Advances over the past 25 years have revealed much about how the structural properties of membranes and associated proteins are linked to the thermodynamics and kinetics of membrane protein (MP) ...folding. At the same time biochemical progress has outlined how cellular proteostasis networks mediate MP folding and manage misfolding in the cell. When combined with results from genomic sequencing, these studies have established paradigms for how MP folding and misfolding are linked to the molecular etiologies of a variety of diseases. This emerging framework has paved the way for the development of a new class of small molecule “pharmacological chaperones” that bind to and stabilize misfolded MP variants, some of which are now in clinical use. In this review, we comprehensively outline current perspectives on the folding and misfolding of integral MPs as well as the mechanisms of cellular MP quality control. Based on these perspectives, we highlight new opportunities for innovations that bridge our molecular understanding of the energetics of MP folding with the nuanced complexity of biological systems. Given the many linkages between MP misfolding and human disease, we also examine some of the exciting opportunities to leverage these advances to address emerging challenges in the development of therapeutics and precision medicine.
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A protein folding intermediate pulls its weight Schlebach, Jonathan P.
Journal of biological chemistry/The Journal of biological chemistry,
08/2020, Volume:
295, Issue:
33
Journal Article
Peer reviewed
Open access
Proteins must acquire and maintain a specific fold to execute their biochemical function(s). In solution, unfolded proteins typically find this native structure through a biased sampling of preferred ...intermediate conformations. However, the initial search for these structures begins during protein synthesis, and it is unclear how much interactions between the ribosome and nascent polypeptide skew folding pathways. In this issue, Jensen and colleagues use a ribosomal force–profiling assay to show that RNase H forms a similar folding intermediate on and off the ribosome. In conjunction with measurements of the rate of RNase H unfolding on and off the ribosome, their results show that ribosomal interactions have little impact on the folding pathway of RNase H. These findings suggest that the ribosome itself does not necessarily rewire protein folding reactions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. During translation of the ...alphavirus structural polyprotein, the efficiency of −1PRF is coordinated by a ‘slippery’ sequence in the transcript, an adjacent RNA stem–loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4530 mutations on −1PRF by deep mutational scanning. While most mutations within the slip-site and stem–loop reduce the efficiency of −1PRF, the effects of mutations upstream of the slip-site are far more variable. We identify several regions where modifications of the amino acid sequence of the nascent polypeptide impact the efficiency of −1PRF. Molecular dynamics simulations of polyprotein biogenesis suggest the effects of these mutations primarily arise from their impacts on the mechanical forces that are generated by the translocon-mediated cotranslational folding of the nascent polypeptide chain. Finally, we provide evidence suggesting that the coupling between cotranslational folding and −1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how −1PRF is coordinated by features within both the transcript and nascent chain.
Graphical Abstract
Graphical Abstract
Nascent chain interactions that occur during translocon-mediated cotranslational folding generate mechanical forces that influence the decoding of a slippery sequence within the subgenomic RNA of the alphavirus structural polyprotein.
Membrane proteins are prone to misfolding and degradation within the cell, yet the nature of the conformational defects involved in this process remain poorly understood. The earliest stages of ...membrane protein folding are mediated by the Sec61 translocon, a molecular machine that facilitates the lateral partitioning of the polypeptide into the membrane. Proper membrane integration is an essential prerequisite for folding of the nascent chain. However, the marginal energetic drivers of this reaction suggest the translocon may operate with modest fidelity. In this work, we employed biophysical modeling in conjunction with quantitative biochemical measurements in order to evaluate the extent to which cotranslational folding defects influence membrane protein homeostasis. Protein engineering was employed to selectively perturb the topological energetics of human rhodopsin, and the expression and cellular trafficking of engineered variants were quantitatively compared. Our results reveal clear relationships between topological energetics and the efficiency of rhodopsin biogenesis, which appears to be limited by the propensity of a polar transmembrane domain to achieve its correct topological orientation. Though the polarity of this segment is functionally constrained, we find that its topology can be stabilized in a manner that enhances biogenesis without compromising the functional properties of rhodopsin. Furthermore, sequence alignments reveal this topological instability has been conserved throughout the course of evolution. These results suggest that topological defects significantly contribute to the inefficiency of membrane protein folding in the cell. Additionally, our findings suggest that the marginal stability of rhodopsin may represent an evolved trait.
