Glucagon-like peptide 1 aggregates into low-molecular-weight oligomers off-pathway to fibrillation.

The physical stability of peptide-based drugs is of great interest to the pharmaceutical industry. Glucagon-like peptide 1 (GLP-1) is a 31-amino acid peptide hormone, the analogs of which are frequently used in the treatment of type 2 diabetes. We investigated the physical stability of GLP-1 and its C-terminal amide derivative, GLP-1-Am, both of which aggregate into amyloid fibrils. While off-pathway oligomers have been proposed to explain the unusual aggregation kinetics observed previously for GLP-1 under specific conditions, these oligomers have not been studied in any detail. Such states are important as they may represent potential sources of cytotoxicity and immunogenicity. Here, we identified and isolated stable, low-molecular-weight oligomers of GLP-1 and GLP-1-Am, using size-exclusion chromatography. Under the conditions studied, isolated oligomers were shown to be resistant to fibrillation or dissociation. These oligomers contain between two and five polypeptide chains and they have a highly disordered structure as indicated by a variety of spectroscopic techniques. They are highly stable with respect to time, temperature, or agitation despite their noncovalent character, which was established using liquid chromatography-mass spectrometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results provide evidence of stable, low-molecular-weight oligomers that are formed by an off-pathway mechanism which competes with amyloid fibril formation.

Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition.

The cell wall is a critical extracellular barrier for bacteria and many other organisms. In bacteria, this structural layer consists of peptidoglycan, which maintains cell shape and structural integrity and provides a scaffold for displaying various protein factors. To attach proteins to the cell wall, Gram-positive bacteria utilize sortase enzymes, which are cysteine transpeptidases that recognize and cleave a specific sorting signal, followed by ligation of the sorting signal-containing protein to the peptidoglycan precursor lipid II (LII). This mechanism is the subject of considerable interest as a target for therapeutic intervention and as a tool for protein engineering, where sortases have enabled sortase-mediated ligation or sortagging strategies. Despite these uses, there remains an incomplete understanding of the stereochemistry of substrate recognition and ligation product formation. Here, we solved the first structures of sortase A from Streptococcus pyogenes bound to two substrate sequences, LPATA and LPATS. In addition, we synthesized a mimetic of the product of sortase-mediated ligation involving LII (LPAT-LII) and solved the complex structure in two ligand conformations. These structures were further used as the basis for molecular dynamics simulations to probe sortase A-ligand dynamics and to construct a model of the acyl-enzyme intermediate, thus providing a structural view of multiple key states in the catalytic mechanism. Overall, this structural information provides new insights into the recognition of the sortase substrate motif and LII ligation partner and will support the continued development of sortases for protein engineering applications.

A longitudinal study of theory of mind and listening comprehension: Is preschool theory of mind important?

Theory of mind has been shown to be important for listening comprehension for children at a range of ages. However, there is a lack of longitudinal evidence for a relationship between early theory of mind and later listening comprehension. The aim of this study was to examine whether preschool theory of mind has a longitudinal direct effect on later listening comprehension over and above the effects of concurrent theory of mind. A total of 147 children were tested on measures of theory of mind, working memory, vocabulary, and grammatical knowledge at Time 1 (mean age = 4;1 [years;months]) and Time 2 (mean age = 5;11). In addition, at Time 2 listening comprehension, comprehension monitoring, and inference making measures were taken. Data were fitted to concurrent and longitudinal models of listening comprehension. Concurrent findings at Time 2 showed theory of mind to have a direct effect on listening comprehension. However, longitudinal findings showed that earlier theory of mind in preschool (Time 1) did not have a direct effect on listening comprehension 22 months later; instead, there was only an indirect effect of earlier theory of mind on later listening comprehension via concurrent theory of mind (Time 2). Taken together, the results give further support for the importance of theory of mind for listening comprehension but show that there are limited additional benefits of early theory of mind acquisition. Implications for the development of children's listening comprehension are discussed.

