Emerging evidence suggests that host glycans influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we reveal that the receptor-binding domain (RBD) of the spike (S) ...protein on SARS-CoV-2 recognizes oligosaccharides containing sialic acid (Sia), with preference for monosialylated gangliosides. Gangliosides embedded within an artificial membrane also bind to the RBD. The monomeric affinities (K
= 100-200 μM) of gangliosides for the RBD are similar to another negatively charged glycan ligand of the RBD proposed as a viral co-receptor, heparan sulfate (HS) dp2-dp6 oligosaccharides. RBD binding and infection of SARS-CoV-2 pseudotyped lentivirus to angiotensin-converting enzyme 2 (ACE2)-expressing cells is decreased following depletion of cell surface Sia levels using three approaches: sialyltransferase (ST) inhibition, genetic knockout of Sia biosynthesis, or neuraminidase treatment. These effects on RBD binding and both pseudotyped and authentic SARS-CoV-2 viral entry are recapitulated with pharmacological or genetic disruption of glycolipid biosynthesis. Together, these results suggest that sialylated glycans, specifically glycolipids, facilitate viral entry of SARS-CoV-2.
Folding and functions of knotted proteins Hsu, Shang-Te Danny
Current opinion in structural biology,
December 2023, 2023-12-00, 20231201, Letnik:
83
Journal Article
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Topologically knotted proteins have entangled structural elements within their native structures that cannot be disentangled simply by pulling from the N- and C-termini. Systematic surveys have ...identified different types of knotted protein structures, constituting as much as 1% of the total entries within the Protein Data Bank. Many knotted proteins rely on their knotted structural elements to carry out evolutionarily conserved biological functions. Being knotted may also provide mechanical stability to withstand unfolding-coupled proteolysis. Reconfiguring a knotted protein topology by circular permutation or cyclization provides insights into the importance of being knotted in the context of folding and functions. With the explosion of predicted protein structures by artificial intelligence, we are now entering a new era of exploring the entangled protein universe.
T-Cell Protein Tyrosine Phosphatase (TCPTP, PTPN2) is a non-receptor type protein tyrosine phosphatase that is ubiquitously expressed in human cells. TCPTP is a critical component of a variety of key ...signaling pathways that are directly associated with the formation of cancer and inflammation. Thus, understanding the molecular mechanism of TCPTP activation and regulation is essential for the development of TCPTP therapeutics. Under basal conditions, TCPTP is largely inactive, although how this is achieved is poorly understood. By combining biomolecular nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and chemical cross-linking coupled with mass spectrometry, we show that the C-terminal intrinsically disordered tail of TCPTP functions as an intramolecular autoinhibitory element that controls the TCPTP catalytic activity. Activation of TCPTP is achieved by cellular competition, i.e., the intrinsically disordered cytosolic tail of Integrin-α1 displaces the TCPTP autoinhibitory tail, allowing for the full activation of TCPTP. This work not only defines the mechanism by which TCPTP is regulated but also reveals that the intrinsically disordered tails of two of the most closely related PTPs (PTP1B and TCPTP) autoregulate the activity of their cognate PTPs via completely different mechanisms.
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•UCH-L1 oxidation by H2O2 is dependent on the protonation of its cysteine residues.•M1, M6, M12, C152, and C90 are the most sensitive residues to oxidation.•C152 acts as an endogenous ...antioxidant to protect C90 from oxidation.•Oxidation of catalytic C90 leads to partial unfolding and increased aggregation.
Redox-dependent inactivation of deubiquitinases (DUBs) is a critical factor for attenuating their DUB activity in response to cellular oxidative stress. Ubiquitin C-terminal hydrolase isoform (UCH-L1) is an important DUB that is highly expressed in human neuronal cells and is implicated in a myriad of human diseases such as neurodegenerative diseases and cancer. Increasing evidence suggests an important role of UCH-L1 in redox regulation and the protection of neuronal cells from oxidative stress. In this study, we examined the molecular basis of how UCH-L1 responds to oxidation in a reversible manner. Using H2O2 as a model oxidant, we showed by mass spectrometry that a subset of methionine and cysteine residues, namely (M1, M6, M12, C90, and C152) were more susceptible to oxidation. Spectroscopic analysis showed that oxidation of C90 can lead to profound structural changes in addition to the loss of function. Importantly, we further demonstrated that C152, which is located at the substrate recognition cross-over loop, serves as a reactive oxygen species (ROS) scavenger to protect catalytic C90 from oxidation under moderate oxidative conditions. Hydrogen-deuterium exchange mass spectrometry analysis provided detailed structural mapping of the destabilizing effect of H2O2-mediated oxidation, which resulted in global destabilization far beyond the oxidation sites. These perturbations may be responsible for irreversible aggregation when subject to prolonged oxidative stress.
