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American Journal of Physiology. Cell... Dec 2021Chemotherapeutic agents (CAs) are first-line antineoplastic treatments against a wide variety of cancers. Despite their effectiveness in halting tumor progression, side...
Chemotherapeutic agents (CAs) are first-line antineoplastic treatments against a wide variety of cancers. Despite their effectiveness in halting tumor progression, side effects associated with CAs promote muscle loss by incompletely understood mechanisms. To address this problem, we first identified how oxidative stress impairs protein synthesis in C2C12 myotubes. Transient elevations in reactive oxygen species (ROS) resulted in protein synthesis deficits and reduced ribosomal (r)RNA levels. Oxidative stress did not reduce rRNA gene (rDNA) transcription, but it caused an increase in rRNA and protein oxidation. To determine whether CAs affect protein synthesis independent of oxidative stress, we exposed myotubes to Paclitaxel (PTX), Doxorubicin (DXR), or Marizomib (Mzb) at doses that did result in elevated ROS levels (sub-ROS). Exposure to CAs reduced protein synthesis and rRNA levels, but unlike oxidative stress, sub-ROS exposures impaired rDNA transcription. These results indicate that although oxidative stress disrupts protein synthesis by compromising ribosomal quantity and quality, CAs at sub-ROS doses compromise protein synthesis and ribosomal capacity, at least in part, by reducing rDNA transcription. Therefore, CAs negatively impact protein synthesis by causing oxidative stress in addition to directly reducing the ribosomal capacity of myotubes in a ROS-independent manner.
Topics: Animals; Antineoplastic Agents; Cell Line; Hydrogen Peroxide; Mice; Muscle Fibers, Skeletal; Oxidative Stress; Protein Biosynthesis; Ribosomes
PubMed: 34705587
DOI: 10.1152/ajpcell.00116.2021 -
Proceedings of the National Academy of... Jun 2020Split inteins are privileged molecular scaffolds for the chemical modification of proteins. Though efficient for in vitro applications, these polypeptide ligases have...
Split inteins are privileged molecular scaffolds for the chemical modification of proteins. Though efficient for in vitro applications, these polypeptide ligases have not been utilized for the semisynthesis of proteins in live cells. Here, we biochemically and structurally characterize the naturally split intein VidaL. We show that this split intein, which features the shortest known N-terminal fragment, supports rapid and efficient protein -splicing under a range of conditions, enabling semisynthesis of modified proteins both in vitro and in mammalian cells. The utility of this protein engineering system is illustrated through the traceless assembly of multidomain proteins whose biophysical properties render them incompatible with a single expression system, as well as by the semisynthesis of dual posttranslationally modified histone proteins in live cells. We also exploit the domain swapping function of VidaL to effect simultaneous modification and translocation of the nuclear protein HP1α in live cells. Collectively, our studies highlight the VidaL system as a tool for the precise chemical modification of cellular proteins with spatial and temporal control.
Topics: Cell Engineering; Inteins; Protein Biosynthesis; Protein Engineering; Protein Splicing
PubMed: 32424098
DOI: 10.1073/pnas.2003613117 -
Cellular and Molecular Life Sciences :... Nov 2016Candida albicans is a major human fungal pathogen responsible for both systemic and mucosal infections in a wide variety of immunocompromised individuals. Because the... (Review)
Review
Candida albicans is a major human fungal pathogen responsible for both systemic and mucosal infections in a wide variety of immunocompromised individuals. Because the ability of C. albicans to undergo a reversible morphological transition from yeast to filaments is important for virulence, significant research efforts have focused on mechanisms that control this transition. While transcriptional and post-translational mechanisms have been well-studied, considerably less is known about the role of post-transcriptional mechanisms. However, in recent years several discoveries have begun to shed light on this important, but understudied, area. Here, I will review a variety of post-transcriptional mechanisms that have recently been shown to control C. albicans morphology, virulence and/or virulence-related processes, including those involving alternative transcript localization, mRNA stability and translation. I will also discuss the role that these mechanisms play in other pathogens as well as the potential they may hold to serve as targets for new antifungal strategies. Ultimately, gaining a better understanding of C. albicans post-transcriptional mechanisms will significantly improve our knowledge of how morphogenesis and virulence are controlled in fungal pathogens and open new avenues for the development of novel and more effective antifungals.
Topics: Alternative Splicing; Antifungal Agents; Candida albicans; Protein Biosynthesis; RNA Stability; Transcription, Genetic
PubMed: 27312239
DOI: 10.1007/s00018-016-2294-y -
The Journal of Neuroscience : the... Apr 2019Secreted amyloid precursor protein-alpha (sAPPα) has growth factor-like properties and can modulate long-term potentiation (LTP) and memory. Here, we demonstrate that...
