scholarly journals Distributed synthesis of sarcolemmal and sarcoplasmic reticulum membrane proteins in cardiac myocytes

2021 ◽  
Vol 116 (1) ◽  
Author(s):  
Vladimir Bogdanov ◽  
Andrew M. Soltisz ◽  
Nicolae Moise ◽  
Galina Sakuta ◽  
Benjamin Hernandez Orengo ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Cardiac Myocytes ◽  
Single Molecule ◽  
Secretory Pathway ◽  
Cardiac Myocyte ◽  
Mrna Translation ◽  
Wide Distribution ◽  
Specific Protein

AbstractIt is widely assumed that synthesis of membrane proteins, particularly in the heart, follows the classical secretory pathway with mRNA translation occurring in perinuclear regions followed by protein trafficking to sites of deployment. However, this view is based on studies conducted in less-specialized cells, and has not been experimentally addressed in cardiac myocytes. Therefore, we undertook direct experimental investigation of protein synthesis in cardiac tissue and isolated myocytes using single-molecule visualization techniques and a novel proximity-ligated in situ hybridization approach for visualizing ribosome-associated mRNA molecules for a specific protein species, indicative of translation sites. We identify here, for the first time, that the molecular machinery for membrane protein synthesis occurs throughout the cardiac myocyte, and enables distributed synthesis of membrane proteins within sub-cellular niches where the synthesized protein functions using local mRNA pools trafficked, in part, by microtubules. We also observed cell-wide distribution of membrane protein mRNA in myocardial tissue from both non-failing and hypertrophied (failing) human hearts, demonstrating an evolutionarily conserved distributed mechanism from mouse to human. Our results identify previously unanticipated aspects of local control of cardiac myocyte biology and highlight local protein synthesis in cardiac myocytes as an important potential determinant of the heart’s biology in health and disease.

scholarly journals A Novel Method for Single Molecule Visualization of Active Protein Synthesis in Cardiac Myocytes Reveals SERCA2a mRNA Translation is Sarcoplasmic Reticulum Ca2+ Load Dependent

2021 ◽  
Vol 120 (3) ◽  
pp. 53a
Author(s):  
Vladimir Bogdanov ◽  
Andrew M. Soltisz ◽  
Marina S. Ivanova ◽  
Ivan S. Andreev ◽  
Rengasayee Veeraraghavan ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Myocytes ◽  
Single Molecule ◽  
Mrna Translation ◽  
Active Protein ◽  
Novel Method

Expression of an active LKB1 complex in cardiac myocytes results in decreased protein synthesis associated with phenylephrine-induced hypertrophy

2007 ◽  
Vol 292 (3) ◽  
pp. H1460-H1469 ◽  
Author(s):  
Anna A. Noga ◽  
Carrie-Lynn M. Soltys ◽  
Amy J. Barr ◽  
Suzanne Kovacic ◽  
Gary D. Lopaschuk ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cardiac Hypertrophy ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Adaptor Protein ◽  
Neonatal Rat ◽  
Hypertrophic Growth ◽  
Pathological Cardiac Hypertrophy ◽  
Neonatal Rat Cardiac Myocytes ◽  
Rat Cardiac Myocytes

AMP-activated protein kinase (AMPK) is a major metabolic regulator in the cardiac myocyte. Recently, LKB1 was identified as a kinase that regulates AMPK. Using immunoblot analysis, we confirmed high expression of LKB1 in isolated rat cardiac myocytes but show that, under basal conditions, LKB1 is primarily localized to the nucleus, where it is inactive. We examined the role of LKB1 in cardiac myocytes, using adenoviruses that express LKB1, and its binding partners Ste20-related adaptor protein (STRADα) and MO25α. Infection of neonatal rat cardiac myocytes with all three adenoviruses substantially increased LKB1/STRADα/MO25α expression, LKB1 activity, and AMPKα phosphorylation at its activating phosphorylation site (threonine-172). Since activation of AMPK can inhibit hypertrophic growth and since LKB1 is upstream of AMPK, we hypothesized that expression of an active LKB1 complex would also inhibit protein synthesis associated with hypertrophic growth. Expression of the LKB1/STRADα/MO25α complex in neonatal rat cardiac myocytes inhibited the increase in protein synthesis observed in cells treated with phenylephrine (measured via [3H]phenylalanine incorporation). This was associated with a decreased phosphorylation of p70S6 kinase and its substrate S6 ribosomal protein, key regulators of protein synthesis. In addition, we show that the pathological cardiac hypertrophy in transgenic mice with cardiac-specific expression of activated calcineurin is associated with a significant decrease in LKB1 expression. Together, our data show that increased LKB1 activity in the cardiac myocyte can decrease hypertrophy-induced protein synthesis and suggest that LKB1 activation may be a method for the prevention of pathological cardiac hypertrophy.

