Folding Sensor Research Articles

The ER folding sensor UGGT1 acts on TAPBPR-chaperoned peptide-free MHC I.

Adaptive immune responses are triggered by antigenic peptides presented on major histocompatibility complex class I (MHC I) at the surface of pathogen-infected or cancerous cells. Formation of stable peptide-MHC I complexes is facilitated by tapasin and TAPBPR, two related MHC I-specific chaperones that catalyze selective loading of suitable peptides onto MHC I in a process called peptide editing or proofreading. On their journey to the cell surface, MHC I complexes must pass a quality control step performed by UGGT1, which senses the folding status of the transiting N-linked glycoproteins in the endoplasmic reticulum (ER). UGGT1 reglucosylates non-native glycoproteins and thereby allows them to revisit the ER folding machinery. Here, we describe a reconstituted in-vitro system of purified human proteins that enabled us to delineate the function of TAPBPR during the UGGT1-catalyzed quality control and reglucosylation of MHC I. By combining glycoengineering with liquid chromatography-mass spectrometry, we show that TAPBPR promotes reglucosylation of peptide-free MHC I by UGGT1. Thus, UGGT1 cooperates with TAPBPR in fulfilling a crucial function in the quality control mechanisms of antigen processing and presentation.

Squaryl group-modified UDP analogs as inhibitors of the endoplasmic reticulum-resident folding sensor enzyme UGGT.

UDP-Glc:glycoprotein glucosyltransferase (UGGT) has a central role to retain quality control of correctly folded N-glycoprotein in the endoplasmic reticulum (ER). A selective and potent inhibitor against UGGT could lead to elucidation of UGGT-related events, but such a molecule has not been identified so far. Examples of small molecules with UGGT inhibitory activity are scarce. Here, we report squaryl group-modified UDP analogs as a promising UGGT inhibitor. Among these, the compound possessing a 2'-amino group of the uridine moiety and hydroxyethyl-substituted squaramide exhibited the highest potency, suggesting its relevance as a molecule for further optimization.

Analysis of Selenoprotein F Binding to UDP-Glucose:Glycoprotein Glucosyltransferase (UGGT) by a Photoreactive Crosslinker.

In the endoplasmic reticulum glycoprotein quality control system, UDP-glucose : glycoprotein glucosyltransferase (UGGT) functions as a folding sensor. Although it is known to form a heterodimer with selenoprotein F (SelenoF), the details of the complex formation remain obscure. A pulldown assay using co-transfected SelenoF and truncated mutants of human UGGT1 (HUGT1) revealed that SelenoF binds to the TRXL2 domain of HUGT1. Additionally, a newly developed photoaffinity crosslinker was selectively introduced into cysteine residues of recombinant SelenoF to determine the spatial orientation of SelenoF to HUGT1. The crosslinking experiments showed that SelenoF formed a covalent bond with amino acids in the TRXL3 region and the interdomain between βS2 and GT24 of HUGT1 via the synthetic crosslinker. SelenoF might play a role in assessing and refining the disulfide bonds of misfolded glycoproteins in the hydrophobic cavity of HUGT1 as it binds to the highly flexible region of HUGT1 to reach its long hydrophobic cavity. Clarification of the SelenoF-binding domain of UGGT and its relative position will help predict and reveal the function of SelenoF from a structural perspective.

Goblet cells need some stress.

The intestinal tract is protected by epithelium-covering mucus, which is constantly renewed by goblet cells, a specialized type of epithelial cell. Mucus is largely composed of MUC2 mucin, an enormous molecule that poses a high demand on the endoplasmic reticulum (ER) for proper folding and protein assembly, creating a challenge for the secretory machinery in goblet cells. In this issue of the JCI, Grey et al. reveal that the ER resident protein and folding sensor ERN2 (also known as IRE1β) was instrumental for goblet cells to produce sufficient amounts of mucus to form a protective mucus layer. In the absence of ERN2, mucus production was reduced, impairing the mucus barrier, which allowed bacteria to penetrate and cause an epithelial cell stress response. This study emphasizes the importance of a controlled unfolded protein response (UPR) for goblet cell secretion.