Interconversion of Unexpected Thiol States Affects the Stability, Structure, and Dynamics of Antibody Engineered for Site-Specific Conjugation.

Antibody-drug conjugates have become one of the most actively developed classes of drugs in recent years. Their great potential comes from combining the strengths of large and small molecule therapeutics: the exquisite specificity of antibodies and the highly potent nature of cytotoxic compounds. More recently, the approach of engineering antibody-drug conjugate scaffolds to achieve highly controlled drug to antibody ratios has focused on substituting or inserting cysteines to facilitate site-specific conjugation. Herein, we characterize an antibody scaffold engineered with an inserted cysteine that formed an unexpected disulfide bridge during manufacture. A combination of mass spectrometry and biophysical techniques have been used to understand how the additional disulfide bridge forms, interconverts, and changes the stability and structural dynamics of the antibody intermediate. This quantitative and structurally resolved model of the local and global changes in structure and dynamics associated with the engineering and subsequent disulfide-bonded variant can assist future engineering strategies.

The Amyloid Fibril-Forming ß-Sheet Regions of Amyloid ß and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ.

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid ß (Aß) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer's and Parkinson's diseases, respectively, a process that is intimately linked to the diseases' progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aß (Aß40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aß40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aß40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt ß-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aß40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.

Knotting and unknotting of a protein in single molecule experiments.

Spontaneous folding of a polypeptide chain into a knotted structure remains one of the most puzzling and fascinating features of protein folding. The folding of knotted proteins is on the timescale of minutes and thus hard to reproduce with atomistic simulations that have been able to reproduce features of ultrafast folding in great detail. Furthermore, it is generally not possible to control the topology of the unfolded state. Single-molecule force spectroscopy is an ideal tool for overcoming this problem: by variation of pulling directions, we controlled the knotting topology of the unfolded state of the 52-knotted protein ubiquitin C-terminal hydrolase isoenzyme L1 (UCH-L1) and have therefore been able to quantify the influence of knotting on its folding rate. Here, we provide direct evidence that a threading event associated with formation of either a 31 or 52 knot, or a step closely associated with it, significantly slows down the folding of UCH-L1. The results of the optical tweezers experiments highlight the complex nature of the folding pathway, many additional intermediate structures being detected that cannot be resolved by intrinsic fluorescence. Mechanical stretching of knotted proteins is also of importance for understanding the possible implications of knots in proteins for cellular degradation. Compared with a simple 31 knot, we measure a significantly larger size for the 52 knot in the unfolded state that can be further tightened with higher forces. Our results highlight the potential difficulties in degrading a 52 knot compared with a 31 knot.

Ubiquitin chain conformation regulates recognition and activity of interacting proteins.

Mechanisms of protein recognition have been extensively studied for single-domain proteins, but are less well characterized for dynamic multidomain systems. Ubiquitin chains represent a biologically important multidomain system that requires recognition by structurally diverse ubiquitin-interacting proteins. Ubiquitin chain conformations in isolation are often different from conformations observed in ubiquitin-interacting protein complexes, indicating either great dynamic flexibility or extensive chain remodelling upon binding. Using single-molecule fluorescence resonance energy transfer, we show that Lys 63-, Lys 48- and Met 1-linked diubiquitin exist in several distinct conformational states in solution. Lys 63- and Met 1-linked diubiquitin adopt extended 'open' and more compact 'closed' conformations, and ubiquitin-binding domains and deubiquitinases (DUBs) select pre-existing conformations. By contrast, Lys 48-linked diubiquitin adopts predominantly compact conformations. DUBs directly recognize existing conformations, but may also remodel ubiquitin chains to hydrolyse the isopeptide bond. Disruption of the Lys 48-diubiquitin interface changes conformational dynamics and affects DUB activity. Hence, conformational equilibria in ubiquitin chains provide an additional layer of regulation in the ubiquitin system, and distinct conformations observed in differently linked polyubiquitin may contribute to the specificity of ubiquitin-interacting proteins.