scholarly journals Quantitative Proteomics Links the LRRC59 Interactome to mRNA Translation on the ER Membrane

2020 ◽  
Vol 19 (11) ◽  
pp. 1826-1849
Author(s):  
Molly M. Hannigan ◽  
Alyson M. Hoffman ◽  
J. Will Thompson ◽  
Tianli Zheng ◽  
Christopher V. Nicchitta
Keyword(s):  
Protein Synthesis ◽  
Steady State ◽  
Membrane Protein ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Translation ◽  
Integral Membrane Protein ◽  
Translation Factors ◽  
Er Membrane

Protein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of numerous cellular components. Together, resident ER membrane proteins, cytoplasmic translation factors, and both integral membrane and cytosolic RNA-binding proteins operate in concert with membrane-associated ribosomes to facilitate ER-localized translation. Little is known, however, regarding the spatial organization of ER-localized translation. This question is of growing significance as it is now known that ER-bound ribosomes contribute to secretory, integral membrane, and cytosolic protein synthesis alike. To explore this question, we utilized quantitative proximity proteomics to identify neighboring protein networks for the candidate ribosome interactors SEC61β (subunit of the protein translocase), RPN1 (oligosaccharyltransferase subunit), SEC62 (translocation integral membrane protein), and LRRC59 (ribosome binding integral membrane protein). Biotin labeling time course studies of the four BioID reporters revealed distinct labeling patterns that intensified but only modestly diversified as a function of labeling time, suggesting that the ER membrane is organized into discrete protein interaction domains. Whereas SEC61β and RPN1 reporters identified translocon-associated networks, SEC62 and LRRC59 reporters revealed divergent protein interactomes. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, with the latter likely representing proximity to an ER luminal chaperone reflux pathway. In contrast, the LRRC59 interactome is highly enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins, uncovering a functional link between LRRC59 and mRNA translation regulation. Importantly, analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Moreover, [35S]-methionine incorporation assays revealed that siRNA silencing of LRRC59 expression reduced steady state translation levels on the ER by ca. 50%, and also impacted steady state translation levels in the cytosol compartment. Collectively, these data reveal a functional domain organization for the ER and identify a key role for LRRC59 in the organization and regulation of local translation.

Specific protein–lipid interactions in membrane proteins

2005 ◽  
Vol 33 (5) ◽  
pp. 938-942 ◽  
Author(s):  
C. Hunte
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Integrity ◽  
Protein Structures ◽  
Specific Protein ◽  
Lipid Binding ◽  
Functional Roles ◽  
Lipid Interactions ◽  
Head Groups ◽  
Lipid Species

Many membrane proteins selectively bind defined lipid species. This specificity has an impact on correct insertion, folding, structural integrity and full functionality of the protein. How are these different tasks achieved? Recent advances in structural research of membrane proteins provide new information about specific protein–lipid interactions. Tightly bound lipids in membrane protein structures are described and general principles of the binding interactions are deduced. Lipid binding is stabilized by multiple non-covalent interactions from protein residues to lipid head groups and hydrophobic tails. Distinct lipid-binding motifs have been identified for lipids with defined head groups in membrane protein structures. The stabilizing interactions differ between the electropositive and electronegative membrane sides. The importance of lipid binding for vertical positioning and tight integration of proteins in the membrane, for assembly and stabilization of oligomeric and multisubunit complexes, for supercomplexes, as well as for functional roles are pointed out.

scholarly journals Occupancy distributions of membrane proteins in heterogeneous liposome populations

