Search

This review discusses the various types of noncovalent interactions in which sulfur atoms participate and their effects on protein stability, structure, folding and bioactivity. Current approaches and recommendations for modelling these noncovalent interactions (in terms of both geometries and interaction energies) are highlighted.

Calculating changes in protein stability (ΔΔG) has been shown to be central for predicting the consequences of single amino acid substitutions in protein engineering as well as interpretation of genomic variants for disease risk. Structure-based calculations are considered most accurate, however the tools used to calculate ΔΔGs have been developed on experimentally resolved structures. Extending those calculations to homology models based on related proteins would greatly extend their applicability as large parts of e.g. the human proteome are not structurally resolved. In this study we aim to investigate the accuracy of ΔΔG values predicted on homology models compared to crystal structures. Specifically, we identified four proteins with a large number of experimentally tested ΔΔGs and templates for homology modeling across a broad range of sequence identities, and selected three methods for ΔΔG calculations to test. We find that ΔΔG-values predicted from homology models compare equally well to experimental ΔΔGs as those predicted on experimentally established crystal structures, as long as the sequence identity of the model template to the target protein is at least 40%. In particular, the Rosetta cartesian_ddg protocol is robust against the small perturbations in the structure which homology modeling introduces. In an independent assessment, we observe a similar trend when using ΔΔGs to categorize variants as low or wild-type-like abundance. Overall, our results show that stability calculations performed on homology models can substitute for those on crystal structures with acceptable accuracy as long as the model is built on a template with sequence identity of at least 40% to the target protein. Calculating changes in protein stability (ΔΔG) has been shown to be central for predicting the consequences of single amino acid substitutions in protein engineering as well as interpretation of genomic variants for disease risk. Structure-based calculations are considered most accurate, however the tools used to calculate ΔΔGs have been developed on experimentally resolved structures. Extending those calculations to homology models based on related proteins would greatly extend their applicability as large parts of e.g. the human proteome are not structurally resolved. In this study we aim to investigate the accuracy of ΔΔG values predicted on homology models compared to crystal structures. Specifically, we identified four proteins with a large number of experimentally tested ΔΔGs and templates for homology modeling across a broad range of sequence identities, and selected three methods for ΔΔG calculations to test. We find that ΔΔG-values predicted from homology models compare equally well to experimental ΔΔGs as those predicted on experimentally established crystal structures, as long as the sequence identity of the model template to the target protein is at least 40%. In particular, the Rosetta cartesian_ddg protocol is robust against the small perturbations in the structure which homology modeling introduces. In an independent assessment, we observe a similar trend when using ΔΔGs to categorize variants as low or wild-type-like abundance. Overall, our results show that stability calculations performed on homology models can substitute for those on crystal structures with acceptable accuracy as long as the model is built on a template with sequence identity of at least 40% to the target protein.

Triple-negative breast cancer (TNBC) is a heterogeneous malignancy in women. It is associated with poor prognosis, aggressive malignant behavior, and limited treatment options. In the ubiquitin‒proteasome system (UPS), deubiquitinases (DUBs) are potential therapeutic targets for various tumors. In this study, by performing unbiased siRNA screening, we identified STAMBP, a JAMM metalloprotease in the DUB family, as a driver of human TNBC tumor growth. Functionally, the knockdown of STAMBP inhibited the proliferation, migration, and invasion of multiple TNBC cell lines. Immunoprecipitation–mass spectrometry combined with functional and morphological analysis verified the interaction between STAMBP and the actin-binding protein RAI14. Mechanistically, STAMBP stabilized the RAI14 protein by suppressing the K48-linked ubiquitination of RAI14 and thus prevented its proteasomal degradation. Therefore, knocking down STAMBP resulted in the reduction in RAI14 protein levels and suppression of tumor growth in vitro and in vivo. Importantly, high levels of STAMBP were correlated with poor prognosis in TNBC patients. In summary, we reveal a previously unrecognized DUB pathway that promotes TNBC progression and provides a rationale for potential therapeutic interventions for the treatment of TNBC.

