Protein Variants Research Articles

Mass Spectrometry-Based Top-Down Proteomics in Nanomedicine: Proteoform-Specific Measurement of Protein Corona.

Conventional mass spectrometry (MS)-based bottom-up proteomics (BUP) analysis of the protein corona [i.e., an evolving layer of biomolecules, mostly proteins, formed on the surface of nanoparticles (NPs) during their interactions with biomolecular fluids] enabled the nanomedicine community to partly identify the biological identity of NPs. Such an approach, however, fails to pinpoint the specific proteoforms─distinct molecular variants of proteins in the protein corona. The proteoform-level information could potentially advance the prediction of the biological fate and pharmacokinetics of nanomedicines. Recognizing this limitation, this study pioneers a robust and reproducible MS-based top-down proteomics (TDP) technique for characterizing proteoforms in the protein corona. Our TDP approach has successfully identified about 900 proteoforms in the protein corona of polystyrene NPs, ranging from 2 to 70 kDa, revealing proteoforms of 48 protein biomarkers with combinations of post-translational modifications, signal peptide cleavages, and/or truncations─details that BUP could not fully discern. This advancement in MS-based TDP offers a more advanced approach to characterize NP protein coronas, deepening our understanding of NPs' biological identities. We, therefore, propose using both TDP and BUP strategies to obtain more comprehensive information about the protein corona, which, in turn, can further enhance the diagnostic and therapeutic efficacy of nanomedicine technologies.

Gene-based drug therapy for children and youth treated with psychoactive medications.

Psychoactive medications are increasingly used to treat children and youth with mental health conditions, but individual variations in response highlight the need for precision medicine. Pharmacogenetic (PGx) testing is a key component of precision medicine. The number of commercial pharmacogenetic testing companies promoting PGx, with the promise of achieving individualized and effective treatment of mental health conditions, has grown exponentially in recent years. Scientific evidence supporting the use of PGx to manage mental health conditions is limited, especially for paediatric populations. This practice point outlines steps guiding the use and interpretation of PGx testing for psychoactive medications in clinical settings, along with key supportive resources. Practice guidelines have been developed for variants in pharmacogenes encoding cytochrome P450 drug-metabolizing enzymes (e.g., CYP2C19, CYP2D6, CYP2C9) as one determinant of drug concentrations in blood, which can support both drug choice and dosing strategy for certain anti-psychotics, anti-depressants, and anti-epileptics. Adverse drug reactions to some anti-epileptic drugs (e.g., carbamazepine and phenytoin) have been associated with certain human leukocyte antigen types and variants in DNA polymerase gamma (POLG; valproic acid). Evidence remains limited for genetic variants of drug target proteins, making it challenging to identify patients with altered treatment responses at a therapeutic blood concentration.

UT-1 Transporter Expression in the Spiny Dogfish (Squalus acanthias): UT-1 Protein Shows a Different Localization in Comparison to That of Other Sharks.

The original UT-1 transporter gene was initially identified in the spiny dogfish (Squalus acanthias), but localization of the UT-1 protein was not determined. Subsequent UT-1 expression was shown to localize to the collecting tubule (CT) of the shark nephron in other shark species, with expression in a closely related chimaera species also located additionally at a lower level in the intermediate-I segment (IS-I) of the nephron. In spiny dogfish, two UT-1 splice variants are known (UT-1 long and short), and there was also a second UT-1 gene described (here termed Brain UT). In this study, a second splice variant of the second Brain UT gene was discovered. Expression profiles (mRNA) of UT-1 long and short and Brain UT were determined in a number of spiny dogfish tissues. Quantitative PCR in kidney samples showed that the level of the short variant of UT-1 was around 100 times higher than the long variant, which was itself expressed around 10 times higher than Brain UT cDNA/mRNA (in kidney). For the long variant, there was a significantly higher level of mRNA abundance in fish acclimatized to 75% seawater. Ultimately, three UT-1 antibodies were made that could bind to both the UT-1 short and long variant proteins. The first two of these showed bands of appropriate sizes on Western blots of around 52.5 and 46 kDa. The second antibody had some additional lower molecular weight bands. The third antibody was mainly bound to the 46 kDa band with faint 52.5 kDa staining. Both the 52.5 and 46 kDa bands were absent when the antibodies were pre-blocked with the peptide antigens used to make them. Across the three antibodies, there were many similarities in localization but differences in subcellular localization. Predominantly, antibody staining was greatest in the intermediate segment 1 (IS-I) and proximal (PIb) segments of the first sinus zone loop of the nephron, with reasonably strong expression also found at the start and middle of the late distal tubule (LDT; second sinus zone loop). While some expression in the collecting tubule (CT) could not be ruled out, the level of staining seemed to be low or non-existent in convoluted bundle zone nephron segments such as the CT. Hence, this suggests that spiny dogfish have a fundamentally different mode of urea absorption in comparison to that found in other shark species, potentially focused more on the nephron sinus zone loops than the CT.

