Complex Protein Research Articles

Dynamics of water-mediated interaction effects on the stability and transmission of Omicron

SARS-Cov-2 Omicron variant and its highly transmissible sublineages amidst news of emerging hybrid variants strengthen the evidence of its ability to rapidly spread and evolve giving rise to unprecedented future waves. Owing to the presence of isolated RBD, monomeric and trimeric Cryo-EM structures of spike protein in complex with ACE2 receptor, comparative analysis of Alpha, Beta, Gamma, Delta, and Omicron assist in a rational assessment of their probability to evolve as new or hybrid variants in future. This study proposes the role of hydration forces in mediating Omicron function and dynamics based on a stronger interplay between protein and solvent with each Covid wave. Mutations of multiple hydrophobic residues into hydrophilic residues underwent concerted interactions with water leading to variations in charge distribution in Delta and Omicron during molecular dynamics simulations. Moreover, comparative analysis of interacting moieties characterized a large number of mutations lying at RBD into constrained, homologous and low-affinity groups referred to as mutational drivers inferring that the probability of future mutations relies on their function. Furthermore, the computational findings reveal a significant difference in angular distances among variants of concern due 3 amino acid insertion (EPE) in Omicron variant that not only facilitates tight domain organization but also seems requisite for characterization of mutational processes. The outcome of this work signifies the possible relation between hydration forces, their impact on conformation and binding affinities, and viral fitness that will significantly aid in understanding dynamics of drug targets for Covid-19 countermeasures. The emerging scenario is that hydration forces and hydrophobic interactions are crucial variables to probe in mutational analysis to explore conformational landscape of macromolecules and reveal the molecular origins of protein behaviors.

CNOT4 Knockout Induces Proteasome Inhibitor Resistance in Multiple Myeloma Cells

Background: The outcomes of multiple myeloma (MM) patients have been significantly improved with novel therapies including proteasome inhibitors (PI). However, many patients develop PI resistance and becoming refractory to PI. Although several studies have identified the mechanisms of PI resistance, such as mutation to the proteasome β5 subunit, ER stress suppression, autophagy activation, and activation of efflux drug pumps, it remains unclear yet. To investigate new genes that may contribute to PI resistance, we performed a genome-wide CRISPR dropout screening in an MM cell line. Materials and Methods: To identify the genes responsible for PI resistance, we used a CRISPR library (Kosuke Yusa, V3) targeting 18,740 human genes. The knockout cells, in which an CRISPR library lentivirus was introduced into a Cas9-expressing AMO1 MM cell line, were treated with ixazomib (IXA), carfilzomib (CFZ), and DMSO for two weeks. Next, we used next generation sequencing (NGS) to count sgRNA copy numbers of the pre-drug control group and the drug-treated group. NGS analysis of the treated cells showed that inactivated genes which induced higher levels of cell proliferation in the PI group than in the DMSO group were considered candidate genes for PI resistance. Results: We identified 35 genes for PI resistance including the genes encoding subunits of the proteasome 19S complex, consistent with previous reports that shows reducing 19S subunits protects MM cells from bortezomib. Among 35 candidate genes, we chose the top hit gene, CNOT4, to further investigate. CNOT4 is a component of CCR4-NOT complex which removes the mRNA poly(A) tail for degradation of mRNA. In addition, CNOT4 has E3 ubiquitin ligase activity and is reported to control the proteasome complex formation. To further validate the effect of PI resistance with inactivated CNOT4, we generated CNOT4 KO AMO1 cells and performed an MTS assay. We observed a decrease in PI sensitivity compared with AMO1 wildtype (WT) (IXA IC 50: 24.8 nM (KO) vs 13.0 nM (WT), CFZ IC 50: 7.45 nM (KO) vs 2.76 nM (WT)). To investigate the influence of CNOT4 on proteasome complex formation and its activity, we first measured the expression of ubiquitinated proteins by Western blotting. In CNOT4 KO AMO1 cells, the expression of ubiquitinated proteins is increased with or without PI treatment, suggesting that inactivated CNOT4 can suppress the function of degrade ubiquitinated proteins in proteasome 19S complex. To elucidate the mechanism underlying the resistance effect, we next checked the expression of ER stress marker proteins. In CNOT4 KO AMO1 cells, the expression of PERK and IRE1α proteins are decreased with PI treatment, suggesting that inactivated CNOT4 can suppress the ER stress levels in spite of the increases of ubiquitinated proteins. Conclusion: Our CRISPR screen revealed that CNOT4 inactivation induced PI resistance in human MM cells. CNOT4 inactivation suppresses the function of 19S proteasome, which is reported the influence of PI resistance, and downregulates the ER stress signal leading to avoid PI induced cell death. Our findings demonstrate that CNOT4 plays an important role in PI sensitivity.

