On the Structural Diversity and Individuality of Polymorphic Amyloid Protein Assemblies

The prediction of highly ordered three-dimensional structures of amyloid protein fibrils from the amino acid sequences of their monomeric self-assembly precursors constitutes a challenging and unresolved aspect of the classical protein folding problem. Because of the polymorphic nature of amyloid assembly whereby polypeptide chains of identical amino acid sequences under identical conditions are capable of self-assembly into a spectrum of different fibril structures, the prediction of amyloid structures from an amino acid sequence requires a detailed and holistic understanding of its assembly free energy landscape. The full extent of the structure space accessible to the cross-β molecular architecture of amyloid must also be resolved. Here, we review the current understanding of the diversity and the individuality of amyloid structures, and how the polymorphic landscape of amyloid links to biology and disease phenotypes. We present a comprehensive review of structural models of amyloid fibrils derived by cryo-EM, ssNMR and AFM to date, and discuss the challenges ahead for resolving the structural basis and the biological consequences of polymorphic amyloid assemblies.     

Adapting protein sequences for optimized therapeutic efficacy

Therapeutic proteins alleviate disease pathology by supplementing missing or defective native proteins, sequestering superfluous proteins, or by acting through designed non-natural mechanisms. Although therapeutic proteins often have the same amino acid sequence as their native counterpart, their maturation paths from expression to the site of physiological activity are inherently different, and optimizing protein sequences for properties that 100s of millions of years of evolution did not need to address presents an opportunity to develop better biological treatments. Because therapeutic proteins are inherently non-natural entities, optimization for their desired function should be considered analogous to that of small molecule drug candidates, which are optimized through expansive combinatorial variation by the medicinal chemist. Here, we review recent successes and challenges of protein engineering for optimized therapeutic efficacy.     

Intradimeric Walker A ATPases: Conserved Features of A Functionally Diverse Family

Nucleoside-triphosphate hydrolases (NTPases) are a diverse, but essential group of enzymes found in all living organisms. NTPases that have a G-X-X-X-X-G-K-[S/T] consensus sequence (where X is any amino acid), known as the Walker A or P-loop motif, constitute a superfamily of P-loop NTPases. A subset of ATPases within this superfamily contains a modified Walker A motif, X-K-G-G-X-G-K-[S/T], wherein the first invariant lysine residue is essential to stimulate nucleotide hydrolysis. Although the proteins in this subset have vastly differing functions, ranging from electron transport during nitrogen fixation to targeting of integral membrane proteins to their correct membranes, they have evolved from a shared ancestor and have thus retained common structural features that affect their functions. These commonalities have only been disparately characterized in the context of their individual proteins systems, but have not been generally annotated as features that unite the members of this family. In this review, we report an analysis based on the sequences, structures, and functions of several members in this family that highlight their remarkable similarities. A principal feature of these proteins is their dependence on homodimerization. Since their functionalities are heavily influenced by changes that happen in conserved elements at the dimer interface, we refer to the members of this subclass as intradimeric Walker A ATPases.     

Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

Whole exome sequencing,in silico and functional studies confirm the association of the GJB2 mutation p.Cys169Tyr with deafness and suggest a role for the TMEM59 gene in the hearing process

The development of next generation sequencing techniques has facilitated the detection of mutations at an unprecedented rate. These efficient tools have been particularly beneficial for extremely heterogeneous disorders such as autosomal recessive non-syndromic hearing loss, the most common form of genetic deafness. GJB2 mutations are the most common cause of hereditary hearing loss. Amongst them the NM_004004.5: c.506G > A (p.Cys169Tyr) mutation has been associated with varying severity of hearing loss with unclear segregation patterns. In this study, we report a large consanguineous Emirati family with severe to profound hearing loss fully segregating the GJB2 missense mutation p.Cys169Tyr. Whole exome sequencing (WES), in silico, splicing and expression analyses ruled out the implication of any other variants and confirmed the implication of the p.Cys169Tyr mutation in this deafness family. We also show preliminary murine expression analysis that suggests a link between the TMEM59 gene and the hearing process. The present study improves our understanding of the molecular pathogenesis of hearing loss. It also emphasizes the significance of combining next generation sequencing approaches and segregation analyses especially in the diagnosis of disorders characterized by complex genetic heterogeneity.     