Left Ventricular Systolic Dysfunction in NBCe1-B/C-Knockout Mice.

Congenital proximal renal tubular acidosis (pRTA) is a rare systemic disease caused by mutations in the SLC4A4 gene that encodes the electrogenic sodium bicarbonate cotransporter, NBCe1. The major NBCe1 protein variants are designated NBCe1-A, NBCe1-B, and NBCe1-C. NBCe1-A expression is kidney-specific, NBCe1-B is broadly expressed and is the only NBCe1 variant expressed in the heart, and NBCe1-C is a splice variant of NBCe1-B that is expressed in the brain. No cardiac manifestations have been reported from patients with pRTA, but studies in adult rats with virally induced reduction in cardiac NBCe1-B expression indicate that NBCe1-B loss leads to cardiac hypertrophy and prolonged QT intervals in rodents. NBCe1-null mice die shortly after weaning, so the consequence of congenital, global NBCe1 loss on the heart is unknown. To circumvent this issue, we characterized the cardiac function of NBCe1-B/C-null (KOb/c) mice that survive up to 2 months of age and which, due to the uninterrupted expression of NBCe1-A, do not exhibit the confounding acidemia of the globally null mice. In contrast to the viral knockdown model, cardiac hypertrophy was not present in KOb/c mice as assessed by heart-weight-to-body-weight ratios and cardiomyocyte cross-sectional area. However, echocardiographic analysis revealed reduced left ventricular ejection fraction, and intraventricular pressure-volume measurements demonstrated reduced load-independent contractility. We also observed increased QT length variation in KOb/c mice. Finally, using the calcium indicator Fura-2 AM, we observed a significant reduction in the amplitude of Ca2+ transients in paced KOb/c cardiomyocytes. These data indicate that congenital, global absence of NBCe1-B/C leads to impaired cardiac contractility and increased QT length variation in juvenile mice. It remains to be determined whether the cardiac phenotype in KOb/c mice is influenced by the absence of NBCe1-B/C from neuronal and endocrine tissues.

Hyperactivation of ATF4/TGF-β1 signaling contributes to the progressive cardiac fibrosis in Arrhythmogenic cardiomyopathy caused by DSG2 Variant

BackgroundArrhythmogenic cardiomyopathy (ACM) is an inherited cardiomyopathy characterized with progressive cardiac fibrosis and heart failure. However, the exact mechanism driving the progression of cardiac fibrosis and heart failure in ACM remains elusive. This study aims to investigate the underlying mechanisms of progressive cardiac fibrosis in ACM caused by newly identified Desmoglein-2 (DSG2) variation.MethodsWe identified homozygous DSG2F531C variant in a family with 8 ACM patients using whole-exome sequencing and generated Dsg2F536C knock-in mice. Neonatal and adult mouse ventricular myocytes isolated from Dsg2F536C knock-in mice were used. We performed functional, transcriptomic and mass spectrometry analyses to evaluate the mechanisms of ACM caused by DSG2F531C variant.ResultsAll eight patients with ACM were homozygous for DSG2F531C variant. Dsg2F536C/F536C mice displayed cardiac enlargement, dysfunction, and progressive cardiac fibrosis in both ventricles. Mechanistic investigations revealed that the variant DSG2-F536C protein underwent misfolding, leading to its recognition by BiP within the endoplasmic reticulum, which triggered endoplasmic reticulum stress, activated the PERK-ATF4 signaling pathway and increased ATF4 levels in cardiomyocytes. Increased ATF4 facilitated the expression of TGF-β1 in cardiomyocytes, thereby activating cardiac fibroblasts through paracrine signaling and ultimately promoting cardiac fibrosis in Dsg2F536C/F536C mice. Notably, inhibition of the PERK-ATF4 signaling attenuated progressive cardiac fibrosis and cardiac systolic dysfunction in Dsg2F536C/F536C mice.ConclusionsHyperactivation of the ATF4/TGF-β1 signaling in cardiomyocytes emerges as a novel mechanism underlying progressive cardiac fibrosis in ACM. Targeting the ATF4/TGF-β1 signaling may be a novel therapeutic target for managing ACM.