Chaperome screening leads to identification of Grp94/Gp96 and FKBP4/52 as modulators of the α-synuclein-elicited immune response.

We have investigated the potential role of molecular chaperones as modulators of the immune response by using α-synuclein (αSyn) as an aggregation-prone model protein. We first performed an in vitro immunoscreening with 21 preselected candidate chaperones and selected 2 from this set as displaying immunological activity with differential profiles, Grp94/Gp96 and FKBP4/52. We then immunized mice with both chaperone/α-synuclein combinations using monomeric or oligomeric α-synuclein (MαSyn or OαSyn, respectively), and we characterized the immune response generated in each case. We found that Grp94 promoted αSyn-specific T-helper (Th)1/Th17 and IgG1 antibody responses (up to a 3-fold increase) with MαSyn and OαSyn, respectively, coupled to a Th2-type general phenotype (generating 2.5-fold higher IgG1/IgG2 levels). In addition, we observed that FKBP4 favored a Th1-skewed phenotype with MαSyn but strongly supported a Th2-type phenotype with OαSyn (with a 3-fold higher IL-10/IFN-γ serum levels). Importantly, results from adoptive transfer of splenocytes from immunized animals in a Parkinson's disease mouse model indicates that these effects are robust, stable in time, and physiologically relevant. Taken together, Grp94 and FKBP4 are able to generate differential immune responses to α-synuclein-based immunizations, depending both on the nature of the chaperone and on the aggregation state of α-synuclein. Our work reveals that several chaperones are potential modulators of the immune response and suggests that different chaperones could be exploited to redirect the amyloid-elicited immunity both for basic studies of the immunological processes associated with neurodegeneration and for immunotherapy of pathologies associated with protein misfolding and aggregation.

Characterization of the Folding of a 52-Knotted Protein Using Engineered Single-Tryptophan Variants.

An increasing number of proteins that contain topological knots have been identified over the past two decades; however, their folding mechanisms are still not well understood. UCH-L1 has a 52-knotted topology. Here, we constructed a series of variants that contain a single tryptophan at different locations along the polypeptide chain. A study of the thermodynamic properties of the variants shows that the structure of UCH-L1 is remarkably tolerant to incorporation of bulky tryptophan side chains. Comprehensive kinetic studies of the variants reveal that they fold via parallel pathways on which there are two intermediate states very similar to wild-type UCH-L1. The structures of the intermediate states do not change significantly with mutation and therefore occupy local minima on the energy landscape that have relatively narrow basins. The kinetic studies also establish that there are considerably more tertiary interactions in the intermediate states than results from previous NMR studies suggested. The two intermediates differ from each other in the extent to which tertiary interactions between the highly stable central ß-sheet and flanking α-helices and loop regions are formed. There is strong evidence that these states are aggregation prone. The transition states from both I1 and I2 to the native state are plastic and change with mutation and denaturant concentration. Previous studies indicated that the threading event that creates the 52 knot occurs during these steps, suggesting that there is a broad energy barrier consistent with the chain undergoing some searching of conformational space as the threading takes place.

A pH-Induced Switch in Human Glucagon-like Peptide-1 Aggregation Kinetics.