Secreted amyloid precursor protein-alpha (sAPPα) has growth factor-like properties and can modulate long-term potentiation (LTP) and memory. Here, we demonstrate that exposure to sAPPα converts short-lasting LTP into protein-synthesis-dependent late LTP in hippocampal slices from male rats. sAPPβ had no discernable effect. We hypothesized that sAPPα facilitated LTP via regulated glutamate receptor trafficking and protein synthesis. We found using a linear mixed model that sAPPα stimulated trafficking of GluA2-lacking AMPARs, as well as NMDARs to the extrasynaptic cell surface, in a calcium/calmodulin-dependent kinase II and protein kinase G-dependent manner. Both cell surface receptor accumulation and LTP facilitation were present even after sAPPα washout and inhibition of receptor trafficking or protein synthesis prevented all these effects. Direct visualization of newly synthesized proteins (FUNCAT-PLA) confirmed the ability of sAPPα to stimulate protein synthesis and revealed GluA1 as one of the upregulated proteins. Therefore, sAPPα generates a coordinated synthesis and trafficking of glutamate receptors to the cell surface that facilitate LTP. Secreted amyloid precursor protein-alpha (sAPPα) is a neurotrophic and neuroprotective protein that can promote synaptic plasticity and memory, yet the molecular mechanisms underlying these effects are still not well understood. Here, we show that sAPPα facilitates long-term potentiation (LTP) in a concentration-dependent fashion through cellular processes involving protein synthesis and trafficking of both GluA2-lacking AMPARs and NMDARs to the extrasynaptic cell surface. sAPPα also enhances GluA1, but not GluA2, synthesis. The trafficking effects, along with the LTP facilitation, persist after sAPPα washout, revealing a metaplastic capability of exogenous sAPPα administration. sAPPα thus facilitates LTP through coordinated activation of protein synthesis and trafficking of glutamate receptors to the cell surface, where they are positioned for priming LTP.
Topics: Amyloid beta-Protein Precursor; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cyclic GMP-Dependent Protein Kinases; Hippocampus; Long-Term Potentiation; Male; Protein Biosynthesis; Protein Transport; Rats; Rats, Sprague-Dawley; Receptors, Glutamate
PubMed: 30804097
DOI: 10.1523/JNEUROSCI.1826-18.2019 -
Journal of Applied Physiology... Aug 2019
Topics: Animals; Humans; Muscle Proteins; Muscle, Skeletal; Muscular Diseases; Myocardium; Protein Biosynthesis; Protein Processing, Post-Translational
PubMed: 31446832
DOI: 10.1152/japplphysiol.00497.2019 -
ACS Synthetic Biology Sep 2023The development of mRNA-based therapeutics centers around the natural functioning of mRNA molecules to provide the genetic information required for protein translation.... (Review)
Review
The development of mRNA-based therapeutics centers around the natural functioning of mRNA molecules to provide the genetic information required for protein translation. To improve the efficacy of these therapeutics and minimize side effects, researchers can focus on the features of mRNA itself or the properties of the delivery agent to achieve the desired response. The tools considered for mRNA manipulation can be improved in terms of targetability, tunability, and translatability to medicine. While ongoing studies are dedicated to improving conventional approaches, innovative approaches can also be considered to unleash the full potential of mRNA-based therapeutics. Here, we discuss the opportunities that emerged from introducing synthetic biology to mRNA therapeutics. It includes a discussion of modular self-assembled mRNA nanoparticles, logic gates on a single mRNA molecule, and other possibilities.
Topics: Humans; Synthetic Biology; Nanoparticles; Protein Biosynthesis; RNA, Messenger; Research Personnel
PubMed: 37672348
DOI: 10.1021/acssynbio.3c00253 -
ACS Synthetic Biology Jan 2023In living cells, proteins often exert their functions by interacting with other proteins forming protein complexes. Obtaining homogeneous samples of protein complexes...
In living cells, proteins often exert their functions by interacting with other proteins forming protein complexes. Obtaining homogeneous samples of protein complexes with correct fold and stoichiometry is critical for its biochemical and biophysical characterization as well as functional investigation. Here, we developed a Ribozyme-Assisted Polycistronic co-expression system (pRAP) for heterologous co-production and in vivo assembly of multi-subunit complexes. In the pRAP system, a polycistronic mRNA transcript is co-transcriptionally converted into individual mono-cistrons in vivo. Each cistron can initiate translation with comparable efficiency, resulting in balanced production for all subunits, thus permitting faithful protein complex assembly. With pRAP polycistronic co-expression, we have successfully reconstituted large functional multi-subunit complexes involved in mammalian translation initiation. Our invention provides a valuable tool for studying the molecular mechanisms of biological processes.