Comparison of Variants of Uncertain Significance in Three Regions of the Human Glucokinase Protein Using In Vitro and In Silico Analyses.

There is a growing field of research focusing on the bioinformatic analysis of human genetic variation and the associated diseases. To study how well in vitro testing of purified proteins compares to bioinformatic variant prediction, we chose to analyze glucokinase (GCK) missense variations between residues 119-132, 257-262, and 412-427. These regions contained a large number of variants of uncertain significance (VUS) as well as a few pathogenic variants to use for comparison. We compared experimentally produced Vmax values from purified GCK variant proteins to predictive methods such as molecular dynamics simulation, ConSurf, iStable, the evolutionary model of variant effect (EVE), PredictSNP, and calculated binding energy. After determining which variants are pathogenic or benign based on experimental results or previous genetic studies, we found that ConSurf was the best at predicting pathogenicity. Interestingly, one VUS, D262N, showed an increase in activity and thus was difficult to interpret as pathogenic or benign. This study is an attempt to provide a framework for the utility of missense variant predictive programs.

Conformationally Constrained Isoquinolinones as Orally Efficacious Hepatitis B Capsid Assembly Modulators.

Isoquinolinone-based HBV capsid assembly modulators that bind at the dimer:dimer interface of HBV core protein have been shown to suppress viral replication in chronic hepatitis B patients. Analysis of their binding mode by protein X-ray crystallography has identified a region of the small molecule where the application of a constraint can lock the preferred binding conformation and has allowed for further optimization of this class of compounds. Key analogues demonstrated single digit nM EC50 values in reducing HBV DNA in a HepDE19 cellular assay in addition to favorable ADME and pharmacokinetic properties, leading to a high degree of oral efficacy in a relevant in vivo hydrodynamic injection mouse model of HBV infection, with 12e effecting a 3 log10 decline in serum HBV DNA levels at a once daily dose of 1 mg/kg. Additionally, maintenance of activity was observed in clinically relevant HBV core protein variants T33N and I105T.

Beta Spike-Presenting SARS-CoV-2 Virus-like Particle Vaccine Confers Broad Protection against Other VOCs in Mice.

Virus-like particles (VLPs) are non-infectious and serve as promising vaccine platforms because they mimic the membrane-embedded conformations of fusion glycoproteins on native viruses. Here, we employed SARS-CoV-2 VLPs (SMEN) presenting ancestral, Beta, or Omicron spikes to identify the variant spike that elicits potent and cross-protective immune responses in the highly sensitive K18-hACE2 challenge mouse model. A combined intranasal and intramuscular SMEN vaccine regimen generated the most effective immune responses to significantly reduce disease burden. Protection was primarily mediated by antibodies, with minor but distinct contributions from T cells in reducing virus spread and inflammation. Immunization with SMEN carrying ancestral spike resulted in 100, 75, or 0% protection against ancestral, Delta, or Beta variant-induced mortality, respectively. However, SMEN with an Omicron spike provided only limited protection against ancestral (50%), Delta (0%), and Beta (25%) challenges. By contrast, SMEN with Beta spikes offered 100% protection against the variants used in this study. Thus, the Beta variant not only overcame the immunity produced by other variants, but the Beta spike also elicited diverse and effective humoral immune responses. Our findings suggest that leveraging the Beta variant spike protein can enhance SARS-CoV-2 immunity, potentially leading to a more comprehensive vaccine against emerging variants.

Chemical induction of the interaction between AIMP2-DX2 and Siah1 to enhance ubiquitination.