BRCC3-Mediated NLRP3 Deubiquitylation Promotes Inflammasome Activation and Atherosclerosis in Tet2 Clonal Hematopoiesis.

Clonal hematopoiesis (CH) has emerged as an independent risk factor for atherosclerotic cardiovascular disease, with activation of macrophage inflammasomes as a potential underlying mechanism. The NLRP3 (NLR family pyrin domain containing 3) inflammasome has a key role in promoting atherosclerosis in mouse models of Tet2 CH, whereas inhibition of the inflammasome product interleukin-1β appeared to particularly benefit patients with TET2 CH in CANTOS (Cardiovascular Risk Reduction Study [Reduction in Recurrent Major CV Disease Events]). TET2 is an epigenetic modifier that decreases promoter methylation. However, the mechanisms underlying macrophage NLRP3 inflammasome activation in TET2 (Tet methylcytosine dioxygenase 2) deficiency and potential links with epigenetic modifications are poorly understood. We used cholesterol-loaded TET2-deficient murine and embryonic stem cell-derived isogenic human macrophages to evaluate mechanisms of NLRP3 inflammasome activation in vitro and hypercholesterolemic Ldlr-/- mice modeling TET2 CH to assess the role of NLRP3 inflammasome activation in atherosclerosis. Tet2 deficiency in murine macrophages acted synergistically with cholesterol loading in cell culture and with hypercholesterolemia in vivo to increase JNK1 (c-Jun N-terminal kinase 1) phosphorylation and NLRP3 inflammasome activation. The mechanism of JNK (c-Jun N-terminal kinase) activation in TET2 deficiency was increased promoter methylation and decreased expression of the JNK-inactivating dual-specificity phosphatase Dusp10. Active Tet1-deadCas9-targeted editing of Dusp10 promoter methylation abolished cholesterol-induced inflammasome activation in Tet2-deficient macrophages. Increased JNK1 signaling led to NLRP3 deubiquitylation and activation by the deubiquitinase BRCC3 (BRCA1/BRCA2-containing complex subunit 3). Accelerated atherosclerosis and neutrophil extracellular trap formation (NETosis) in Tet2 CH mice were reversed by holomycin, a BRCC3 deubiquitinase inhibitor, and also by hematopoietic deficiency of Abro1, an essential scaffolding protein in the BRCC3-containing cytosolic complex. Human TET2-/- macrophages displayed increased JNK1 and NLRP3 inflammasome activation, especially after cholesterol loading, with reversal by holomycin treatment, indicating human relevance. Hypercholesterolemia and TET2 deficiency converge on a common pathway of NLRP3 inflammasome activation mediated by JNK1 activation and BRCC3-mediated NLRP3 deubiquitylation, with potential therapeutic implications for the prevention of cardiovascular disease in TET2 CH.

Title: Single-Cell Transcriptome Analyses Further Elucidate the Mechanism of Action of Eed Inhibition on HbF Induction