This study addresses well-known shortcomings of poly(ethylene glycol) (PEG)-based conjugates. PEGylation is by far the most common method employed to overcome immunogenicity and suboptimal pharmacokinetics of, for example, therapeutic proteins but has significant drawbacks. First, PEG offers no protection from denaturation during lyophilization, storage, or oxidation (e.g., by biological oxidants, reactive oxygen species); second, PEG's inherent immunogenicity, leading to hypersensitivity and accelerated blood clearance (ABC), is a growing concern. We have here developed an active-stealth' polymer, poly(thioglycidyl glycerol)(PTGG), which in human plasma is less immunogenic than PEG (35% less complement activation) and features a reactive oxygen species-scavenging and anti-inflammatory action (50% less TNF-α in LPS-stimulated macrophages at only 0.1 mg/mL). PTGG was conjugated to proteins via a one-pot process; molar mass- and grafting density-matched PTGG-lysozyme conjugates were superior to their PEG analogues in terms of enzyme activity and stability against freeze-drying or oxidation; the latter is due to sacrificial oxidation of methionine-mimetic PTGG chains. Both in mice and rats, PTGG-ovalbumin displayed circulation half-lives up to twice as long as PEG-ovalbumin, but most importantly and differently from PEG without any associated ABC effect seen either in the time dependency of blood concentration, in the liver/splenic accumulation, or in antipolymer IgM/IgG titers. Furthermore, similar pharmacokinetic results were obtained with PTGGylated/PEGylated liposomal nanocarriers. PTGG's active-stealth' character therefore makes it a highly promising alternative to PEG for conjugation to biologics or nanocarriers.

Many of the diseases that plague society today are driven by a loss of protein quality. One method to quantify protein quality is to measure the protein folding stability (PFS). Here, we present a novel mass spectrometry (MS)-based approach for PFS measurement, iodination protein stability assay (IPSA). IPSA quantifies the PFS by tracking the surface-accessibility differences of tyrosine, histidine, methionine, and cysteine under denaturing conditions. Relative to current methods, IPSA increases protein coverage and granularity to track the PFS changes of a protein along its sequence. To our knowledge, this study is the first time the PFS of human serum proteins has been measured in the context of the blood serum (in situ). We show that IPSA can quantify the PFS differences between different transferrin iron-binding states in near

Cyclin F, unlike canonical and transcriptional cyclins, does not bind or activate any cyclin-dependent kinases. Instead, it harbors an F-box motif and primarily functions as the substrate recognition subunit of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCF

Most membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes docked at the translocon, a heterogeneous ensemble of transmembrane factors operating on the nascent chain1,2. How the translocon coordinates the actions of these factors to accommodate its different substrates is not well understood. Here we define the composition, function and assembly of a translocon specialized for multipass membrane protein biogenesis3. This ‘multipass translocon’ is distinguished by three components that selectively bind the ribosome–Sec61 complex during multipass protein synthesis: the GET- and EMC-like (GEL), protein associated with translocon (PAT) and back of Sec61 (BOS) complexes. Analysis of insertion intermediates reveals how features of the nascent chain trigger multipass translocon assembly. Reconstitution studies demonstrate a role for multipass translocon components in protein topogenesis, and cells lacking these components show reduced multipass protein stability. These results establish the mechanism by which nascent multipass proteins selectively recruit the multipass translocon to facilitate their biogenesis. More broadly, they define the ER translocon as a dynamic assembly whose subunit composition adjusts co-translationally to accommodate the biosynthetic needs of its diverse range of substrates.

A nuclear serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) is a critical regulator of development and DNA damage response. HIPK2 can induce apoptosis under cellular stress conditions and thus its protein level is maintained low by constant proteasomal degradation. In the present study, we present evidence that TNF receptor-associated factor 2 (TRAF2) regulates the protein stability of HIPK2. Overexpression of TRAF2 decreased while its knockdown increased the HIPK2 protein level. The TRAF2-mediated decrease in HIPK2 protein expression was blocked by proteasomal inhibitor. In addition, TRAF2 decreased the protein half-life of HIPK2. We found that HIPK2 and TRAF2 co-immunoprecipitated. Interestingly, the co-immunoprecipitation was reduced while HIPK2 protein level increased following TNFα treatment, suggesting TNFα induced dissociation of TRAF2 from HIPK2 to accumulate HIPK2. Inhibition of HIPK2 partially suppressed TNFα-induced cell death, indicating that the accumulated HIPK2 may contribute to the TNFα-induced cell death. Our results suggest that TRAF2 can regulate proapoptotic function of HIPK2 by promoting proteasomal degradation.