AIMP2-DX2 (hereafter DX2) is an oncogenic variant of aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2) that mediates tumorigenic interactions with various factors involved in cancer. Reducing the levels of DX2 can effectively inhibit tumorigenesis. We previously reported that DX2 can be degraded through Siah1-mediated ubiquitination. In this study, we identified a compound, SDL01, which enhanced the interaction between DX2 and Siah1, thereby facilitating the ubiquitin-dependent degradation of DX2. SDL01 was found to bind to the pocket surrounding the N-terminal flexible region and GST domain of DX2, causing a conformational change that stabilized its interaction with Siah1. Our findings demonstrate that protein-protein interactions (PPIs) can be modulated through chemically induced conformational changes.

Surface-mutagenesis strategies to enable structural biology crystallization platforms.

A key prerequisite for the successful application of protein crystallography in drug discovery is to establish a robust crystallization system for a new drug-target protein fast enough to deliver crystal structures when the first inhibitors have been identified in the hit-finding campaign or, at the latest, in the subsequent hit-to-lead process. The first crucial step towards generating well folded proteins with a high likelihood of crystallizing is the identification of suitable truncation variants of the target protein. In some cases an optimal length variant alone is not sufficient to support crystallization and additional surface mutations need to be introduced to obtain suitable crystals. In this contribution, four case studies are presented in which rationally designed surface modifications were key to establishing crystallization conditions for the target proteins (the protein kinases Aurora-C, IRAK4 and BUB1, and the KRAS-SOS1 complex). The design process which led to well diffracting crystals is described and the crystal packing is analysed to understand retrospectively how the specific surface mutations promoted successful crystallization. The presented design approaches are routinely used in our team to support the establishment of robust crystallization systems which enable structure-guided inhibitor optimization for hit-to-lead and lead-optimization projects in pharmaceutical research.

Establishment of Star-edited Y1 cells as a novel in vitro functional assay for STAR.

Genetic variants involving steroidogenic acute regulatory protein cause lipoid congenital adrenal hyperplasia, which is characterized by impaired steroidogenesis in the adrenal glands and gonads. Functional assessment of variant STAR proteins is necessary for accurate genetic diagnosis. Ideally, steroidogenic cells should be used to assess the functionality of STAR proteins, but the presence of endogenous STARs in steroidogenic cells precludes such a method. Here, we generated Star-edited cells from steroidogenic Y1 mouse adrenocortical tumor cells by genome editing. Star-edited Y1 cells exhibited very low but measurable cAMP-dependent pregnenolone production. Furthermore, stimulation of the cAMP pathway for two weeks resulted in the formation of lipid droplets in the cytoplasm of Star-edited Y1 cells, which resembled the histology of the adrenal glands of patients with lipoid congenital adrenal hyperplasia. The steroidogenic defect of Star-edited Y1 cells can be restored by transient over-expression of mouse Star. We found that human STAR can also restore defective steroidogenesis of Star-edited Y1 cells, and were able to construct a novel in vitro system to evaluate human STAR variants. Collectively, we established Star-edited Y1 cells that retain the steroidogenic pathway downstream the Star protein. Star-edited Y1 cells recapitulate the functional and morphological changes of lipoid congenital adrenal hyperplasia, and can be used to evaluate the functionality of human STAR variants.

Comparison of two crystal polymorphs of NowGFP reveals a new conformational state trapped by crystal packing.

Crystal polymorphism serves as a strategy to study the conformational flexibility of proteins. However, the relationship between protein crystal packing and protein conformation often remains elusive. In this study, two distinct crystal forms of a green fluorescent protein variant, NowGFP, are compared: a previously identified monoclinic form (space group C2) and a newly discovered orthorhombic form (space group P212121). Comparative analysis reveals that both crystal forms exhibit nearly identical linear assemblies of NowGFP molecules interconnected through similar crystal contacts. However, a notable difference lies in the stacking of these assemblies: parallel in the monoclinic form and perpendicular in the orthorhombic form. This distinct mode of stacking leads to different crystal contacts and induces structural alteration in one of the two molecules within the asymmetric unit of the orthorhombic crystal form. This new conformational state captured by orthorhombic crystal packing exhibits two unique features: a conformational shift of the β-barrel scaffold and a restriction of pH-dependent shifts of the key residue Lys61, which is crucial for the pH-dependent spectral shift of this protein. These findings demonstrate a clear connection between crystal packing and alternative conformational states of proteins, providing insights into how structural variations influence the function of fluorescent proteins.