Folding state as the Decision Maker in Detecting Misfolded Proteins by Hsp70

Maintaining cellular proteostasis is challenging due to the constantly changing and crowded cellular environment. Mutations, transcriptional and translational errors, and chemical and pathological stresses in a cell lead to protein misfolding and eventual formation of insoluble toxic aggregates. These aggregates typically result in loss of native protein function and are often associated with conformational diseases such as Alzheimer's, Parkinson's, Huntington's, and various types of cancer. The cellular protein quality control systems are essential for creating a stable cellular environment by resolving the misfolded proteins to uphold a stable proteome. The triage decision for a misfolded protein is regulated by Heat shock protein 70 (Hsp70), the central hub of protein quality control machinery, along with the E3 ubiquitin ligase C‐terminus of Hsc70‐interacting protein (CHIP). Hsp70 itself helps misfolded “client” proteins to refold and directs protein clients to downstream refolding pathways, whereas Hsp70 interaction with CHIP can lead to CHIP‐mediated ubiquitination, ultimately directing the proteins to proteasomal degradation. Understanding the molecular mechanisms that enable the Hsp70/CHIP complex to triage client proteins would allow for the identification of new avenues of treatment for the above‐mentioned diseases. With an objective to illustrate how the varying degrees of folding in a model client protein dictate the interaction between Hsp70 and the client, we have designed a spectrum of client proteins we named folding sensor TPRs (FSTPRs). We initially hypothesized that the more unfolded the client higher the affinity towards Hsp70. Circular dichroism (CD) spectroscopy was used to assess the folding states of each FSTPR variant, and biolayer interferometry (BLI) was utilized to quantify the affinities of each FSTPR towards the Hsp70 substrate‐binding domain (SBD). During our BLI experiments, we observed a range of threshold unfolding percentages that correspond to the highest affinity. Above and below the threshold range, the affinity decreased. This behavior was recapitulated with a disulfide‐locked FSTPR that is folded in the oxidized state and predominantly unfolded in the reduced state. Even though support from further data is necessary, these studies provide us preliminary insight into how Hsp70 interacts with a misfolded client and the role played by folding state in dictating Hsp70/client interactions.

Study on the binding mode of aptamer to ampicillin and its electrochemical response behavior in two different reaction media.

A study was carried out to investigate the binding mode of aptamer to ampicillin (AMP) and its electrochemical response behavior. The binding mode was confirmed using the molecular dynamics (MD) simulation method to obtain the corresponding binding dynamic change process. Following the confirmed binding mode, a qualitative elucidation was provided on the electrochemical response characteristics of a single-probe aptamer-based folding sensor. The results show that there exist two different binding modes in two different solution systems, Phys2 and H2O (0.1M NaCl). These two binding modes can respectively induce two different contraction changes, thereby driving the methylene blue (MB)-modified aptamer probe to show a "close-to-interface" convergence behavior with different degrees on the actual electrode surface, which validates two apparently different electrochemical response behavior characteristics of "signal-on" for the sensor. By contrast, H2O (0.1M NaCl) as the reaction medium is more conducive to the formation of a stable aptamer/AMP complex and the development of a high-sensitivity analytical method with a low detection limit of 0.033μM. The simulation results effectively support the experimental results, which is helpful in gaining a deeper understanding of the relationship between the signaling mechanism and practical analytical performance for aptamer-based folding sensors at the molecular level.