The PHY Domain Dimer Interface of Bacteriophytochromes Mediates Cross-talk between Photosensory Modules and Output Domains

Protein dynamics play a major role for the catalytic function of enzymes, the interaction of protein complexes or signal integration in regulatory proteins. In the context of multi-domain proteins involved in light-regulation of enzymatic effectors, the central role of conformational dynamics is well established. Light activation of sensory modules is followed by long-range signal transduction to different effectors; rather than domino-style structural rearrangements, a complex interplay of functional elements is required to maintain functionality. One family of such sensor-effector systems are red-light-regulated phytochromes that control diguanylate cyclases involved in cyclic-dimeric-GMP formation. Based on structural and functional studies of one prototypic family member, the central role of the coiled-coil sensor-effector linker was established. Interestingly, subfamilies with different linker lengths feature strongly varying biochemical characteristics. The dynamic interplay of the domains involved, however, is presently not understood. Here we show that the PHY domain dimer interface plays an essential role in signal integration, and that a functional coupling with the coiled-coil linker element is crucial. Chimaeras of two biochemically different family members highlight the phytochrome-spanning helical spine as an essential structural element involved in light-dependent upregulation of enzymatic turnover. However, isolated structural elements can frequently not be assigned to individual characteristics, which further emphasises the importance of global conformational dynamics. Our results provide insights into the intricate processes at play during light signal integration and transduction in these photosensory systems and thus provide additional guidelines for a more directed design of novel sensor-effector combinations with potential applications as optogenetic tools.     

Improving Statistical Certainty of Glycosylation Similarity between Influenza A Virus Variants Using Data-Independent Acquisition Mass Spectrometry

Amino acid sequences of immunodominant domains of hemagglutinin (HA) on the surface of influenza A virus (IAV) evolve rapidly, producing viral variants. HA mediates receptor recognition, binding and cell entry, and serves as the target for IAV vaccines. Glycosylation, a post-translational modification that places large branched polysaccharide molecules on proteins, can modulate the function of HA and shield antigenic regions allowing for viral evasion from immune responses. Our previous work showed that subtle changes in the HA protein sequence can have a measurable change in glycosylation. Thus, being able to quantitatively measure glycosylation changes in variants is critical for understanding how HA function may change throughout viral evolution. Moreover, understanding quantitatively how the choice of viral expression systems affects glycosylation can help in the process of vaccine design and manufacture. Although IAV vaccines are most commonly expressed in chicken eggs, cell-based vaccines have many advantages, and the adoption of more cell-based vaccines would be an important step in mitigating seasonal influenza and protecting against future pandemics. Here, we have investigated the use of data-independent acquisition (DIA) mass spectrometry for quantitative glycoproteomics. We found that DIA improved the sensitivity of glycopeptide detection for four variants of A/Switzerland/9715293/2013 (H3N2): WT and mutant, each expressed in embryonated chicken eggs and Madin–Darby canine kidney cells. We used the Tanimoto similarity metric to quantify changes in glycosylation between WT and mutant and between egg-expressed and cell-expressed virus. Our DIA site-specific glycosylation similarity comparison of WT and mutant expressed in eggs confirmed our previous analysis while achieving greater depth of coverage. We found that sequence variations and changing viral expression systems affected distinct glycosylation sites of HA. Our methods can be applied to track glycosylation changes in circulating IAV variants to bolster genomic surveillance already being done, for a more complete understanding of IAV evolution.     

Extending the Horizon of Homology Detection with Coevolution-based Structure Prediction