A Humanized Yeast Model for Studying TRAPP Complex Mutations; Proof-of-Concept Using Variants from an Individual with a TRAPPC1-Associated Neurodevelopmental Syndrome.

Variants in membrane trafficking proteins are known to cause rare disorders with severe symptoms. The highly conserved transport protein particle (TRAPP) complexes are key membrane trafficking regulators that are also involved in autophagy. Pathogenic genetic variants in specific TRAPP subunits are linked to neurological disorders, muscular dystrophies, and skeletal dysplasias. Characterizing these variants and their phenotypes is important for understanding the general and specialized roles of TRAPP subunits as well as for patient diagnosis. Patient-derived cells are not always available, which poses a limitation for the study of these diseases. Therefore, other systems, like the yeast Saccharomyces cerevisiae, can be used to dissect the mechanisms at the intracellular level underlying these disorders. The development of CRISPR/Cas9 technology in yeast has enabled a scar-less editing method that creates an efficient humanized yeast model. In this study, core yeast subunits were humanized by replacing them with their human orthologs, and TRAPPC1, TRAPPC2, TRAPPC2L, TRAPPC6A, and TRAPPC6B were found to successfully replace their yeast counterparts. This system was used for studying the first reported individual with an autosomal recessive disorder caused by biallelic TRAPPC1 variants, a girl with a severe neurodevelopmental disorder and myopathy. We show that the maternal variant (TRAPPC1 p.(Val121Alafs*3)) is non-functional while the paternal variant (TRAPPC1 p.(His22_Lys24del)) is conditional-lethal and affects secretion and non-selective autophagy in yeast. This parallels defects seen in fibroblasts derived from this individual which also showed membrane trafficking defects and altered Golgi morphology, all of which were rescued in the human system by wild-type TRAPPC1. This study suggests that humanized yeast can be an efficient means to study TRAPP subunit variants in the absence of human cells and can assign significance to variants of unknown significance (VUS). This study lays the foundation for characterizing further TRAPP variants through this system, rapidly contributing to disease diagnosis.

Design of intrinsically disordered protein variants with diverse structural properties.

Intrinsically disordered proteins (IDPs) perform a broad range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Computational design of IDPs with specific conformational properties has, however, been difficult because of their substantial dynamics and structural complexity. We describe a general algorithm for designing IDPs with specific structural properties. We demonstrate the power of the algorithm by generating variants of naturally occurring IDPs that differ in compaction, long-range contacts, and propensity to phase separate. We experimentally tested and validated our designs and analyzed the sequence features that determine conformations. We show how our results are captured by a machine learning model, enabling us to speed up the algorithm. Our work expands the toolbox for computational protein design and will facilitate the design of proteins whose functions exploit the many properties afforded by protein disorder.

Targeting SARS-CoV2 spike glycoprotein: molecular insights into phytocompounds binding interactions – in-silico molecular docking

This study utilized small molecular characterization and docking study to evaluate the binding affinity of seven antiviral phytocompounds with the SARS CoV-2 variants (SARS-CoV-2 Spike Glycoprotein, SARS-CoV-2 Spike Protein Variant in 1-RBD, Alpha Variant SARS-CoV2- Spike Protein). The results revealed that five of seven compounds, possesses excellent drug lead property reveled through in-silico ADMET analysis. In addition, six of seven except D-Glucosamine, exhibited excellent binding affinity. Six ligands possess significant binding affinity towards SARS-CoV-2 variants 6VXX, 7LWV and 7R13, which is certainly greater than Remdesivir. Fagaronine found to be the best drug candidate against SARS-CoV-2 variants, It was found that −7.4, −5.6 and −6.3 is the docking score respectively. Aranotin, Beta aescin, Gliotoxin, and Fagaronine formed hydrogen bonds with specific amino acids and exhibited significant binding interactions. These findings suggest that these phytocompounds could be promising candidates for developing antiviral therapies against SARS-CoV-2. Moreover, the study underscores the importance of molecular docking in understanding protein-ligand interactions and its role in drug discovery. The documented pharmacological properties of these compounds in the literature further support their potential therapeutic relevance in various diseases.

An integrated picture of chronic pancreatitis derived by mapping variants in multiple disease genes onto pathogenic pathways.