Using Folding Structure to Enhance Measurement Range, Sensitivity of the Flexible Sensors: A Simple, Eco‐Friendly, and Effective Method

AbstractFlexible sensing technology has shown great application value in motion monitoring and physiological signal detection. In many applications, the detected signal has the characteristics of low strength and wide range, thus improving the sensitivity and measurement range of sensors becomes extremely important. An enhancing method based on folding structure is proposed in this paper. Through the analysis of folding structure characteristics, the theoretical model of the folding structure effect in flexible sensors is established and the enhancement effect of sensitivity and measurement range is testified through three kinds of commonly used sensors, resistive, capacitive, and inductive sensors. Through changing the number of folding units, the sensitivity and measurement range of sensors can achieve to be adjusted. Folding sensors also have good hysteresis and repeatability properties and show the great performance in the application of human motion monitoring, such as limbs bending and rotating, muscle powering. The proposed method has the characteristics of simple, economical, environment friendly, and widely suitable, meanwhile avoiding the complexity of other ways to improve performance. This work has potential to enhance the sensitivity and measurement range for more kinds of flexible sensors and is of great value to fabricate high performance sensors based on folding structure.

Chemical-Synthesis-Based Approach to Glycoprotein Functions in the Endoplasmic Reticulum.

The introduction of Asn-linked glycans to nascent polypeptides occurs in the lumen of the endoplasmic reticulum of eukaryotic cells. After the removal of specific sugar residues, glycoproteins acquire signals in the glycoprotein quality control (GPQC) system and enter the folding cycle composed of lectin-chaperones calnexin (CNX) and calreticulin (CRT), glucosidase II (G-II), and UDP-Glc:glycoprotein glucosyltransferase (UGGT). G-II initiates glycoproteins' entry and exit from the cycle, and UGGT serves as the "folding sensor". This account summarizes our effort to analyze the properties of enzymes and lectins that play important roles in GPQC, especially those involved in the CNX/CRT cycle. To commence our study, general methods for the synthesis of high-mannose-type glycans and glycoproteins were established. Based on these, various substrates to analyze components of the GPQC were created, and properties of CRT, G-II, and UGGT have been clarified.

A metabolite binding protein moonlights as a bile-responsive chaperone.

Bile salts are secreted into the gastrointestinal tract to aid in the absorption of lipids. In addition, bile salts show potent antimicrobial activity in part by mediating bacterial protein unfolding and aggregation. Here, using a protein folding sensor, we made the surprising discovery that the Escherichia coli periplasmic glycerol-3-phosphate (G3P)-binding protein UgpB can serve, in the absence of its substrate, as a potent molecular chaperone that exhibits anti-aggregation activity against bile salt-induced protein aggregation. The substrate G3P, which is known to accumulate in the later compartments of the digestive system, triggers a functional switch between UgpB's activity as a molecular chaperone and its activity as a G3P transporter. A UgpB mutant unable to bind G3P is constitutively active as a chaperone, and its crystal structure shows that it contains a deep surface groove absent in the G3P-bound wild-type UgpB. Our work illustrates how evolution may be able to convert threats into signals that first activate and then inactivate a chaperone at the protein level in a manner that bypasses the need for ATP.

Glycan dependent refolding activity of ER glucosyltransferase (UGGT)

BackgroundIn the endoplasmic reticulum (ER), folding of glycoproteins is assisted by a combined action of enzymes and chaperones that leads them to biologically functional structures. In this system, UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) plays an essential role as the “folding sensor” by virtue of its ability to discriminate folding states of client glycoproteins. However, besides its transferase activity, whether UGGT1 possesses any chaperone activity that facilitates protein folding is yet to be addressed. MethodsWe prepared oligomannose-type glycan modified RNase (M9GN2-RNase) by chemoenzymatic means using M9GN-oxazoline and glycan truncated RNase B and analyzed the effect of human UGGT1 (HUGT1) for refolding of the denatured M9GN2-RNase. Refolding was evaluated based on the RNase activity which was measured by the cleavage of the RNA substrate. ResultsHUGT1 slightly accelerated the folding of M9GN2-RNase and non-glycosylated RNase A as the same extent. However, HUGT1 remarkably accelerated the folding of M9GN2-RNase in the presence of UDP-Glc. In contrast, neither UDP nor UDP-Gal was effective in enhancing the folding. Additionally, an HUGT1 mutant which lacks the glucosyltransferase activity did not accelerate the protein folding of M9GN2-RNase. ConclusionsHUGT1has the ability to promote the refolding of denatured protein and the effect would be enhanced when HUGT1 tightly interacts with the client protein via glycan recognition. General significanceOur study provides a possibility that HUGT1 play a role not only in sensing the misfolded glycoprotein but also in promoting folding of glycoproteins in the endoplasmic reticulum glycoprotein quality control.