Mutations in the gene encoding the hemoglobin β (HBβ) subunit lead to red blood cell disorders such as Sickle Cell Disease (SCD) and β-thalassemia. Hemoglobin γ subunit, a fetal counterpart of HBβ, can relieve the disease symptoms by forming a functional complex with hemoglobin a subunit, thereby compensating for the mutated HBβ. The expression of β-like hemoglobins is tightly regulated during development, with a switch from the fetal ortholog (γ) to the adult ortholog (β) shortly after birth. Maintenance of HBγ is therapeutically beneficial for SCD patients and is observed clinically in individuals with mutations for both SCD and hereditary persistent fetal hemoglobin (HPFH) who exhibit less severe or even no SCD-related symptoms. Polycomb repressive complex (PRC) proteins have been shown to regulate the expression of β-like hemoglobins. Our previous research has demonstrated inhibitors targeting the PRC2 protein embryonic ectoderm development (EED) upregulate fetal hemoglobin (HbF) expression, leading to the formation of HbF complexes in both human CD34 + HSC-derived erythroid cells and the Townes SCD mouse model. However, the functional mechanisms of PRC complexes in this regulatory process and their target genes remain to be clarified. In our study, we conducted single-cell transcriptome analyses on erythroid cells derived from CD34 + HSCs collected at various stages of differentiation. We identified several erythroid subpopulations, from early progenitors to maturing cells, within the CD34 + derived erythroid cell cultures. HbF + and HbF - cell populations were also identified using known markers. To explore the mechanisms underlying HbF upregulation by EED inhibition, we compared single-cell transcriptome profiles of samples treated with and without an EED inhibitor. Our findings reveal that EED inhibitor treatment consistently leads to increased HbF + populations across multiple donors without impacting erythroid cell differentiation. Differential gene expression analysis revealed sequential changes in the expression of previously identified HbF regulators during differentiation in the presence of EED inhibitor treatment. Through pseudotime analyses, we identified erythroid progenitor cells undergoing fate bifurcation, leading to an increase in the lineage of mature cells that express HBγ. Finally, by comparing the pseudotime trajectories of different samples, we identified genes whose expression changed along the differentiation trajectories in response to EED inhibitor treatment which influence cell fate. Overall, this study provides a deeper understanding of the relevant biological pathways that respond to EED inhibition and potential for the identification of novel molecular targets for HbF induction.

Novel Clinically Useful Inhibitor of Mediator Complex, RVU120, Relives Differentiation Block in MDS/AML

Background: Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemias (AML) are characterized by a block in hematopoietic differentiation that can often be traced to aberrant stage-specific transcription. The kinase module of the mediator complex, consisting of CDK8, its paralog CDK19, Cyclin C, MED12, and MED13, have been identified as a molecular switch that regulates gene expression. A variety of cancers have exploited this mechanism to maintain stemness and an undifferentiated state. RVU120 is a first-in-class, specific, and selective inhibitor of CDK8/19, currently in Phase Ib clinical trial in patients with AML or High-Risk MDS (HR-MDS) (NCT04021368). RVU120 has been shown to stimulate erythroid differentiation in transformed CD34+ cord blood (CB) cells and in CD34+ cells from Diamond-Blackfan Anemia (DBA). To evaluate the further therapeutic potential of CDK8/19 inhibition, we confirmed aberrant expression of mediator complex components and erythroid-stimulating activity in a collection of CD34+ primary samples of AML and MDS. Results: We investigated the expression of mediator complex components in a large database of MDS CD34+ marrow samples and age-matched controls. Our findings revealed that MED12, a critical component of the mediator complex, was significantly overexpressed in MDS samples from refractory anemia with excess blasts (RAEB), a higher risk subset of MDS(p=0.018) (Fig A). Furthermore, we observed that this overexpression of MED12 was associated with a higher rate of transformation to AML. To delve deeper, we examined the expression of MED12 in sorted leukemic stem cells from primary AML samples and found significantly elevated levels in Long Term Hematopoietic Stem Cells from AML samples with both normal karyotype and complex karyotypes compared to healthy controls. Next, we conducted an evaluation of the efficacy of the mediator kinase module inhibition in MDS-derived cell line (MDS-L). Inhibition of CDK8/19 with RVU120 led to increased erythroid differentiation in this model which confirmed our observations in transformed CD34+ CB cells in CB cells, these phenotypic changes were correlated with the inhibition of CDK8-dependent phosphorylation marker S726 on STAT5 and a profound increase in the expression of genes involved in erythroid commitment and hemoglobin metabolism. In order to confirm therapeutic potential of CDK8/CDK19 inhibition in MDS and AML, we performed colony-forming assay on primary samples of MDS and AML (n= 15). The treatment with RVU120 resulted in increased erythroid differentiation in a majority of samples, evident from an augmented number of colonies. Detailed analysis indicated changes in the expression of erythroid differentiation markers, including increased CD71 and Glycophorin A expression on colonies, as assessed by FACS analysis (Figure B, shows representative sample). Conclusion Our data highlight that inhibition of the overexpressed mediator complex proteins can alleviate the differentiation blocks seen in MDS/AML. These results provide a compelling rationale for further developing the CDK8/19 inhibitor RVU120 for transfusion- dependent MDS/AML patients.

