicient vesicle formation and transport. Describe the function of v-SNARE, t-SNARE & NSF in vesicle-membrane fusion. v-SNARE (vesicle-SNARE) proteins on transport vesicles recognize and bind to complementary t-SNARE (target-SNARE) proteins on the target membrane. This interaction ensures that the vesicle fuses with the correct target membrane. NSF (N-ethylmaleimide-sensitive factor) is an ATPase that plays a crucial role in disassembling the SNARE complex after membrane fusion. This disassembly is essential for recycling the SNARE proteins for subsequent rounds of vesicle fusion. Discuss the role of the KDEL sequence & KDEL receptor in resident ER protein localization The KDEL sequence is a specific amino acid sequence (Lys-Asp-Glu-Leu) found at the C-terminal end of proteins that reside in the ER. The KDEL receptor, located in the Golgi apparatus, recognizes and binds to the KDEL sequence of ER-resident proteins. This binding triggers the packaging of the receptor- protein complex into COPI-coated vesicles, which then return to the ER. This mechanism ensures that proteins essential for ER function are retrieved and maintained within the ER lumen. LDL (low-density lipoprotein) particles, carrying cholesterol, bind to speciﬁc LDL receptors on the cell surface. These receptors cluster in coated pits, which invaginate and pinch oN to form clathrin-coated vesicles containing the LDL particles. The vesicles fuse with lysosomes, where the LDL particles are degraded, releasing cholesterol for cellular use. Defects in any of the steps involved in receptor-mediated endocytosis can lead to impaired LDL uptake. For example: Mutations in the LDL receptor gene can result in non-functional receptors or receptors that cannot bind LDL properly. This leads to reduced LDL uptake and can cause familial hypercholesterolemia, a condition characterized by very high levels of cholesterol in the blood. Defects in clathrin, adaptor proteins, or dynamin can hinder coated pit formation and vesicle budding, also aNecting LDL uptake. Problems with vesicle traNicking or fusion with lysosomes can prevent the degradation of LDL particles, even if they are successfully endocytosed. To interpret defects in LDL uptake from primary data, you would need information about the speciﬁc experiment and the data collected. For example, if the data shows an accumulation of LDL in the bloodstream and reduced cellular cholesterol levels, it could indicate a defect in LDL receptor binding or receptor-mediated endocytosis. Please note: This response provides a general explanation of potential defects in LDL uptake. Analyzing speciﬁc primary data would require further information about the experiment and the observed results. NUCLEUS AND MITOCHONDRIA Assess the role of Ran and the Ran gradient in nuclear import. Ran is a GTP-binding protein that exists in two states: Ran-GTP (active) and Ran-GDP (inactive). The Ran gradient is crucial for nuclear import and export because Ran-GTP is predominantly found in the nucleus, while Ran-GDP is mainly in the cytoplasm. This gradient is maintained by Ran-GEF (Guanine nucleotide Exchange Factor), which is located in the nucleus and promotes the exchange of GDP for GTP on Ran, and Ran-GAP (GTPase Activating Protein), which is located in the cytoplasm and stimulates the hydrolysis of GTP to GDP on Ran. The Ran gradient plays a crucial role in the directionality of nuclear transport by regulating the interactions between cargo proteins, importins, and exportins. Identify the mechanism of nuclear import & export and the proteins involved. 1. Cargo Recognition and Binding: Proteins destined for the nucleus contain a Nuclear Localization Signal (NLS), a short sequence of amino acids rich in lysine and proline. Importin α recognizes and binds to the NLS on the cargo protein. Importin β then binds to importin α, forming a cargo-receptor complex. 2. Translocation through the Nuclear Pore Complex (NPC): The cargo-receptor complex interacts with nucleoporins, proteins that make up the NPC. Nucleoporins contain FG repeats (phenylalanine-glycine repeats) that interact with importin β, facilitating the translocation of the complex through the NPC. The complex moves through the pore in its native state, meaning the protein does not need to unfold. 3. Cargo Release in the Nucleus: Once inside the nucleus, Ran-GTP binds to importin β with a higher aTinity than the cargo protein. This binding causes a conformational change in importin β, leading to the release of the cargo protein into the nucleus. 