Traditional sequence analysis algorithms fail to identify distant homologies when they lie beyond a detection horizon. In this review, we discuss how co-evolution-based contact and distance prediction methods are pushing back this homology detection horizon, thereby yielding new functional insights and experimentally testable hypotheses. Based on correlated substitutions, these methods divine three-dimensional constraints among amino acids in protein sequences that were previously devoid of all annotated domains and repeats. The new algorithms discern hidden structure in an otherwise featureless sequence landscape. Their revelatory impact promises to be as profound as the use, by archaeologists, of ground-penetrating radar to discern long-hidden, subterranean structures. As examples of this, we describe how triplicated structures reflecting longin domains in MON1A-like proteins, or UVR-like repeats in DISC1, emerge from their predicted contact and distance maps. These methods also help to resolve structures that do not conform to a “beads-on-a-string” model of protein domains. In one such example, we describe CFAP298 whose ubiquitin-like domain was previously challenging to perceive owing to a large sequence insertion within it. More generally, the new algorithms permit an easier appreciation of domain families and folds whose evolution involved structural insertion or rearrangement. As we exemplify with α1-antitrypsin, coevolution-based predicted contacts may also yield insights into protein dynamics and conformational change. This new combination of structure prediction (using innovative co-evolution based methods) and homology inference (using more traditional sequence analysis approaches) shows great promise for bringing into view a sea of evolutionary relationships that had hitherto lain far beyond the horizon of homology detection.     

Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

Multiple Reaction Monitoring-Mass Spectrometry Enables Robust Quantitation of Plasma Proteins Regardless of Whole Blood Processing Delays That May Occur in the Clinic

Plasma is an important biofluid for clinical research and diagnostics. In the clinic, unpredictable delays—from minutes to hours—between blood collection and plasma generation are often unavoidable. These delays can potentially lead to protein degradation and modification and might considerably affect intact protein measurement methods such as sandwich enzyme-linked immunosorbent assays that bind proteins on two epitopes to increase specificity, thus requiring largely intact protein structures. Here, we investigated, using multiple reaction monitoring mass spectrometry (MRM-MS), how delays in plasma processing affect peptide-centric “bottom-up” proteomics. We used validated assays for proteotypic peptide surrogates of 270 human proteins to analyze plasma generated after whole blood had been kept at room temperature from 0 to 40 h to mimic delays that occur in the clinic. Moreover, we evaluated the impact of different plasma-thawing conditions on MRM-based plasma protein quantitation. We demonstrate that >90% of protein concentration measurements were unaffected by the thawing procedure and by up to 40-h delayed plasma generation, reflected by relative standard deviations (RSDs) of <30%. Of the 159 MRM assays that yielded quantitative results in 60% of the measured time points, 139 enabled a stable protein quantitation (RSD <20%), 14 showed a slight variation (RSD 20–30%), and 6 appeared unstable/irreproducible (RSD > 30%). These results demonstrate the high robustness and thus the potential for MRM-based plasma-protein quantitation to be used in a clinical setting. In contrast to enzyme-linked immunosorbent assay, peptide-based MRM assays do not require intact three-dimensional protein structures for an accurate and precise quantitation of protein concentrations in the original sample.     

Chromatin Sensing by the Auxiliary Domains of KDM5C Regulates Its Demethylase Activity and Is Disrupted by X-linked Intellectual Disability Mutations

The H3K4me3 chromatin modification, a hallmark of promoters of actively transcribed genes, is dynamically removed by the KDM5 family of histone demethylases. The KDM5 demethylases have a number of accessory domains, two of which, ARID and PHD1, lie between the segments of the catalytic domain. KDM5C, which has a unique role in neural development, harbors a number of mutations adjacent to its accessory domains that cause X-linked intellectual disability (XLID). The roles of these accessory domains remain unknown, limiting an understanding of how XLID mutations affect KDM5C activity. Through in vitro binding and kinetic studies using nucleosomes, we find that while the ARID domain is required for efficient nucleosome demethylation, the PHD1 domain alone has an inhibitory role in KDM5C catalysis. In addition, the unstructured linker region between the ARID and PHD1 domains interacts with PHD1 and is necessary for nucleosome binding. Our data suggests a model in which the PHD1 domain inhibits DNA recognition by KDM5C. This inhibitory effect is relieved by the H3 tail, enabling recognition of flanking DNA on the nucleosome. Importantly, we find that XLID mutations adjacent to the ARID and PHD1 domains break this regulation by enhancing DNA binding, resulting in the loss of specificity of substrate chromatin recognition and rendering demethylase activity lower in the presence of flanking DNA. Our findings suggest a model by which specific XLID mutations could alter chromatin recognition and enable euchromatin-specific dysregulation of demethylation by KDM5C.     