Quantitative Label-Free Mass Spectrometry Identifies Molecular Content and Signaling Differences between Neonatal and Adult Platelets

Observational and randomized studies have associated platelet transfusions with increased morbidity and mortality in preterm neonates. Clinical practice changes have been hindered by a limited understanding of why adult donor-derived platelets might be inappropriate or harmful to infants. Neonatal and adult platelets differ in reactivity and function, but we lack an understanding of underlying molecular differences. As proteins and kinase-driven phosphorylation signaling dictate platelet biology, we aimed to characterize altered protein and phosphoprotein content between neonatal and adult platelets to help explain differences in platelet reactivity and function. We isolated resting platelets from full term cord blood (n=9) and adult peripheral blood (n=7) and used mass spectrometry-based platforms to ascertain protein and phosphoprotein content. We identified 4745 high-confidence proteins in adult and neonatal platelets, including 331 differentially abundant proteins (≥1.5-fold change, p<0.05). Neonatal platelets had increased metabolic and ribosomal proteins, consistent with transcriptional data. Adult platelets were enriched for inflammatory proteins, including complement components, consistent with pro-inflammatory function. We also identified 2115 phospho-proteins (17852 unique phosphopeptides) across all samples. These impacted actin cytoskeletal biology, cell adhesion, and GTPase signaling that regulate platelet functions. Kinase enrichment analysis identified FYN, SRC, ABL1, and AKT1 as kinases most responsible for platelet phosphorylation activities. Granule trafficking and degranulation proteins were among 1183 differentially abundant phosphoproteins in neonatal vs adult samples (p<0.05). For example, 31 different Reticulon 1 (RTN1) phosphopeptides were enriched in adult platelets (p<0.05). RTN1 regulates membrane trafficking and participates in SNARE-mediated exocytosis. Increased RTN1-directed membrane trafficking may promote increased activation and degranulation in adult vs neonatal platelets. Indeed, SNARE complex protein deficiency was previously suggested to mediate hyporeactive degranulation in neonatal platelets. We detected 2 phosphopeptides in adult samples that were absent from any neonatal sample. We reasoned that these could represent developmental stage-specific differences that exist only in adult platelets. One of the adult-specific phosphopeptides was in Rap1GAP2 (phospho-S588). Rap1GAP2 is a highly phosphorylated GTPase-activating protein that inhibits Rap1, a small guanine-nucleotide-binding protein that facilitates integrin activation in platelets. Rap1GAP2 binds synaptotagmin-like protein 1 (Slp1) to regulate dense granule secretion. These findings may link altered developmentally regulated Rap1GAP2 activities with platelet degranulation. A Platelet Cell Adhesion Molecule (PECAM) phosphopeptide (phospho-S726) was also found exclusively in adult platelets. PECAM facilitates inflammation and leukocyte transendothelial migration. PECAM phosphorylation in adult platelets may reflect an enhanced ability to participate in these processes. These findings reveal key differences in protein content and signaling that underlie differential neonatal vs adult platelet reactivity and function, including pro-inflammatory activities and enhanced activation and/or degranulation propensity in adult platelets. RTN1-mediated SNARE complex function and Rap1 signaling activities represent novel and potentially modifiable targets to manipulate platelet activation and/or degranulation. Our results also help provide a biological rationale for the higher morbidity and mortality observed in preterm infants transfused with adult platelets, including the inadvertent introduction of serum complement and other pro-inflammatory molecules within transfusions of donated adult platelets.

Influence of Guluronic and Mannuronic Groups in Sodium Alginate Blends with Silk Fibroin: Phase Equilibrium and Thermodynamic Modeling

Silk fibroin (SF) and sodium alginate (SA) are natural polymers with interesting properties to produce biomaterials. Blends of these polymers form a complex protein–polysaccharide system where phase separation can be observed. Therefore, the thermodynamic analysis of this system is important to understand the interaction between the polymers and predict the final state and composition of the phases found in these blends. This study explored blends with a different initial composition of SF, SA, and water at 25°C and neutral pH. The influence of the proportion between mannuronic and guluronic acids on SA composition was investigated. After phase separation, two phases were identified, and the equilibrium data were fitted on three different thermodynamic models: Flory–Huggins, non-random two-liquids, and universal quasichemical. Cohn equation was also used to investigate the potential of SA to precipitate the SF in solution. The results show that the proportion of mannuronic and guluronic acids on the SA can significantly influence on equilibrium data and on the SF/SA interaction parameter, hence, becoming a variability factor if this parameter is not under control in formulations.

