Immunogenic potential of neopeptides depends on parent protein subcellular location

Antigen presentation via the major histocompatibility complex (MHC) is essential for anti-tumor immunity, however the rules that determine what tumor-derived peptides will be immunogenic are still incompletely understood. Here we investigate whether protein subcellular location driven constraints on accessibility of peptides to the MHC associate with potential for peptide immunogenicity. Analyzing over 380,000 of peptides from studies of MHC presentation and peptide immunogenicity, we find clear spatial biases in both eluted and immunogenic peptides. We find that including parent protein location improves prediction of peptide immunogenicity in multiple datasets. In human immunotherapy cohorts, location was associated with response to a neoantigen vaccine, and immune checkpoint blockade responders generally had a higher burden of neopeptides from accessible locations. We conclude that protein subcellular location adds important information for optimizing immunotherapies.

“Out of Pocket” Protein Binding—A Dilemma of Epitope Imprinted Polymers Revealed for Human Hemoglobin

The epitope imprinting approach applies exposed peptides as templates to synthesize Molecularly Imprinted Polymers (MIPs) for the recognition of the parent protein. While generally the template protein binding to such MIPs is considered to occur via the epitope-shaped cavities, unspecific interactions of the analyte with non-imprinted polymer as well as the detection method used may add to the complexity and interpretation of the target rebinding. To get new insights on the effects governing the rebinding of analytes, we electrosynthesized two epitope-imprinted polymers using the N-terminal pentapeptide VHLTP-amide of human hemoglobin (HbA) as the template. MIPs were prepared either by single-step electrosynthesis of scopoletin/pentapeptide mixtures or electropolymerization was performed after chemisorption of the cysteine extended VHLTP peptide. Rebinding of the target peptide and the parent HbA protein to the MIP nanofilms was quantified by square wave voltammetry using a redox probe gating, surface enhanced infrared absorption spectroscopy, and atomic force microscopy. While binding of the pentapeptide shows large influence of the amino acid sequence, all three methods revealed strong non-specific binding of HbA to both polyscopoletin-based MIPs with even higher affinities than the target peptides.

Increased alpha-helicity of a supercharged coiled-coil protein increases siRNA delivery efficiency of protein-lipid hybrid vehicle

Gene therapy has the potential to treat various diseases and has recently gained new interest due to the deployment nucleic acid based vaccines for COVID-19. Despite these developments, there still remains a need for further development of gene delivery vehicles to increase their safety and efficacy.. We have recently developed a lipoproteoplex (LPP) consisting of a super-charged coiled-coil protein (CSP) and a cationic liposomal carrier, that has the ability to condense nucleic acids and deliver them in vivo. The LPP is distinct from other liposomal gene delivery systems in that it utilizes a modular protein component to drive transfection activity as opposed to relying on the passive effects of the cationic lipids. A CSP library has been rationally designed to improve the efficacy of the LPP compared to the parent protein via improved alpha-helical structure and increased nucleic acid binding through the use of extended histidine tags and increased positive charge. The secondary structure and nucleic acid binding ability of each library member was assessed, then compared to functional transfection data in NIH-3T3 mouse fibroblasts. Structural and functional data suggests that increasing alpha-helicity of the protein component of the LPP compared to the parent sequence doubles nucleic acid binding affinity and increases transfection activity almost 3-fold with a favorable safety profile.

A sequence-based method for predicting extant fold switchers that undergo α-helix β-strand transitions