Aggregation and amyloid fibril formation of peptides and proteins is a widespread phenomenon. It has serious implications in a range of areas from biotechnological and pharmaceutical applications to medical disorders. The aim of this study was to develop a better understanding of the mechanism of aggregation and amyloid fibrillation of an important pharmaceutical, human glucagon-like peptide-1 (GLP-1). GLP-1 is a 31-residue hormone peptide that plays an important role regulating blood glucose levels, analogues of which are used for treatment of type 2 diabetes. Amyloid fibril formation of GLP-1 was monitored using thioflavin T fluorescence as a function of peptide concentration between pH 7.5 and 8.2. Results from these studies establish that there is a highly unusual pH-induced switch in GLP-1 aggregation kinetics. At pH 8.2, the kinetics are consistent with a nucleation-polymerization mechanism for fibril formation. However, at pH 7.5, highly unusual kinetics are observed, where the lag time increases with increasing peptide concentration. We attribute this result to the formation of off-pathway species together with an initial slow, unimolecular step where monomer converts to a different monomeric form that forms on-pathway oligomers and ultimately fibrils. Estimation of the pKa values of all the ionizable groups in GLP-1 suggest it is the protonation/deprotonation of the N-terminus that is responsible for the switch with pH. In addition, a range of biophysical techniques were used to characterize (1) the start point of the aggregation reaction and (2) the structure and stability of the fibrils formed. These results show that the off-pathway species form under conditions where GLP-1 is most prone to form oligomers.

Knotted fusion proteins reveal unexpected possibilities in protein folding.

Proteins that contain a distinct knot in their native structure are impressive examples of biological self-organization. Although this topological complexity does not appear to cause a folding problem, the mechanisms by which such knotted proteins form are unknown. We found that the fusion of an additional protein domain to either the amino terminus, the carboxy terminus, or to both termini of two small knotted proteins did not affect their ability to knot. The multidomain constructs remained able to fold to structures previously thought unfeasible, some representing the deepest protein knots known. By examining the folding kinetics of these fusion proteins, we found evidence to suggest that knotting is not rate limiting during folding, but instead occurs in a denatured-like state. These studies offer experimental insights into when knot formation occurs in natural proteins and demonstrate that early folding events can lead to diverse and sometimes unexpected protein topologies.

Bayesian inference of accurate population sizes and FRET efficiencies from single diffusing biomolecules.

It is of significant biophysical interest to obtain accurate intramolecular distance information and population sizes from single-molecule Förster resonance energy transfer (smFRET) data obtained from biomolecules in solution. Experimental methods of increasing cost and complexity are being developed to improve the accuracy and precision of data collection. However, the analysis of smFRET data sets currently relies on simplistic, and often arbitrary methods, for the selection and denoising of fluorescent bursts. Although these methods are satisfactory for the analysis of simple, low-noise systems with intermediate FRET efficiencies, they display systematic inaccuracies when applied to more complex systems. We have developed an inference method for the analysis of smFRET data from solution studies based on rigorous model-based Bayesian techniques. We implement a Monte Carlo Markov chain (MCMC) based algorithm that simultaneously estimates population sizes and intramolecular distance information directly from a raw smFRET data set, with no intermediate event selection and denoising steps. Here, we present both our parametric model of the smFRET process and the algorithm developed for data analysis. We test the algorithm using a combination of simulated data sets and data from dual-labeled DNA molecules. We demonstrate that our model-based method systematically outperforms threshold-based techniques in accurately inferring both population sizes and intramolecular distances.

Heterogeneity and dynamics in the assembly of the heat shock protein 90 chaperone complexes.

The Hsp90 cycle depends on the coordinated activity of a range of cochaperones, including Hop, Hsp70 and peptidyl-prolyl isomerases such as FKBP52. Using mass spectrometry, we investigate the order of addition of these cochaperones and their effects on the stoichiometry and composition of the resulting Hsp90-containing complexes. Our results show that monomeric Hop binds specifically to the Hsp90 dimer whereas FKBP52 binds to both monomeric and dimeric forms of Hsp90. By preforming Hsp90 complexes with either Hop, followed by addition of FKBP52, or with FKBP52 and subsequent addition of Hop, we monitor the formation of a predominant asymmetric ternary complex containing both cochaperones. This asymmetric complex is subsequently able to interact with the chaperone Hsp70 to form quaternary complexes containing all four proteins. Monitoring the population of these complexes during their formation and at equilibrium allows us to model the complex formation and to extract 14 different K(D) values. This simultaneous calculation of the K(D)s from a complex system with the same method, from eight deferent datasets under the same buffer conditions delivers a self-consistent set of values. In this case, the K(D) values afford insights into the assembly of ten Hsp90-containing complexes and provide a rationale for the cellular heterogeneity and prevalence of intermediates in the Hsp90 chaperone cycle.