NUP85 as a Neurodevelopmental Gene: From Podocyte to Neuron.

Pathogenic gene variants encoding nuclear pore complex (NPC) proteins were previously implicated in the pathogenesis of steroid-resistant nephrotic syndrome (SRNS). The NUP85 gene, encoding nucleoporin, is related to a very rare form of SRNS with limited genotype-phenotype information. We identified an Italian boy affected with an SRNS associated with severe neurodevelopmental impairment characterized by microcephaly, axial hypotonia, lack of achievement of motor milestones, and refractory seizures with an associated hypsarrhythmic pattern on electroencephalography. Brain magnetic resonance imaging (MRI) showed hypoplasia of the corpus callosum and a simplified gyration of the cerebral cortex. Since the age of 3 years, the boy was followed up at our Pediatric Nephrology Department for an SRNS, with a focal segmental glomerulosclerosis at renal biopsy. The boy died 32 months after SRNS onset, and a Whole-Exome Sequencing analysis revealed a novel compound heterozygous variant in NUP85 (NM_024844.5): 611T>A (p.Val204Glu), c.1904T>G (p.Leu635Arg), inherited from the father and mother, respectively. We delineated the clinical phenotypes of NUP85-related disorders, reviewed the affected individuals so far reported in the literature, and overall expanded both the phenotypic and the molecular spectrum associated with this ultra-rare genetic condition. Our study suggests a potential occurrence of severe neurological phenotypes as part of the NUP85-related clinical spectrum and highlights an important involvement of nucleoporin in brain developmental processes and neurological function.

Melatonin-Mediated Enhancement of Photosynthetic Capacity and Photoprotection Improves Salt Tolerance in Wheat

The role of melatonin in plant growth and response to environmental stress has been widely demonstrated. However, the physiological and molecular regulation of salt tolerance in wheat seedlings by melatonin remains unclear. In this study, we investigated changes in phenotype, physiology, photosynthetic parameters, and transcript levels in wheat seedlings to reveal the role of melatonin in the regulation of salt tolerance in wheat. The results indicate that the application of exogenous melatonin significantly alleviates growth inhibition, reactive oxygen species accumulation, and membrane oxidative damage induced by salt stress in wheat. Additionally, exogenous melatonin increased antioxidant enzyme activity and regulated photosynthetic gas exchange. Transcriptomic data showed a significant up-regulation of genes encoding light-harvesting chlorophyll protein complex proteins in photosynthesis and genes related to chlorophyll and carotenoid biosynthesis under the influence of melatonin. These results suggest that exogenous melatonin improves salt tolerance in wheat seedlings by enhancing the antioxidant, photoprotective, and photosynthesis activities.

Genome-Wide Analysis of the Universal Stress Protein Gene Family in Blueberry and Their Transcriptional Responses to UV-B Irradiation and Abscisic Acid

Universal stress proteins (USPs) play essential roles in plant development, hormonal regulation, and abiotic stress responses. However, the characteristics and functional divergence of USP family members have not been studied in blueberry (Vaccinium corymbosum). In this study, we identified 72 VcUSP genes from the Genome Database for Vaccinium. These VcUSPs could be divided into five groups based on their phylogenetic relationships. VcUSPs from groups Ⅰ, Ⅳ, and Ⅴ each possess one UspA domain; group Ⅰ proteins also contain an ATP-binding site that is not present in group Ⅳ and Ⅴ proteins. Groups Ⅱ and Ⅲ include more complex proteins possessing one to three UspA domains and UspE or UspF domains. Prediction of cis-regulatory elements in the upstream sequences of VcUSP genes indicated that their protein products are likely involved in phytohormone signaling pathways and abiotic stress responses. Analysis of RNA deep sequencing data showed that 21 and 7 VcUSP genes were differentially expressed in response to UV-B radiation and exogenous abscisic acid (ABA) treatments, respectively. VcUSP41 and VcUSP68 expressions responded to both treatments, and their encoded proteins may integrate the UV-B and ABA signaling pathways. Weighted gene co-expression network analysis revealed that VcUSP22, VcUSP26, VcUSP67, VcUSP68, and VcUSP41 were co-expressed with many transcription factor genes, most of which encode members of the MYB, WRKY, zinc finger, bHLH, and AP2 families, and may be involved in plant hormone signal transduction, circadian rhythms, the MAPK signaling pathway, and UV-B-induced flavonoid biosynthesis under UV-B and exogenous ABA treatments. Our study provides a useful reference for the further functional analysis of VcUSP genes and blueberry molecular breeding.