The ER glycoprotein folding sensor UDP-Glc: glycoprotein glucosyltransferase is broadly expressed in C. elegans hermaphrodite.

The ER glycoprotein folding sensor UDP-Glc: glycoprotein glucosyltransferase is broadly expressed in C. elegans hermaphrodite.

Prion Protein Translocation Mechanism Revealed by Pulling Force Studies

The mammalian prion protein (PrP) engages with the ribosome–Sec61 translocation channel complex to generate different topological variants that are either physiological, or involved in neurodegenerative diseases. Here, we describe cotranslational folding and translocation mechanisms of PrP coupled to an Xbp1-based arrest peptide as folding sensor, to measure forces acting on PrP nascent chain. Our data reveal two main pulling events followed by a minor third one exerted on the nascent chains during their translocation.Using those force landscapes, we show that a specific sequence within an intrinsically disordered region, containing a polybasic and glycine-proline rich residues, modulates the second pulling event by interacting with TRAP complex. This work also delineates the sequence of events involved in generation of PrP toxic transmembrane topologies during its synthesis. Our results shed new insight into the folding of such a topological complex protein, where marginal pulling by the signal sequence, together with the flanking downstream sequence in the mature domain, primarily drives an overall inefficient translocation resulting in the nascent chain to adopt alternative topologies.

Biophysical Folding Sensors for the Detection of Misfolded Proteins by Hsp70

Chaperoned ubiquitination complexes play a defining role in responding to chemical and pathological stresses and an understanding of how these complexes are regulated on a molecular level offers the opportunity to identify new avenues of treatment for a wide range of diseases. Hsp70 participates in chaperoned ubiquitination by recruiting and attempting to refold misfolded protein “clients” in an ATP‐dependent process. Misfolded clients interact with a binding cleft within the substrate binding domain (SBD) formed by a beta‐sheet subdomain (SBD‐beta) and helical lid subdomain. Understanding how Hsp70 recruits clients and positions clients relative to the E3 ligase CHIP is critical for understanding the core interactions between CHIP and Hsp70 that dictate the outcome of chaperoned ubiquitination. Herein we describe a set of folding sensors representing a spectrum of folded and unfolded states. When utilized in ubiquitination reactions this system provides a tunable folded/unfolded client with an Hsp70‐SBD binding site that provides control over the distance from the model unfolded client to the E2~Ub active site bound to the E3 ligase CHIP, thereby providing a measure of how folding state dictates degree of ubiquitination.Support or Funding InformationNIGMS R35 GM128595

How Do O‐Fucose Glycans Stabilize Thrombospondin Type 1 Repeats?