Extant fold-switching proteins remodel their secondary structures and change their functions in response to cellular stimuli, regulating biological processes and affecting human health. In spite of their biological importance, these proteins remain understudied. Few representative examples of fold switchers are available in the Protein Data Bank, and they are difficult to predict. In fact, all 96 experimentally validated examples of extant fold switchers were stumbled upon by chance. Thus, predictive methods are needed to expedite the process of discovering and characterizing more of these shapeshifting proteins. Previous approaches require a solved structure or all-atom simulations, greatly constraining their use. Here, we propose a high-throughput sequence-based method for predicting extant fold switchers that transition from α-helix in one conformation to β-strand in the other. This method leverages two previous observations: (1) α-helix <-> β-strand prediction discrepancies from JPred4 are a robust predictor of fold switching, and (2) the fold-switching regions (FSRs) of some extant fold switchers have different secondary structure propensities when expressed in isolation (isolated FSRs) than when expressed within the context of their parent protein (contextualized FSRs). Combining these two observations, we ran JPred4 on the sequences of isolated and contextualized FSRs from 14 known extant fold switchers and found α-helix <->β-strand prediction discrepancies in every case. To test the overall robustness of this finding, we randomly selected regions of proteins not expected to switch folds (single-fold proteins) and found significantly fewer α-helix <-> β-strand prediction discrepancies (p < 4.2*10−20, Kolmogorov-Smirnov test). Combining these discrepancies with the overall percentage of predicted secondary structure, we developed a classifier that often robustly identifies extant fold switchers (Matthews Correlation Coefficient of 0.70). Although this classifier had a high false negative rate (6/14), its false positive rate was very low (1/211), suggesting that it can be used to predict a subset of extant fold switchers from billions of available genomic sequences.

Commonalities between Copper Neurotoxicity and Alzheimer’s Disease

Alzheimer’s disease, a highly prevalent form of dementia, targets neuron function beginning from the hippocampal region and expanding outwards. Alzheimer’s disease is caused by elevated levels of heavy metals, such as lead, zinc, and copper. Copper is found in many areas of daily life, raising a concern as to how this metal and Alzheimer’s disease are related. Previous studies have not identified the common pathways between excess copper and Alzheimer’s disease etiology. Our review corroborates that both copper and Alzheimer’s disease target the hippocampus, cerebral cortex, cerebellum, and brainstem, affecting motor skills and critical thinking. Additionally, Aβ plaque formation was analyzed beginning from synthesis at the APP parent protein site until Aβ plaque formation was completed. Structural changes were also noted. Further analysis revealed a relationship between amyloid-beta plaques and copper ion concentration. As copper ion levels increased, it bound to the Aβ monomer, expediting the plaque formation process, and furthering neurodegeneration. These conclusions can be utilized in the medical community to further research on the etiology of Alzheimer’s disease and its relationships to copper and other metal-induced neurotoxicity.

Elucidating the Biological Activity of Fish-Derived Collagen and Gelatine Hydrolysates using Animal Cell Culture-A review

: A large percentage of a fish's weight is generally discarded during fish processing. Reducing waste in products of marine origin is a subject of great interest within the scientific community. Pelagic by-products such as the structural protein collagen, which can be generated during the processing of fish, has been proposed as an alternative to terrestrial, mammalian sources due to advantages including high availability and low risk of zoonotic disease transmission. Gelatine has multiple possible applications, ranging from nutraceutical applications to cosmetics and has the advantage of being generally regarded as safe. In this multidisciplinary review, the chemistry of gelatine and its parent protein collagen, the chemical reactions to generate their hydrolysates and studies on their biological activities using animal cell culture are discussed.

Antimicrobial and Amyloidogenic Activity of Peptides Synthesized on the Basis of the Ribosomal S1 Protein from Thermus Thermophilus

Controlling the aggregation of vital bacterial proteins could be one of the new research directions and form the basis for the search and development of antibacterial drugs with targeted action. Such approach may be considered as an alternative one to antibiotics. Amyloidogenic regions can, like antibacterial peptides, interact with the “parent” protein, for example, ribosomal S1 protein (specific only for bacteria), and interfere with its functioning. The aim of the work was to search for peptides based on the ribosomal S1 protein from T. thermophilus, exhibiting both aggregation and antibacterial properties. The biological system of the response of Gram-negative bacteria T. thermophilus to the action of peptides was characterized. Among the seven studied peptides, designed based on the S1 protein sequence, the R23I (modified by the addition of HIV transcription factor fragment for bacterial cell penetration), R23T (modified), and V10I (unmodified) peptides have biological activity that inhibits the growth of T. thermophilus cells, that is, they have antimicrobial activity. But, only the R23I peptide had the most pronounced activity comparable with the commercial antibiotics. We have compared the proteome of peptide-treated and intact T. thermophilus cells. These important data indicate a decrease in the level of energy metabolism and anabolic processes, including the processes of biosynthesis of proteins and nucleic acids. Under the action of 20 and 50 μg/mL R23I, a decrease in the number of proteins in T. thermophilus cells was observed and S1 ribosomal protein was absent. The obtained results are important for understanding the mechanism of amyloidogenic peptides with antimicrobial activity and can be used to develop new and improved analogues.