2019 ◽  
Author(s):  
Lucy Cliff ◽  
Rahul Chadda ◽  
Janice L. Robertson
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Skewed Distribution ◽  
Liposome Size ◽  
Protein Incorporation ◽  
Size Heterogeneity ◽  
The Right ◽  
And Function

AbstractMeasurements of membrane protein structure and function often rely on reconstituting the protein into lipid bilayers through the formation of liposomes. Many measurements conducted in proteoliposomes, e.g. transport rates, single-molecule dynamics, monomer-oligomer equilibrium, require some understanding of the occupancy statistics of the liposome population for correct interpretation of the results. In homogenous liposomes, this is easy to calculate as the act of protein incorporation can be described by the Poisson distribution. However, in reality, liposomes are heterogeneous, which alters the statistics of occupancy in several ways. Here, we determine the liposome occupancy distribution for membrane protein reconstitution while taking into account liposome size heterogeneity. We calculate the protein occupancy for a homogenous population of liposomes with radius r = 200 nm, representing an idealization of vesicles extruded through 400 nm pores and compare it to the right-skewed distribution of 400 nm 2:1 POPE:POPG vesicles. As is the case for E. coli polar lipids, this synthetic composition yields a sub-population of small liposomes, ∼25 nm in radius with a long tail of larger vesicles. Previously published microscopy data of the co-localization of the CLC-ec1 Cl-/H+ transporter with liposomes, and vesicle occupancy measurements using functional transport assays, shows agreement with the heterogeneous 2:1 POPE:POPG population. Next, distributions of 100 nm and 30 nm extruded 2:1 POPE:POPG liposomes are measured by cryo-electron microscopy, demonstrating that extrusion through smaller pores does not shift the peak, but reduces polydispersity arising from large liposomes. Single-molecule photobleaching analysis of CLC-ec1-Cy5 shows the 30 nm extruded population increases the ‘Poisson-dilution’ range, reducing the probability of vesicles with more than one protein at higher protein/lipid densities. These results demonstrate that the occupancy distributions of membrane proteins into vesicles can be accurately predicted in heterogeneous populations with experimental knowledge of the liposome size distribution.

scholarly journals Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles

2020 ◽  
Author(s):  
Jia Hui Li ◽  
Paula Santos-Otte ◽  
Braedyn Au ◽  
Jakob Rentsch ◽  
Stephan Block ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Magnetic Nanoparticles ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Single Molecule ◽  
Super Resolution ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Single Molecule Level ◽  
Membrane Components

AbstractThe plasma membrane is the interface through which cells interact with their environment. Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. However, few methods are available for nanoscale manipulation of membrane protein location at the single molecule level. Here, we report the use of fluorescent magnetic nanoparticles (FMNPs) to track membrane molecules and to manipulate their movement. FMNPs allow single-particle tracking (SPT) at 10 nm spatial and 5 ms temporal resolution, and using a magnetic needle, we pull membrane components laterally through the membrane with femtonewton-range forces. In this way, we successfully dragged lipid-anchored and transmembrane proteins over the surface of living cells. Doing so, we detected submembrane barriers and in combination with super-resolution microscopy could localize these barriers to the actin cytoskeleton. We present here a versatile approach to probe membrane processes in live cells via the magnetic control of membrane protein motion.

From atoms to cells: bridging the gap between potency, efficacy, and safety of small molecules directed at a membrane protein

2021 ◽  
Author(s):  
Rodrigo Aguayo-Ortiz ◽  
Jeffery Creech ◽  
Eric N. Jimenez-Vazquez ◽  
Guadalupe Guerrero-Serna ◽  
Nulang Wang ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Single Molecule ◽  
Protein Function ◽  
Drug Targets ◽  
Transmembrane Protein ◽  
Protein Domains ◽  
Cardiac Cells ◽  
Therapeutic Drug ◽  
Efficacy And Safety