Thrombospondin Type 1 Repeats (TSRs) are small cysteine‐rich domains found in multiple cell‐surface and secreted proteins. TSRs containing the consensus sequence Cxx(S/T)C are typically modified on the serine or threonine with an O‐linked Glcβ1‐3Fuc disaccharide. The O‐fucose is added by Protein O‐fucosyltransferase 2 (POFUT2), which is then elongated by β1‐3glucosyltransferase (B3GLCT). Elimination of Pofut2 in mice results in early embryonic lethality, and human mutations in B3GLCT cause Peters Plus Syndrome (PPS), a Congenital Disorder of Glycosylation. POFUT2 and B3GLCT are localized in the endoplasmic reticulum (ER) ‐ the folding compartment for proteins in the secretory pathway, and POFUT2 only adds fucose to properly folded TSRs. Thus, POFUT2 appears to function as a folding sensor for TSRs, and both POFUT2 and B3GLCT have been proposed to assist in the folding of TSR‐containing proteins. Interestingly, loss of POFUT2 causes secretion defects for most TSR‐containing proteins, while loss of B3GLCT only effects secretion of a subset of these proteins. The mechanisms behind why POFUT2 is required for section of most TSR‐containing proteins but B3GLCT is required for only a few is not understood. We hypothesize that this unique disaccharide on TSRs is interacting with amino acids in close proximity to the sugars thereby stabilizing the folded state of TSRs and ultimately the protein as a whole, and that the fucose has the major stabilizing effect, while the glucose is only required for some TSRs. To test this hypothesis, we designed a Reductive Unfolding Assay to monitor the effects of the sugars on the stability of TSRs. We have identified amino acids affected by the presence of the fucose or glucose using NMR and molecular dynamics simulations. We have mutated several of these amino acids to determine if they reduce the stabilizing effects of the sugars using the reductive unfolding assay. One such mutant, P547A in TSR3 from human thrombospondin‐1, decreases the sugar‐mediated stabilization of TSR3, providing evidence that the disaccharide stabilizes the folded state by interacting with certain amino acids in close proximity. We are examining the stabilizing effects of the fucose and glucose on several other TSRs as well. These results provide further evidence that POFUT2 and B3GLCT act as quality control enzymes inside the ER.Support or Funding InformationThis work was supported by NIH grant HD090156.

Monitoring Proteome Stress in Live Cells Using HaloTag-Based Fluorogenic Sensor.

Fluorescent folding sensor is a powerful tool to detect proteome stresses, including heat, osmotic, oxidative, and drug induced stresses. Monitoring proteome stress using these sensors allows us to dissect the mechanism of cellular stress and find therapeutics that ameliorate stress related diseases. Here we present a HaloTag-based fluorogenic proteome stress sensor (AgHalo) to robustly detect and quantify proteome stresses in live cells. We describe how proteome stresses are monitored in both bacterial and mammalian live cells using fluorescence confocal microscope and fluorescence plate reader.

All-in-one cellulose based triboelectric nanogenerator for electronic paper using simple filtration process

Cellulose is one of the emerging materials as a natural polymer. In this work, cellulose nanofiber is utilized as a dielectric layer in triboelectric nanogenerator. To retain the flexibility of the fabricated paper, Ag nanowires layer is employed for electrode material and counter triboelectric layer simultaneously. This triboelectric nanogenerator is fabricated by a filtration process with different cellulose nanofiber and Ag nanowire condition. This triboelectric nanogenerator operates by contacting and separating of only two same bilayer paper consisting of cellulose nanofiber and Ag nanowire. Cellulose nanofiber and Ag nanowires layer acting as not only triboelectric layer and counter-triboelectric layer but substrate and electrode respectively. The case of cellulose nanofiber solution homogenized with 20 passes in 1000 bar and Ag nanowire with 0.1 wt% solution shows the optimal peak corresponding to 21 V of open-circuit voltage and 2.5 µA of short-circuit current. The maximum power output represents the value of 693 mW/m2 for a 10 MΩ external resistance. Fabricated device is dispersed and completely dissolved in deionized water by using sonicator for 30 min and returns to original ingredient without producing any pollutant. This nanogenerator plays roles of not only harvesting ambient energy but folding sensor, humidity sensor, and e-paper-like device due to the paper-like characteristics and nano-porous of cellulose nanofiber and Ag nanowires.

Substrate Recognition of Glycoprotein Folding Sensor UGGT Analyzed by Site-Specifically 15N-Labeled Glycopeptide and Small Glycopeptide Library Prepared by Parallel Native Chemical Ligation

UDP-glucose:glycoprotein glucosyltransferase (UGGT) distinguishes glycoproteins in non-native conformations from those in native conformations and glucosylates from only non-native glycoproteins. To analyze how UGGT recognizes non-native glycoproteins, we chemically synthesized site-specifically 15N-labeled interleukin 8 (IL-8) C-terminal (34-72) glycopeptides bearing a Man9GlcNAc2 (M9) oligosaccharide. Chemical shift perturbation mapping NMR experiments suggested that Phe65 of the glycopeptide specifically interacts with UGGT. To analyze this interaction, we constructed a glycopeptide library by varying Phe65 with 10 other natural amino acids, via parallel native chemical ligation between a glycopeptide-α-thioester and a peptide library consisting of 11 peptides. UGGT assay against the glycopeptide library revealed that, although less hydrophobic glycopeptides could be used as substrates for UGGT, hydrophobic glycopeptides are preferred.