Improved characterization of trastuzumab deruxtecan with PTCR and internal fragments implemented in middle-down MS workflows.

Antibody-drug conjugates (ADCs) are highly complex proteins mainly due to the structural microvariability of the mAb, along with the additional heterogeneity afforded by the bioconjugation process. Top-down (TD) and middle-down (MD) strategies allow the straightforward fragmentation of proteins to elucidate the conjugated amino acid residues. Nevertheless, these spectra are very crowded with multiple overlapping and unassigned ion fragments. Here we report on the use of dedicated software (ClipsMS) and application of proton transfer charge reduction (PTCR), to respectively expand the fragment ion search space to internal fragments and improve the separation of overlapping fragment ions for a more comprehensive characterization of a recently approved ADC, trastuzumab deruxtecan (T-DXd). Subunit fragmentation allowed between 70 and 90% of sequence coverageto be obtained. Upon addition of internal fragment assignment, the three subunits were fully sequenced, although internal fragments did not contribute significantly to the localization of the payloads. Finally, the use of PTCR after subunit fragmentation provided a moderate sequence coverage increase between 2 and 13%. The reaction efficiently decluttered the fragmentation spectra allowing increasing the number of fragment ions characteristic of the conjugation site by 1.5- to 2.5-fold. Altogether, these results show the interest in the implementation of internal fragment ion searches and more particularly the use of PTCR reactions to increase the number of signature ions to elucidate the conjugation sites and enhance the overall sequence coverage of ADCs, making this approach particularly appealing for its implementation in R&D laboratories.

A Proposal for a Consolidated Structural Model of the CagY Protein of Helicobacter pylori

CagY is the largest and most complex protein from Helicobacter pylori’s (Hp) type IV secretion system (T4SS), playing a critical role in the modulation of gastric inflammation and risk for gastric cancer. CagY spans from the inner to the outer membrane, forming a channel through which Hp molecules are injected into human gastric cells. Yet, a tridimensional structure has been reported for only short segments of the protein. This intricate protein was modeled using different approaches, including homology modeling, ab initio, and deep learning techniques. The challengingly long middle repeat region (MRR) was modeled using deep learning and optimized using equilibrium molecular dynamics. The previously modeled segments were assembled into a 1595 aa chain and a 14-chain CagY multimer structure was assembled by structural alignment. The final structure correlated with published structures and allowed to show how the multimer may form the T4SS channel through which CagA and other molecules are translocated to gastric cells. The model confirmed that MRR, the most polymorphic and complex region of CagY, presents numerous cysteine residues forming disulfide bonds that stabilize the protein and suggest this domain may function as a contractile region playing an essential role in the modulating activity of CagY on tissue inflammation.

Recent advances in expression and purification strategies for plant made vaccines

Plants have been explored as a platform to produce pharmaceutical proteins for over 20 years. Important features such as the cost-effectiveness of production, the ease of scaling up to manufacturing capacity, the lack of cold chain requirements and the ability to produce complex therapeutic proteins which are biologically and functionally identical to their mammalian counterparts, make plants a strong alternative for vaccine production. This review article focuses on both the expression as well as the downstream purification processes for plant made vaccines. Expression strategies including transgenic, transient and cell suspension cultures are outlined, and various plant tissues targeted such as leaves and seeds are described. The principal components used for downstream processing of plant made vaccines are examined. The review concludes with a reflection of the future benefits of plant production platforms for vaccine production.