Membrane proteins constitute a substantial fraction of the human proteome, thus representing a vast source of therapeutic drug targets. Indeed, newly devised technologies now allow targeting "undruggable" regions of membrane proteins to modulate protein function in the cell. Despite the advances in technology, the rapid translation of basic science discoveries into potential drug candidates targeting transmembrane protein domains remains challenging. We address this issue by harmonizing single molecule-based and ensemble-based atomistic simulations of ligand-membrane interactions with patient-derived induced pluripotent stem cell (iPSC)-based experiments to gain insights into drug delivery, cellular efficacy, and safety of molecules directed at membrane proteins. In this study, we interrogated the pharmacological activation of the cardiac Ca2+ pump (Sarcoplasmic reticulum Ca2+-ATPase, SERCA2a) in human iPSC-derived cardiac cells as a proof-of-concept model. The combined computational-experimental approach serves as a platform to explain the differences in the cell-based activity of candidates with similar functional profiles, thus streamlining the identification of drug-like candidates that directly target SERCA2a activation in human cardiac cells. Systematic cell-based studies further showed that a direct SERCA2a activator does not induce cardiotoxic pro-arrhythmogenic events in human cardiac cells, demonstrating that pharmacological stimulation of SERCA2a activity is a safe therapeutic approach targeting the heart. Overall, this novel platform encompasses organ-specific drug potency, efficacy, and safety, and opens new avenues to accelerate the bench-to-patient research aimed at designing effective therapies directed at membrane protein domains.

scholarly journals All aboard!: The secretory pathway

2003 ◽  
Vol 25 (3) ◽  
pp. 10-12
Author(s):  
Stephen High ◽  
Samuel G. Crawshaw
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Synthesis ◽  
Membrane Protein ◽  
Cell Surface ◽  
Secretory Pathway ◽  
Entry Point ◽  
Eukaryotic Cells ◽  
Secretory Cells ◽  
Intracellular Membrane ◽  
Biosynthetic Activity

The endoplasmic reticulum (ER) is a major subcellular feature of most eukaryotic cells, and in specialized secretory cells, like those of the pancreas, it densely packs most of the cell. It is the de facto entry point into the secretory pathway and one of its key functions is to provide an extensive intracellular membrane network that supports protein synthesis (Figure 1). This biosynthetic activity is highlighted by the large number of ribosomes that are bound tightly to much of its surface, and it is these ribosomes that synthesize the secretory and membrane protein cargoes that are ultimately destined for export from the ER to the cell surface via the Golgi complex1,2.

scholarly journals Quantitative proteomics links the LRRC59 interactome to mRNA translation on the ER membrane

2020 ◽  
Author(s):  
Molly M. Hannigan ◽  
Alyson M. Hoffman ◽  
J. Will Thompson ◽  
Tianli Zheng ◽  
Christopher V. Nicchitta
Keyword(s):  
Protein Synthesis ◽  
Membrane Proteins ◽  
Redox Regulation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Translation ◽  
Functional Domain ◽  
Translation Factors ◽  
Er Membrane

AbstractProtein synthesis on the endoplasmic reticulum (ER) requires the dynamic coordination of resident membrane proteins and cytoplasmic translation factors. While ER membrane proteins functioning in ribosome association, mRNA anchoring, and protein translocation, have been identified, little is known regarding the higher order organization of ER-localized translation. Here we utilized proximity proteomics to identify neighboring protein networks for the ribosome interactors SEC61β, RPN1, SEC62, and LRRC59. Whereas the SEC61β and RPN1 BioID reporters revealed translocon-associated networks, the SEC62 and LRRC59 reporters identified divergent interactome networks of previously unexplored functions. Notably, the SEC62 interactome is enriched in redox-linked proteins and ER luminal chaperones, whereas the LRRC59 interactome is enriched in SRP pathway components, translation factors, and ER-localized RNA-binding proteins. Analysis of the LRRC59 interactome by native immunoprecipitation identified similar protein and functional enrichments. Combined, these data reveal a functional domain organization for the ER and suggest a key role for LRRC59 in the organization of mRNA translation on the ER.SummaryHannigan et al. characterize the protein interactomes of four ER ribosome-binding proteins, providing evidence that ER-bound ribosomes reside in distinct molecular environments. Their data link SEC62 to ER redox regulation and chaperone trafficking, and suggest a role for LRRC59 in SRP-coupled protein synthesis.

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