Energy Landscapes and Structural Ensembles of Glucagon-like Peptide-1 Monomers.

While GLP-1 and its analogues are important pharmaceutical agents in the treatment of type 2 diabetes and obesity, their susceptibility to aggregate into amyloid fibrils poses a significant safety issue. Many factors may contribute to the aggregation propensity, including pH. While it is known that the monomeric structure of GLP-1 has a strong impact on primary nucleation, probing its diverse structural ensemble is challenging. Here, we investigated the monomer structural ensembles at pH 3, 4, and 7.5 using state-of-the-art computational methods in combination with experimental data. We found significant stabilization of ß-strand structures and destabilization of helical structures at lower pH, correlating with observed aggregation lag times, which are lower under these conditions. We further identified helical defects at pH 4, which led to the fastest observed aggregation, in agreement with our far-UV circular dichroism data. The detailed atomistic structures that result from the computational studies help to rationalize the experimental results on the aggregation propensity of GLP-1. This work provides a new insight into the pH-dependence of monomeric structural ensembles of GLP-1 and connects them to experimental observations.

A unique binding mode of P1' Leu-containing target sequences for Streptococcus pyogenes sortase A results in alternative cleavage.

Sortase enzymes are cysteine transpeptidases that attach environmental sensors, toxins, and other proteins to the cell surface in Gram-positive bacteria. The recognition motif for many sortases is the cell wall sorting signal (CWSS), LPXTG, where X = any amino acid. Recent work from ourselves and others has described recognition of additional amino acids at a number of positions in the CWSS, specifically at the Thr (or P1) and Gly (or P1') positions. In addition, although standard cleavage occurs between these two residues (P1/P1'), we previously observed that the SrtA enzyme from Streptococcus pneumoniae will cleave after the P1' position when its identity is a Leu or Phe. The stereochemical basis of this alternative cleavage is not known, although homologs, e.g., SrtA from Listeria monocytogenes or Staphylococcus aureus do not show alternative cleavage to a significant extent. Here, we use protein biochemistry, structural biology, and computational biochemistry to predict an alternative binding mode that facilitates alternative cleavage. We use Streptococcus pyogenes SrtA (spySrtA) as our model enzyme, first confirming that it shows similar standard/alternative cleavage ratios for LPATL, LPATF, and LPATY sequences. Molecular dynamics simulations suggest that when P1' is Leu, this amino acid binds in the canonical S1 pocket, pushing the P1 Thr towards solvent. The P4 Leu (L̲PATL) binds as it does in standard binding, resulting in a puckered binding conformation. We use P1 Glu-containing peptides to support our hypotheses, and present the complex structure of spySrtA-LPALA to confirm favorable accommodation of Leu in the S1 pocket. Overall, we structurally characterize an alternative binding mode for spySrtA and specific target sequences, expanding the potential protein engineering possibilities in sortase-mediated ligation applications.

Influence of donor-recipient sex on engraftment of normal and leukemia stem cells in xenotransplantation.

Immunodeficient mouse models are widely used for the assessment of human normal and leukemic stem cells. Despite the advancements over the years, reproducibility, as well as the differences in the engraftment of human cells in recipient mice remains to be fully resolved. Here, we used various immunodeficient mouse models to characterize the effect of donor-recipient sex on the engraftment of the human leukemic and healthy cells. Donor human cells and recipient immunodeficient mice demonstrate sex-specific engraftment levels with significant differences observed in the lineage output of normal CD34+ hematopoietic stem and progenitor cells upon xenotransplantation. Intriguingly, human female donor cells display heightened sensitivity to the recipient mice's gender, influencing their proliferation and resulting in significantly increased engraftment in female recipient mice. Our study underscores the intricate interplay taking place between donor and recipient characteristics, shedding light on important considerations for future studies, particularly in the context of pre-clinical research.