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Most biological processes require the production and degradation of proteins, a task that weighs heavily on the cell. Mutations that compromise the conformational stability of proteins place both ...specific and general burdens on cellular protein homeostasis (proteostasis) in ways that contribute to numerous diseases. Efforts to elucidate the chain of molecular events responsible for diseases of protein folding address one of the foremost challenges in biomedical science. However, relatively little is known about the processes by which mutations prompt the misfolding of α-helical membrane proteins, which rely on an intricate network of cellular machinery to acquire and maintain their functional structures within cellular membranes. In this review, we summarize the current understanding of the physical principles that guide membrane protein biogenesis and folding in the context of mammalian cells. Additionally, we explore how pathogenic mutations that influence biogenesis may differ from those that disrupt folding and assembly, as well as how this may relate to disease mechanisms and therapeutic intervention. These perspectives indicate an imperative for the use of information from structural, cellular, and biochemical studies of membrane proteins in the design of novel therapeutics and in personalized medicine.
Despite broad biochemical relevance, our understanding of the physiochemical reactions that limit the assembly and cellular trafficking of integral membrane proteins remains superficial. In this ...work, we report the first experimental assessment of the relationship between the conformational stability of a eukaryotic membrane protein and the degree to which it is retained by cellular quality control in the secretory pathway. We quantitatively assessed both the conformational equilibrium and cellular trafficking of 12 variants of the α-helical membrane protein peripheral myelin protein 22 (PMP22), the intracellular misfolding of which is known to cause peripheral neuropathies associated with Charcot–Marie–Tooth disease (CMT). We show that the extent to which these mutations influence the energetics of Zn(II)-mediated PMP22 folding is proportional to the observed reduction in cellular trafficking efficiency. Strikingly, quantitative analyses also reveal that the reduction of motor nerve conduction velocities in affected patients is proportional to the extent of the mutagenic destabilization. This finding provides compelling evidence that the effects of these mutations on the energetics of PMP22 folding lie at the heart of the molecular basis of CMT. These findings highlight conformational stability as a key factor governing membrane protein biogenesis and suggest novel therapeutic strategies for CMT.
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The US faces an unprecedented surge in fatal drug overdoses. Naloxone, the only antidote for opiate overdose, competes at the mu opioid receptor (μOR) orthosteric site. Naloxone struggles against ...fentanyl-class synthetic opioids that now cause ∼80% of deaths. Negative allosteric modulators (NAMs) targeting secondary sites may noncompetitively downregulate μOR activation. (−)-Cannabidiol ((−)-CBD) is a candidate μOR NAM. To explore its therapeutic potential, we evaluated the structure–activity relationships among CBD analogs to identify NAMs with increased potency. Using a cyclic AMP assay, we characterize reversal of μOR activation by 15 CBD analogs, several of which proved more potent than (−)-CBD. Comparative docking investigations suggest that potent compounds interact with a putative allosteric pocket to stabilize the inactive μOR conformation. Finally, these compounds enhance naloxone displacement of fentanyl from the orthosteric site. Our results suggest that CBD analogs offer considerable potential for the development of next-generation antidotes for opioid overdose.
Aberrant protein folding and assembly contribute to a number of diseases, and efforts to rationalize how pathogenic mutations cause this phenomenon represent an important imperative in biochemical ...research. However, for α-helical membrane proteins, this task is complicated by the fact that membrane proteins require intricate machinery to achieve structural and functional maturity under cellular conditions. In this work, we utilized the Δ
G
predictor algorithm (
www.dgpred.cbr.su.se
) to survey 470 known pathogenic mutations occurring in five misfolding-prone α-helical membrane proteins for their predicted effects on the translocon-mediated membrane integration of transmembrane helices, a critical step in biosynthesis and folding of nascent membrane proteins. The results suggest that about 10 % of these mutations are likely to have adverse effects on the topogenesis of nascent membrane proteins. These results suggest that the misfolding of a modest but nonetheless significant subset of pathogenic variants may begin at the translocon. Potential implications for therapeutic design and personalized medicine are discussed.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of “temperature-sensitive” variants is ...also typically sensitive to corrector molecules that bind and stabilize the native conformation. There are many examples of temperature-sensitive rhodopsin variants, the misfolding of which is associated with the molecular basis of retinitis pigmentosa. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and 9-cis-retinal, an investigational corrector, on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures correlates with the response to 9-cis-retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants appear to disrupt a native helical kink within this transmembrane domain. By comparison, mutants that alter the structure of a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest that this observed insensitivity cannot be attributed to a single variable, but likely arises from the composite effects of mutations on the energetics of membrane integration, the stability of the native conformation, and the integrity of the retinal-binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings highlight several biophysical constraints that appear to influence the sensitivity of genetic variants to temperature and small-molecule correctors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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