Topics: Animals; Protein Biosynthesis; RNA, Catalytic; Proteins; Protein Processing, Post-Translational; Mammals
PubMed: 36512506
DOI: 10.1021/acssynbio.2c00416 -
Physiological Reviews Oct 2007A substantial fraction of eukaryotic gene products are synthesized by ribosomes attached at the cytosolic face of the endoplasmic reticulum (ER) membrane. These... (Review)
Review
A substantial fraction of eukaryotic gene products are synthesized by ribosomes attached at the cytosolic face of the endoplasmic reticulum (ER) membrane. These polypeptides enter cotranslationally in the ER lumen, which contains resident molecular chaperones and folding factors that assist their maturation. Native proteins are released from the ER lumen and are transported through the secretory pathway to their final intra- or extracellular destination. Folding-defective polypeptides are exported across the ER membrane into the cytosol and destroyed. Cellular and organismal homeostasis relies on a balanced activity of the ER folding, quality control, and degradation machineries as shown by the dozens of human diseases related to defective maturation or disposal of individual polypeptides generated in the ER.
Topics: Endoplasmic Reticulum; Humans; Metabolic Diseases; Molecular Chaperones; Protein Biosynthesis; Protein Folding
PubMed: 17928587
DOI: 10.1152/physrev.00050.2006 -
Open Biology Dec 2020Protein synthesis from mRNA is an energy-intensive and tightly controlled cellular process. Translation elongation is a well-coordinated, multifactorial step in... (Review)
Review
Protein synthesis from mRNA is an energy-intensive and tightly controlled cellular process. Translation elongation is a well-coordinated, multifactorial step in translation that undergoes dynamic regulation owing to cellular state and environmental determinants. Recent studies involving genome-wide approaches have uncovered some crucial aspects of translation elongation including the mRNA itself and the nascent polypeptide chain. Additionally, these studies have fuelled quantitative and mathematical modelling of translation elongation. In this review, we provide a comprehensive overview of the key determinants of translation elongation. We discuss consequences of ribosome stalling or collision, and how the cells regulate translation in case of such events. Next, we review theoretical approaches and widely used mathematical models that have become an essential ingredient to interpret complex molecular datasets and study translation dynamics quantitatively. Finally, we review recent advances in live-cell reporter and related analysis techniques, to monitor the translation dynamics of single cells and single-mRNA molecules in real time.
Topics: Animals; Eukaryotic Cells; Humans; Models, Biological; Peptide Chain Elongation, Translational; Protein Biosynthesis; RNA, Messenger; RNA, Transfer; Ribosomes
PubMed: 33292102
DOI: 10.1098/rsob.200292 -
FEMS Yeast Research Dec 2010Errors occur randomly and at low frequency during the translation of mRNA. However, such errors may also be programmed by the sequence and structure of the mRNA. These... (Review)
Review
Errors occur randomly and at low frequency during the translation of mRNA. However, such errors may also be programmed by the sequence and structure of the mRNA. These programmed events are called 'recoding' and are found mostly in viruses, in which they are usually essential for viral replication. Translational errors at a stop codon may also be induced by drugs, raising the possibility of developing new treatment protocols for genetic diseases on the basis of nonsense mutations. Many studies have been carried out, but the molecular mechanisms governing these events remain largely unknown. Studies on the yeast Saccharomyces cerevisiae have contributed to characterization of the HIV-1 frameshifting site and have demonstrated that frameshifting is conserved from yeast to humans. Yeast has also proved a particularly useful model organism for deciphering the mechanisms of translation termination in eukaryotes and identifying the factors required to obtain a high level of natural suppression. These findings open up new possibilities for large-scale screening in yeast to identify new drugs for blocking HIV replication by inhibiting frameshifting or restoring production of the full-length protein from a gene inactivated by a premature termination codon. We explore these two aspects of the contribution of yeast studies to human medicine in this review.
Topics: Frameshifting, Ribosomal; Humans; Peptide Chain Termination, Translational; Protein Biosynthesis; RNA, Messenger; Saccharomyces cerevisiae; Suppression, Genetic
PubMed: 20955199
DOI: 10.1111/j.1567-1364.2010.00684.x