Structure-Function Studies of Two Yeast Homing Endonucleases that Evolved to Cleave Identical Targets with Dissimilar Rates and Specificities

The LAGLIDADG family of homing endonucleases (LHEs) bind to and cleave their DNA recognition sequences with high specificity. Much of our understanding for how these proteins evolve their specificities has come from studying LHE homologues. To gain insight into the molecular basis of LHE specificity, we characterized I-WcaI, the homologue of the Saccharomyces cerevisiae I-SceI LHE found in Wickerhamomyces canadensis. Although I-WcaI and I-SceI cleave the same recognition sequence, expression of I-WcaI, but not I-SceI, is toxic in bacteria. Toxicity suppressing mutations frequently occur at I-WcaI residues critical for activity and I-WcaI cleaves many more non-cognate sequences in the Escherichia coli genome than I-SceI, suggesting I-WcaI endonuclease activity is the basis of toxicity. In vitro, I-WcaI is a more active and a less specific endonuclease than I-SceI, again accounting for the observed toxicity in vivo. We determined the X-ray crystal structure of I-WcaI bound to its cognate target site and found that I-WcaI and I-SceI use residues at different positions to make similar base-specific contacts. Furthermore, in some regions of the DNA interface where I-WcaI specificity is lower, the protein makes fewer DNA contacts than I-SceI. Taken together, these findings demonstrate the plastic nature of LHE site recognition and suggest that I-WcaI and I-SceI are situated at different points in their evolutionary pathways towards acquiring target site specificity.     

Emergence,evolution, and vaccine production approaches of SARS-CoV-2 virus: Benefits of getting vaccinated and common questions

The emergence of coronavirus disease 2019 (COVID-19) pandemic in Wuhan city, China at the end of 2019 made it urgent to identify the origin of the causal pathogen and its molecular evolution, to appropriately design an effective vaccine. This study analyzes the evolutionary background of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or SARS-2) in accordance with its close relative SARS-CoV (SARS-1), which was emerged in 2002. A comparative genomic and proteomic study was conducted on SARS-2, SARS-1, and Middle East respiratory syndrome coronavirus (MERS), which was emerged in 2012. In silico analysis inferred the genetic variability among the tested viruses. The SARS-1 genome harbored 11 genes encoding 12 proteins, while SARS-2 genome contained only 10 genes encoding for 10 proteins. MERS genome contained 11 genes encoding 11 proteins. The analysis also revealed a slight variation in the whole genome size of SARS-2 comparing to its siblings resulting from sequential insertions and deletions (indels) throughout the viral genome particularly ORF1AB, spike, ORF10 and ORF8. The effective indels were observed in the gene encoding the spike protein that is responsible for viral attachment to the angiotensin-converting enzyme 2 (ACE2) cell receptor and initiating infection. These indels are responsible for the newly emerging COVID-19 variants αCoV, βCoV, γCoV and δCoV. Nowadays, few effective COVID-19 vaccines developed based on spike (S) glycoprotein were approved and become available worldwide. Currently available vaccines can relatively prevent the spread of COVID-19 and suppress the disease. The traditional (killed or attenuated virus vaccine and antibody-based vaccine) and innovated vaccine production technologies (RNA- and DNA-based vaccines and viral vectors) are summarized in this review. We finally highlight the most common questions related to COVID-19 disease and the benefits of getting vaccinated.     

Cryo-EM Structure of the Full-length hnRNPA1 Amyloid Fibril

Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) is a multifunctional RNA-binding protein that is associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis and multisystem proteinopathy. In this study, we have used cryo-electron microscopy to investigate the three-dimensional structure of amyloid fibrils from full-length hnRNPA1 protein. We find that the fibril core is formed by a 45-residue segment of the prion-like low-complexity domain of the protein, whereas the remaining parts of the protein (275 residues) form a fuzzy coat around the fibril core. The fibril consists of two fibril protein stacks that are arranged into a pseudo-2₁ screw symmetry. The ordered core harbors several of the positions that are known to be affected by disease-associated mutations, but does not encompass the most aggregation-prone segments of the protein. These data indicate that the structures of amyloid fibrils from full-length proteins may be more complex than anticipated by current theories on protein misfolding.