The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K. Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K. As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

The Effect of Oligomerization on A Solid-Binding Peptide Binding to Silica-Based Materials

The bifunctional linker-protein G (LPG) fusion protein comprises a peptide (linker) sequence and a truncated form of Streptococcus strain G148 protein G (protein G). The linker represents a multimeric solid-binding peptide (SBP) comprising 4 × 21-amino acid sequence repeats that display high binding affinity towards silica-based materials. In this study, several truncated derivatives were investigated to determine the effect of the SBP oligomerization on the silica binding function of LPG (for the sake of clarity, LPG will be referred from here on as 4 × LPG). Various biophysical characterization techniques were used to quantify and compare the truncated derivatives against 4 × LPG and protein G without linker (PG). The derivative containing two sequence repeats (2 × LPG) showed minimal binding to silica, while the truncated derivative with only a single sequence (1 × LPG) displayed no binding. The derivative containing three sequence repeats (3 × LPG) was able to bind to silica with a binding affinity of KD = 53.23 ± 4.5 nM, which is 1.5 times lower than that obtained for 4 × LPG under similar experimental conditions. Circular dichroism (CD) spectroscopy and fluorescence spectroscopy studies indicated that the SBP degree of oligomerization has only a small effect on the secondary structure (the linker unravels the beginning of the protein G sequence) and chemical stability of the parent protein G. However, based on quartz crystal microbalance with dissipation monitoring (QCM-D), oligomerization is an important parameter for a strong and stable binding to silica. The replacement of three sequence repeats by a (GGGGS)12 glycine-rich spacer indicated that the overall length rather than the SBP oligomerization mediated the effective binding to silica.

Epitope mapping of an uncertain endogenous antigen implies secretogranin II peptide splicing

Background: The search for a tissue-mass reducing reproductive hormone involved a bioassay-guided physicochemical fractionation of sheep blood plasma. This brought forth a candidate protein whose apparent mass on gels and in mass spectrometry (MS) was 7-8 kDa, implying a polypeptide of ~70 residues. Four purification runs gave Edman N-terminal sequences relating to 1MKPLTGKVKEFNNI14. This is bioinformatically obscure and has been resistant to molecular biological investigation. The sequence was synthesized as the peptide EPL001, against which was raised a goat polyclonal antiserum, G530. Used in an antigen capture campaign, G530 pointed to the existence of a novel derivative of secretogranin II (SgII), the neuroendocrine secretory vesicle helper protein and prohormone. The proposed SgII derivative was dubbed SgII-70, yet the sequence commonality between SgII and EPL001 is essentially NNI. Methods: Immunohistochemical (IHC) labelling with G530 is reported within rat, mouse and human cerebrovasculature and in glandular elements of the mouse intestine. Epitope mapping involved IHC peptide preabsorption, allied to deductive bioinformatics and molecular modelling in silico. Results: G530 is deemed monoepitopic in regard to both its synthetic antigen (EPL001) and its putative endogenous antigen (SgII related). The epitope within EPL001 of the anti-EPL001 antibody is inferred to be the contiguous C-terminal 9KEFNNI14. This is so because the G530 blockade data are consistent with the epitope in the mammalian endogenous antigen being part contiguous, part non-contiguous KE·F·NNI, ex hypothesi. The observed immunostaining is deduced to be due to pre-SgII-70, which has a non-C-terminal NNI, and SgII-70, which has an N-terminal MLKTGEKPV/N and a C-terminal NNI (these two motifs being in the reverse order in the SgII parent protein). Conclusion: The present data are consistent with the hypothesis that the anti-EPL001 antibody binds to an SgII-related epitope. SgII is apparently subject to peptide splicing, as has been reported for the related chromogranin A.