Quantification of biases in predictions of protein-protein binding affinity changes upon mutations.

Understanding the impact of mutations on protein-protein binding affinity is a key objective for a wide range of biotechnological applications and for shedding light on disease-causing mutations, which are often located at protein-protein interfaces. Over the past decade, many computational methods using physics-based and/or machine learning approaches have been developed to predict how protein binding affinity changes upon mutations. They all claim to achieve astonishing accuracy on both training and test sets, with performances on standard benchmarks such as SKEMPI 2.0 that seem overly optimistic. Here we benchmarked eight well-known and well-used predictors and identified their biases and dataset dependencies, using not only SKEMPI 2.0 as a test set but also deep mutagenesis data on the severe acute respiratory syndrome coronavirus 2 spike protein in complex with the human angiotensin-converting enzyme 2. We showed that, even though most of the tested methods reach a significant degree of robustness and accuracy, they suffer from limited generalizability properties and struggle to predict unseen mutations. Interestingly, the generalizability problems are more severe for pure machine learning approaches, while physics-based methods are less affected by this issue. Moreover, undesirable prediction biases toward specific mutation properties, the most marked being toward destabilizing mutations, are also observed and should be carefully considered by method developers. We conclude from our analyses that there is room for improvement in the prediction models and suggest ways to check, assess and improve their generalizability and robustness.

Advances in neuroproteomics for neurotrauma: unraveling insights for personalized medicine and future prospects

Neuroproteomics, an emerging field at the intersection of neuroscience and proteomics, has garnered significant attention in the context of neurotrauma research. Neuroproteomics involves the quantitative and qualitative analysis of nervous system components, essential for understanding the dynamic events involved in the vast areas of neuroscience, including, but not limited to, neuropsychiatric disorders, neurodegenerative disorders, mental illness, traumatic brain injury, chronic traumatic encephalopathy, and other neurodegenerative diseases. With advancements in mass spectrometry coupled with bioinformatics and systems biology, neuroproteomics has led to the development of innovative techniques such as microproteomics, single-cell proteomics, and imaging mass spectrometry, which have significantly impacted neuronal biomarker research. By analyzing the complex protein interactions and alterations that occur in the injured brain, neuroproteomics provides valuable insights into the pathophysiological mechanisms underlying neurotrauma. This review explores how such insights can be harnessed to advance personalized medicine (PM) approaches, tailoring treatments based on individual patient profiles. Additionally, we highlight the potential future prospects of neuroproteomics, such as identifying novel biomarkers and developing targeted therapies by employing artificial intelligence (AI) and machine learning (ML). By shedding light on neurotrauma’s current state and future directions, this review aims to stimulate further research and collaboration in this promising and transformative field.

Unbiased interrogation of functional lysine residues in human proteome

CRISPR screens have empowered the high-throughput dissection of gene functions; however, more explicit genetic elements, such as codons of amino acids, require thorough interrogation. Here, we establish a CRISPR strategy for unbiasedly probing functional amino acid residues at the genome scale. By coupling adenine base editors and barcoded sgRNAs, we target 215,689 out of 611,267 (35%) lysine codons, involving 85% of the total protein-coding genes. We identify 1,572 lysine codons whose mutations perturb human cell fitness, with many of them implicated in cancer. These codons are then mirrored to gene knockout screen data to provide functional insights into the role of lysine residues in cellular fitness. Mining these data, we uncover a CUL3-centric regulatory network in which lysine residues of CUL3 CRL complex proteins control cell fitness by specifying protein-protein interactions. Our study offers a general strategy for interrogating genetic elements and provides functional insights into the human proteome.

Hypertrophic cardiomyopathy is characterized by alterations of the mitochondrial calcium uniporter complex proteins: insights from patients with aortic valve stenosis versus hypertrophic obstructive cardiomyopathy

Hypertrophic cardiomyopathy is characterized by alterations of the mitochondrial calcium uniporter complex proteins: insights from patients with aortic valve stenosis versus hypertrophic obstructive cardiomyopathy

Phanto-IDP: compact model for precise intrinsically disordered protein backbone generation and enhanced sampling.