Hsp90: structure and function.

Hsp90 is a highly abundant and ubiquitous molecular chaperone which plays an essential role in many cellular processes including cell cycle control, cell survival, hormone and other signalling pathways. It is important for the cell's response to stress and is a key player in maintaining cellular homeostasis. In the last ten years, it has become a major therapeutic target for cancer, and there has also been increasing interest in it as a therapeutic target in neurodegenerative disorders, and in the development of anti-virals and anti-protozoan infections. The focus of this review is the structural and mechanistic studies which have been performed in order to understand how this important chaperone acts on a wide variety of different proteins (its client proteins) and cellular processes. As with many of the other classes of molecular chaperone, Hsp90 has a critical ATPase activity, and ATP binding and hydrolysis known to modulate the conformational dynamics of the protein. It also uses a host of cochaperones which not only regulate the ATPase activity and conformational dynamics but which also mediate interactions with Hsp90 client proteins. The system is also regulated by post-translational modifications including phosphorylation and acetylation. This review discusses all these aspects of Hsp90 structure and function.

Knot formation in newly translated proteins is spontaneous and accelerated by chaperonins.

Topological knots are found in a considerable number of protein structures, but it is not clear how they knot and fold within the cellular environment. We investigated the behavior of knotted protein molecules as they are first synthesized by the ribosome using a cell-free translation system. We found that newly translated knotted proteins can spontaneously self-tie and do not require the assistance of molecular chaperones to fold correctly to their trefoil-knotted structures. This process is slow but efficient, and we found no evidence of misfolded species. A kinetic analysis indicates that the knotting process is rate limiting, occurs post-translationally, and is specifically and significantly (P < 0.001) accelerated by the GroEL-GroES chaperonin complex. This demonstrates a new active mechanism for this molecular chaperone and suggests that chaperonin-catalyzed knotting probably dominates in vivo. These results explain how knotted protein structures have withstood evolutionary pressures despite their topological complexity.

Experimental detection of knotted conformations in denatured proteins.

Structures that contain a knot formed by the path of the polypeptide backbone represent some of the most complex topologies observed in proteins. How or why these topological knots arise remains unclear. By developing a method to experimentally trap and detect knots in nonnative polypeptide chains, we find that two knotted methyltransferases, YibK and YbeA, can exist in a trefoil-knot conformation even in their chemically unfolded states. The unique denatured-state topology of these molecules explains their ability to efficiently fold to their native knotted structures in vitro and offers insights into the potential role of knots in proteins. Furthermore, the high prevalence of the denatured-state knots identified here suggests that they are either difficult to untie or that threading of any untied molecules is rapid and spontaneous. The occurrence of such knotted topologies in unfolded polypeptide chains raises the possibility that they could play an important, and as yet unexplored, role in folding and misfolding processes in vivo.

A preliminary study into internet related addictions among adults with dyslexia.

In recent decades, studies have investigated associations between learning disorders such as Autism Spectrum Disorder (ASD) and Attention Deficit Hyperactivity Disorder (ADHD), and the various types of internet addictions, ranging from general internet addiction (GIA) to specific internet addictions such as social media addiction (SMA) and internet gaming disorder (IGD). However, to date, no study has investigated such internet addictions among persons with dyslexia. The present study aimed to investigate whether differences exist between adults with dyslexia and controls in terms of GIA, SMA and IGD. A total of 141 adults with dyslexia and 150 controls (all UK based) were recruited. Controlling for age, gender, marital status, employment, and income levels, it was found that adults with dyslexia had higher levels of GIA and IGD compared to controls. However, these participants did not show any significant difference in terms of SMA. The results indicate that internet addictions may have a larger ambit for learning disorders beyond just ASD and ADHD and could be a hidden problem for these individuals.