TAPBPR bridges UDP-glucose:glycoprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway.

Recently, we revealed that TAPBPR is a peptide exchange catalyst that is important for optimal peptide selection by MHC class I molecules. Here, we asked whether any other co-factors associate with TAPBPR, which would explain its effect on peptide selection. We identify an interaction between TAPBPR and UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1), a folding sensor in the calnexin/calreticulin quality control cycle that is known to regenerate the Glc1Man9GlcNAc2 moiety on glycoproteins. Our results suggest the formation of a multimeric complex, dependent on a conserved cysteine at position 94 in TAPBPR, in which TAPBPR promotes the association of UGT1 with peptide-receptive MHC class I molecules. We reveal that the interaction between TAPBPR and UGT1 facilities the reglucosylation of the glycan on MHC class I molecules, promoting their recognition by calreticulin. Our results suggest that in addition to being a peptide editor, TAPBPR improves peptide optimisation by promoting peptide-receptive MHC class I molecules to associate with the peptide-loading complex.

Folding in the Cell - Ions, Crowders, Osmolytes

Proteins fold and function in the densely crowded and highly heterogeneous cell, which is filled up to a volume of 40% with macromolecules. That under such conditions cells can keep their proteome folded and organized without uncontrollable aggregation is a remarkable aspect of biology. We first discuss how the different cosolutes in the cellular milieu such as ions, crowders and osmolytes govern the protein folding equilibrium. We thereby present a novel classification scheme of cosolute effects based on their thermodynamic fingerprints. This model is of fundamental importance to understand how the proteome stability is modulated by cellular processes, e.g. to understand how osmolytes or chaperones protect the proteome or how most destabilized proteins aggregate under different cell stresses. We further developed folding sensors that probe the different cosolute effects directly in cells. Thereby, we show that cell stress can significantly modulate the folding equilibrium. Remarkably, protective cellular mechanisms such as the heat shock response or the regulatory volume increase are highly adapted to minimize the impact on the proteome.

Functional Rescue of a Misfolded Drosophila melanogaster Dopamine Transporter Mutant Associated with a Sleepless Phenotype by Pharmacological Chaperones

Folding-defective mutants of the human dopamine transporter (DAT) cause a syndrome of infantile dystonia/parkinsonism. Here, we provide a proof-of-principle that the folding deficit is amenable to correction in vivo by two means, the cognate DAT ligand noribogaine and the HSP70 inhibitor, pifithrin-μ. We examined the Drosophila melanogaster (d) mutant dDAT-G108Q, which leads to a sleepless phenotype in flies harboring this mutation. Molecular dynamics simulations suggested an unstable structure of dDAT-G108Q consistent with a folding defect. This conjecture was verified; heterologously expressed dDAT-G108Q and the human (h) equivalent hDAT-G140Q were retained in the endoplasmic reticulum in a complex with endogenous folding sensors (calnexin and HSP70-1A). Incubation of the cells with noribogaine (a DAT ligand selective for the inward-facing state) and/or pifithrin-μ (an HSP70 inhibitor) restored folding of, and hence dopamine transport by, dDAT-G108Q and hDAT-G140Q. The mutated versions of DAT were confined to the cell bodies of the dopaminergic neurons in the fly brain and failed to reach the axonal compartments. Axonal delivery was restored, and sleep time was increased to normal length (from 300 to 1000 min/day) if the dDAT-G108Q-expressing flies were treated with noribogaine and/or pifithrin-μ. Rescuing misfolded versions of DAT by pharmacochaperoning is of therapeutic interest; it may provide opportunities to remedy disorders arising from folding-defective mutants of human DAT and of other related SLC6 transporters.