Chronic pancreatitis (CP) is an etiologically and genetically heterogeneous inflammatory syndrome characterised by progressive damage to the exocrine and endocrine components of the pancreas [ 1]. The multigenic paradigm of CP has sparked research in recent years [ 2]. We aimed to expand the current knowledge of genetic susceptibility of pancreatitis in patients of Indian origin. By employing whole-exome sequencing in an Indian hospital cohort, we dissect the genetic landscape associated with CP or recurrent acute pancreatitis (RAP). Notably, all patients had at least one genetic variant identified in a pancreatitis-risk gene, and most had a co-occurrence of a second variant in an additional risk gene. Based on the presence of both acinar and ductal gene variants in individual patients, we propose a two-hit hypothesis where variants in proteins expressed in both acinar and ductal cells are critical for RAP/CP development.

The Role of WNT3A Protein and Gene Variants in Allergic Rhinitis: A Case-Control Study.

Allergic rhinitis (AR) is a prevalent inflammatory disorder of the upper respiratory tract, driven by allergen exposure. Understanding mechanisms and identifying biomarkers for AR could significantly impact diagnosis and treatment. This study aimed to investigate the association between serum Wingless-Type MMTV Integration Site Family, Member 3A (WNT3A) protein levels, WNT3A polymorphisms, and AR. A cohort of 92 AR patients and 86 healthy controls was recruited. Serum WNT3A levels were measured by enzyme-linked immunosorbent assay (ELISA). WNT3A gene polymorphisms (rs752107 and rs3121310) were analyzed using Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) method. The study revealed significantly higher serum WNT3A levels in AR patients compared to controls (p < 0.0001). The impact of WNT3A in the differential diagnosis of AR was determined to be moderate, with an area under the curve (AUC) value of 0.67 (95% Confidence Interval: 0.59-0.75) based on the receiver operating characteristic (ROC) curve analysis. The rs3121310 polymorphism showed a significant association with the GA genotype more prevalent in controls (p < 0.05). However, no significant relationship was observed between rs3121310 genotypes and clinical parameters of the patients. These findings suggest a role for WNT3A in AR pathogenesis, given the elevated serum levels in patients. Larger cohort studies are needed to validate these findings and explore serum WNT3A levels as a biomarker for AR diagnosis and treatment monitoring.

Transglutaminase 2-mediated histone monoaminylation and its role in cancer.

Transglutaminase 2 (TGM2) has been known as a well-characterized factor regulating the progression of multiple types of cancer, due to its multifunctional activities and the ubiquitous signaling pathways it is involved in. As a member of the transglutaminase family, TGM2 catalyzes protein post-translational modifications (PTMs), including monoaminylation, amide hydrolysis, cross-linking, etc., through the transamidation of variant glutamine-containing protein substrates. Recent discoveries revealed histone as an important category of TGM2 substrates, thus identifying histone monoaminylation as an emerging epigenetic mark, which is highly enriched in cancer cells and possesses significant regulatory functions of gene transcription. In this review, we will summarize recent advances in TGM2-mediated histone monoaminylation as well as its role in cancer and discuss the key research methodologies to better understand this unique epigenetic mark, thereby shedding light on the therapeutic potential of TGM2 as a druggable target in cancer treatment.

Towards Cell-Permeable Hepatitis B Virus Core Protein Variants as Potential Antiviral Agents.

Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli, and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile.

Integrating Large-Scale Protein Structure Prediction into Human Genetics Research.

The last five years have seen impressive progress in deep learning models applied to protein research. Most notably, sequence-based structure predictions have seen transformative gains in the form of AlphaFold2 and related approaches. Millions of missense protein variants in the human population lack annotations, and these computational methods are a valuable means to prioritize variants for further analysis. Here, we review the recent progress in deep learning models applied to the prediction of protein structure and protein variants, with particular emphasis on their implications for human genetics and health. Improved prediction of protein structures facilitates annotations of the impact of variants on protein stability, protein-protein interaction interfaces, and small-molecule binding pockets. Moreover, it contributes to the study of host-pathogen interactions and the characterization of protein function. As genome sequencing in large cohorts becomes increasingly prevalent, we believe that better integration of state-of-the-art protein informatics technologies into human genetics research is of paramount importance.