The biological function of proteins is determined not only by their static structures but also by the dynamic properties of their conformational ensembles. Numerous high-accuracy static structure prediction tools have been recently developed based on deep learning; however, there remains a lack of efficient and accurate methods for exploring protein dynamic conformations. Traditionally, studies concerning protein dynamics have relied on molecular dynamics (MD) simulations, which incur significant computational costs for all-atom precision and struggle to adequately sample conformational spaces with high energy barriers. To overcome these limitations, various enhanced sampling techniques have been developed to accelerate sampling in MD. Traditional enhanced sampling approaches like replica exchange molecular dynamics (REMD) and frontier expansion sampling (FEXS) often follow the MD simulation approach and still cost a lot of computational resources and time. Variational autoencoders (VAEs), as a classic deep generative model, are not restricted by potential energy landscapes and can explore conformational spaces more efficiently than traditional methods. However, VAEs often face challenges in generating reasonable conformations for complex proteins, especially intrinsically disordered proteins (IDPs), which limits their application as an enhanced sampling method. In this study, we presented a novel deep learning model (named Phanto-IDP) that utilizes a graph-based encoder to extract protein features and a transformer-based decoder combined with variational sampling to generate highly accurate protein backbones. Ten IDPs and four structured proteins were used to evaluate the sampling ability of Phanto-IDP. The results demonstrate that Phanto-IDP has high fidelity and diversity in the generated conformation ensembles, making it a suitable tool for enhancing the efficiency of MD simulation, generating broader protein conformational space and a continuous protein transition path.

The Role of Bcl-2 and Beclin-1 Complex in "Switching" between Apoptosis and Autophagy in Human Glioma Cells upon LY294002 and Sorafenib Treatment.

Gliomas are the most malignant tumors of the central nervous system. One of the factors in their high drug resistance is avoiding programmed death (PCD) induction. This is related to the overexpression of intracellular survival pathways: PI3K-Akt/PKB-mTOR and Ras-Raf-MEK-ERK. Apoptosis and autophagy are co-existing processes due to the interactions between Bcl-2 and beclin-1 proteins. Their complex may be a molecular "toggle-switch" between PCD types. The aim of this research was to investigate the role of Bcl-2:beclin-1 complex in glioma cell elimination through the combined action of LY294002 and sorafenib. Drug cytotoxicity was estimated with an MTT test. The type of cell death was evaluated using variant microscopy techniques (fluorochrome staining, immunocytochemistry, and transmission electron microscopy), as well as the Bcl-2:beclin-1 complex formation and protein localization. Molecular analysis of PCD indicators was conducted through immunoblotting, immunoprecipitation, and ELISA testing. SiRNA was used to block Bcl-2 and beclin-1 expression. The results showed the inhibitors used in simultaneous application resulted in Bcl-2:beclin-1 complex formation and apoptosis becoming dominant. This was accompanied by changes in the location of the tested proteins. "Switching" between apoptosis and autophagy using PI3K and Raf inhibitors with Bcl-2:beclin-1 complex formation opens new therapeutic perspectives against gliomas.

Investigation the role of Human Leukocyte Antigen-G in Iraqi patients with papillary thyroid carcinoma

Human leukocyte antigen G (HLA-G) is a non-classical major histocompatibility complex (MHC) protein with well-known immunomodulatory characteristics. It is also believed to be an important sign of immune tolerance in cancer cells. Immune Escape. HLA-G is also associated with disease development and prognosis in cancer patients. The goal of this study was to compare soluble HLA-G (sHLA-G) levels before and after radioactive iodine therapy (RAI) by enzyme immunoassay (ELISA). Immunohistochemistry was used to examine HLA-G protein expression in thyroid tissues from patients with papillary thyroid cancer (PTC) to investigate the relationship between HLA-G expression and patients' clinical variables. Prospective research included 138 blood samples from patients and controls, as well as 25 thyroid paraffin-embedded tissues from individuals suffering from papillary thyroid cancer (PTC) and controls. Our findings demonstrated a significant difference in the means of people who had radioactive iodine therapy against those who did not (Mean SE = 4.19 0.31, 2.216 0.08, respectively). HLA-G staining, on the other hand, was identified in tumor cells in papillary thyroid cancer, all sections were positive, strongly stained and completely membranous, indicating the presence of HLA-G protein in the tested material. These findings suggest that HLA-G may be involved in the pathogenesis of papillary thyroid cancer.