Deubiquitinating enzymes (DUBs) form a large protease family involved in a myriad of biological and pathological processes, including ROS sensors. ROS-mediated inhibition of their DUB activities is ...critical for fine-tuning the stress-activated signaling pathways. Here, we demonstrate that the ubiquitin C-terminal hydrolase (UCH) domain of BAP1 (BAP1-UCH) is highly sensitive to moderate oxidative stress. Oxidation of the catalytic C91 significantly destabilizes BAP1-UCH and increases the population of partially unfolded form, which is prone to aggregation. Unlike other DUBs, the oxidation-induced structural and functional loss of BAP1-UCH cannot be fully reversed by reducing agents. The oligomerization of oxidized BAP1-UCH is attributed to inter-molecular disulfide bond formation. Hydrogen-deuterium mass exchange spectrometry (HDX-MS) reveals increased fluctuations of the central β-sheet upon oxidation. Our findings suggest that oxidation-mediated functional loss and increased aggregation propensity may contribute to oncogenesis associated with BAP1.
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•BAP1-UCH is highly sensitive to mild oxidative stress induced by H2O2.•Oxidized BAP1-UCH forms disulfide bond-mediated higher-order oligomers.•Oxidation of catalytic C91 triggers misfolding and aggregation.•Oxidation-mediated functional loss and aggregation of BAP1 may contribute to oncogenesis.
Galectins are β-galactoside-binding proteins. As carbohydrate-binding proteins, they participate in intracellular trafficking, cell adhesion, and cell-cell signaling. Accumulating evidence indicates ...that they play a pivotal role in numerous physiological and pathological activities, such as the regulation on cancer progression, inflammation, immune response, and bacterial and viral infections. Galectins have drawn much attention as targets for therapeutic interventions. Several molecules have been developed as galectin inhibitors. In particular, TD139, a thiodigalactoside derivative, is currently examined in clinical trials for the treatment of idiopathic pulmonary fibrosis. Herein, we provide an in-depth review on the development of galectin inhibitors, aiming at the dissection of the structure-activity relationship to demonstrate how inhibitors interact with galectin(s). We especially integrate the structural information established by X-ray crystallography with several biophysical methods to offer, not only in-depth understanding at the molecular level, but also insights to tackle the existing challenges.
Proteins can spontaneously tie a variety of intricate topological knots through twisting and threading of the polypeptide chains. Recently developed artificial intelligence algorithms have predicted ...several new classes of topological knotted proteins, but the predictions remain to be authenticated experimentally. Here, we showed by X-ray crystallography and solution-state NMR spectroscopy that Q9PR55, an 89-residue protein from Ureaplasma urealyticum, possesses a novel 71 knotted topology that is accurately predicted by AlphaFold 2, except for the flexible N terminus. Q9PR55 is monomeric in solution, making it the smallest and most complex knotted protein known to date. In addition to its exceptional chemical stability against urea-induced unfolding, Q9PR55 is remarkably robust to resist the mechanical unfolding-coupled proteolysis by a bacterial proteasome, ClpXP. Our results suggest that the mechanical resistance against pulling-induced unfolding is determined by the complexity of the knotted topology rather than the size of the molecule.
Complete coverage of all N-glycosylation sites on the SARS-CoV2 spike protein would require the use of multiple proteases in addition to trypsin. Subsequent identification of the resulting ...glycopeptides by searching against database often introduces assignment errors due to similar mass differences between different permutations of amino acids and glycosyl residues. By manually interpreting the individual MS
spectra, we report here the common sources of errors in assignment, especially those introduced by the use of chymotrypsin. We show that by applying a stringent threshold of acceptance, erroneous assignment by the commonly used Byonic software can be controlled within 15%, which can be reduced further if only those also confidently identified by a different search engine, pGlyco3, were considered. A representative site-specific N-glycosylation pattern could be constructed based on quantifying only the overlapping subset of N-glycopeptides identified at higher confidence. Applying the two complimentary glycoproteomic software in a concerted data analysis workflow, we found and confirmed that glycosylation at several sites of an unstable Omicron spike protein differed significantly from those of the stable trimeric product of the parental D614G variant.
More than one thousand knotted protein structures have been identified so far, but the functional roles of these knots remain elusive. It has been postulated that backbone entanglement may provide ...additional mechanostability. Here, we employed a bacterial proteasome, ClpXP, to mechanically unfold 5
-knotted human ubiquitin C-terminal hydrolase (UCH) paralogs from their C-termini, followed by processive translocation into the proteolytic chamber for degradation. Our results revealed unprecedentedly slow kinetics of ClpXP-mediated proteolysis for the proteasome-associated UCHL5: ten thousand times slower than that of a green fluorescence protein (GFP), which has a comparable size to the UCH domain but much higher chemical and thermal stabilities. The ClpXP-dependent mechanostability positively correlates with the intrinsic unfolding rates of the substrates, spanning over several orders of magnitude for the UCHs. The broad range of mechanostability within the same protein family may be associated with the functional requirements for their differential malleabilities.
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