4. Receptor Recycling: The importin α/β-Ran-GTP complex is exported back to the cytoplasm. In the cytoplasm, Ran-GAP stimulates the hydrolysis of GTP on Ran, converting it to Ran-GDP. This conversion causes the dissociation of the complex, releasing importin α and β to be used for another round of import. CAS (Cellular Apoptosis Susceptibility protein) is an export receptor that speciﬁcally facilitates the recycling of importin α back to the cytoplasm. Nuclear Export Mechanism 1. Cargo Recognition and Binding: Proteins destined for export from the nucleus contain a Nuclear Export Signal (NES). Exportin, such as Crm1 (Chromosomal Region Maintenance 1), recognizes and binds to the NES on the cargo protein. Ran-GTP in the nucleus also binds to the exportin, forming a trimeric complex. 2. Translocation through the NPC: Similar to nuclear import, the export complex interacts with the FG repeats of nucleoporins and moves through the NPC. 3. Cargo Release in the Cytoplasm: Upon reaching the cytoplasm, Ran-GAP stimulates the hydrolysis of GTP to GDP on Ran. This hydrolysis triggers the dissociation of the export complex, releasing the cargo protein into the cytoplasm. 4. Receptor Recycling: Exportin and Ran-GDP are recycled back to the nucleus to participate in further export events. Predict what transporters a protein may go through based upon its ﬁnal desitination in a mitochondrial compartment. The speciﬁc transporters a protein goes through in the mitochondria depend on its ﬁnal destination within the organelle: 1. Matrix Proteins: These proteins need to cross both the outer and inner mitochondrial membranes to reach the matrix. TOM complex (Translocase of the Outer Membrane): The protein ﬁrst passes through the TOM complex located on the outer membrane. TIM23 complex (Translocase of the Inner Membrane 23): Then, the protein moves through the TIM23 complex located on the inner membrane. MPP (Mitochondrial Processing Peptidase): Once inside the matrix, the targeting signal is cleaved oN by MPP. 2. Inner Membrane Proteins: These proteins use various pathways depending on their speciﬁc location and function within the inner membrane. Some inner membrane proteins are ﬁrst imported into the matrix via the TOM and TIM23 complexes and then inserted into the inner membrane by Oxa1, a protein translocase located in the inner membrane. Other inner membrane proteins use the TIM22 complex (Translocase of the Inner Membrane 22), which speciﬁcally inserts proteins with multiple transmembrane domains into the inner membrane. 3. Intermembrane Space Proteins: Proteins destined for the intermembrane space, the region between the outer and inner membranes, use various pathways involving both TOM and TIM complexes. Some proteins are ﬁrst transported into the matrix and then re-translocated to the intermembrane space. Others are directly inserted into the intermembrane space during their import process. 4. Outer Membrane Proteins: Proteins targeted to the outer membrane typically use the TOM complex for insertion. Some outer membrane proteins, particularly β-barrel proteins, use the SAM complex (Sorting and Assembly Machinery) to fold and insert correctly into the outer membrane. Key Points for Mitochondrial Protein Import: Proteins are imported into the mitochondria post-translationally, meaning they are fully synthesized and folded in the cytoplasm before import. Proteins must be unfolded to pass through the TOM and TIM complexes. Chaperone proteins, such as HSP70 (Heat Shock Protein 70), help maintain the unfolded state of the protein during import and facilitate its refolding once inside the mitochondria. The process of mitochondrial protein import requires energy, which is provided by ATP hydrolysis and the membrane potential across the inner membrane. CELL SIGNALING Predict how a signaling pathway will respond to the activity change in a speciﬁc protein Is the protein an activator or an inhibitor? If it's an activator, increasing its activity will likely enhance the signal, while decreasing its activity will weaken it. The opposite holds for an inhibitor. Where in the pathway does the protein act? A change in activity of a protein acting early in the pathway will have a more significant impact than a change in a protein acting downstream. What are the protein's targets? Knowing the targets helps predict the specific downstream effects of the activity change. For example, consider the Ras protein in the MAP kinase pathway. Ras is a small GTP-binding protein that activates MAP kinase kinase kinase (MAPKKK) when bound to GTP. An increase in Ras activity would lead to increased activation of MAPKKK, ultimately amplifying the signal and promoting cell proliferation. Conversely, decreasing Ras activity would weaken the signal and inhibit proliferation. Know the role of heterotrimeric G proteins, PLC and Adenylyl cyclase pathways play in cell communication Heterotrimeric G proteins, PLC (phospholipase C), and adenylyl cyclase are key players in signal transduction pathways initiated by G protein-coupled receptors (GPCRs). Heterotrimeric G proteins act as molecular switches, transducing signals from activated GPCRs to downstream eNector enzymes like adenylyl cyclase and PLC. They consist of α, β, and γ subunits. Upon GPCR activation, the α subunit exchanges GDP for GTP and dissociates from the βγ subunits, activating downstream eNectors. Adenylyl cyclase catalyzes the conversion of ATP to cyclic AMP (cAMP), a second messenger that activates protein kinase A (PKA). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), both second messengers. DAG activates protein kinase C (PKC), while IP3 triggers calcium release from the endoplasmic reticulum. These pathways are involved in various cellular processes, including: Fight-or-ﬂight response: Adrenalin activates a G protein pathway, leading to cAMP production, PKA activation, and glycogen breakdown for energy. Cell growth and diTerentiation: PKC activation by PLC is involved in hormone secretion and cell growth, division, and diNerentiation. Neurotransmitter release: PKC also plays a role in neurotransmitter release. Interpret data on the mechanism of b-arrestin downregulation of GPCR (what do you expect to happen?) Data on β-arrestin downregulation of GPCRs might include: Reduced receptor phosphorylation: β-arrestins facilitate GPCR phosphorylation by kinases, leading to receptor desensitization. Downregulation of β-arrestin would likely result in reduced receptor phosphorylation. Decreased receptor internalization: β-arrestins bind to phosphorylated GPCRs and promote their internalization through clathrin-coated pits. Reduced β-arrestin levels would lead to decreased receptor internalization. Prolonged signaling: With less β-arrestin, GPCRs would remain at the cell surface for longer, leading to prolonged signaling in response to ligand binding. Increased receptor degradation: While β-arrestins can also promote receptor recycling, downregulation might shift the balance toward increased receptor degradation in lysosomes. Overall, downregulation of β-arrestin would likely result in enhanced and prolonged GPCR signaling due to decreased desensitization and internalization. Justify the regulation of b-catenin by phosphorylation and ubiquitination in relation to experimental data β-catenin is a multifunctional protein involved in cell adhesion and Wnt signaling. Its levels and activity are tightly regulated by phosphorylation and ubiquitination. In the absence of Wnt signaling: β-catenin is phosphorylated by a destruction complex containing GSK3 (glycogen synthase kinase 3) and CK1 (casein kinase 1). Phosphorylation creates a binding site for β-TrCP, an E3 ubiquitin ligase, which ubiquitinates β-catenin, marking it for degradation by the proteasome. Upon Wnt signaling activation: The destruction complex is inhibited, preventing β-catenin phosphorylation and degradation. β-catenin accumulates in the cytoplasm and translocates to the nucleus. In the nucleus, β-catenin interacts with transcription factors like TCF (T-cell factor), activating the transcription of target genes involved in cell proliferation and development. Experimental data supporting this regulation might include: Mutations in the phosphorylation sites of β-catenin: These mutations would prevent its degradation, leading to constitutive activation of Wnt signaling. Inhibition of GSK3 or CK1: Blocking these kinases would stabilize β-catenin and enhance Wnt signaling. Proteasome inhibitors: Treating cells with proteasome inhibitors would prevent β- catenin degradation, even in the absence of Wnt signaling. Phosphorylation and ubiquitination are crucial for regulating β-catenin levels and activity, ensuring appropriate Wnt signaling activation and preventing uncontrolled cell proliferation Identify one mechanism to downregulate receptor molecules One mechanism to downregulate receptor molecules is receptor-mediated endocytosis. Ligand binding: Binding of a ligand to its receptor can trigger receptor internalization. Clathrin-coated pits: Receptors accumulate in specialized regions of the plasma membrane called clathrin-coated pits. Internalization: The coated pits invaginate and pinch oN, forming clathrin-coated vesicles containing the receptors. Sorting and degradation: The vesicles fuse with endosomes, where the receptors are sorted. Some receptors are recycled back to the plasma membrane, while others are targeted to lysosomes for degradation. This process is essential for: Attenuating signaling: Removing receptors from the cell surface reduces the number of available binding sites for ligands, dampening the signal. Preventing overstimulation: Continuous receptor activation can be detrimental. Endocytosis helps control the duration and intensity of signaling. Regulating receptor levels: Cells can adjust the rate of receptor endocytosis and degradation to ﬁne-tune their sensitivity to ligands. Receptor-mediated endocytosis is a crucial mechanism for downregulating receptor molecules, controlling signaling strength and preventing excessive or prolonged activation.

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