The significant role of papain-like cysteine proteases, including papain, cathepsin L and SARS-CoV-2 PLpro, in biomedicine and biotechnology makes them interesting model systems for sensor development. These enzymes have a free thiol group that is suitable for many sensor designs including strong binding to gold nanoparticles or low-molecular-weight inhibitors. Focusing on the importance of the preservation of native protein structure for inhibitor-binding and molecular-imprinting, which has been applied in some efficient examples of sensor development, the aim of this work was to examine the effects of the free-thiol-group’s reversible blocking on papain denaturation that is the basis of its activity loss and aggregation. To utilize biophysical methods common in protein structural transitions characterization, such as fluorimetry and high-resolution infrared spectroscopy, low-molecular-weight electrophilic thiol blocking reagent S-Methyl methanethiosulfonate (MMTS) was used in solution. MMTS binding led to a two-fold increase in 8-Anilinonaphthalene-1-sulfonic acid fluorescence, indicating increased hydrophobic residue exposure. A more in-depth analysis showed significant transitions on the secondary structure level upon MMTS binding, mostly characterized by the lowered content of α-helices and unordered structures (either for approximately one third), and the increase in aggregation-specific β-sheets (from 25 to 52%) in a dose-dependant manner. The recovery of this inhibited protein showed that reversibility of inhibition is accompanied by reversibility of protein denaturation. Nevertheless, a 100-fold molar excess of the inhibitor led to the incomplete recovery of proteolytic activity, which can be explained by irreversible denaturation. The structural stability of the C-terminal β-sheet rich domain of the papain-like cysteine protease family opens up an interesting possibility to use its foldamers as a strategy for sensor development and other multiple potential applications that rely on the great commercial value of papain-like cysteine proteases.

We performed molecular dynamics simulations of Reteplase in the presence of different excipients to study the stabilizing mechanisms and to identify the role of excipients during freeze drying. To simulate the freeze-drying process, we divided the process into five distinct steps: (i) protein–excipient formulations at room temperature, (ii) the ice-growth process, (iii)–(iv) the partially solvated and fully dried formulations, and (v) the reconstitution. Furthermore, coarse-grained (CG) simulations were employed to explore the protein-aggregation process in the presence of arginine. By using a coarse-grained representation, we could observe the collective behavior and interactions between protein molecules during the aggregation process. The CG simulations revealed that the presence of arginine prevented intermolecular interactions of the catalytic domain of Reteplase, thus reducing the aggregation propensity. This suggests that arginine played a stabilizing role by interacting with protein-specific regions. From the freeze-drying simulations, we could identify several protein-specific events: (i) collapse of the domain structure, (ii) recovery of the drying-induced damages during reconstitution, and (iii) stabilization of the local aggregation-prone region via direct interactions with excipients. Complementary to the simulations, we employed nanoDSF, size-exclusion chromatography, and CD spectroscopy to investigate the effect of the freeze-drying process on the protein structure and stability.

The importance of studying the structural stability of proteins is determined by the structure–function relationship. Protein stability is influenced by many factors among which are freeze–thaw and thermal stresses. The effect of trehalose, betaine, sorbitol and 2-hydroxypropyl-β-cyclodextrin (HPCD) on the stability and aggregation of bovine liver glutamate dehydrogenase (GDH) upon heating at 50 °C or freeze–thawing was studied by dynamic light scattering, differential scanning calorimetry, analytical ultracentrifugation and circular dichroism spectroscopy. A freeze–thaw cycle resulted in the complete loss of the secondary and tertiary structure, and aggregation of GDH. All the cosolutes suppressed freeze–thaw- and heat-induced aggregation of GDH and increased the protein thermal stability. The effective concentrations of the cosolutes during freeze–thawing were lower than during heating. Sorbitol exhibited the highest anti-aggregation activity under freeze–thaw stress, whereas the most effective agents stabilizing the tertiary structure of GDH were HPCD and betaine. HPCD and trehalose were the most effective agents suppressing GDH thermal aggregation. All the chemical chaperones stabilized various soluble oligomeric forms of GDH against both types of stress. The data on GDH were compared with the effects of the same cosolutes on glycogen phosphorylase b during thermal and freeze–thaw-induced aggregation. This research can find further application in biotechnology and pharmaceutics.

Spermatogenesis accompanied by self-renewal and differentiation of spermatogonia under complicated regulation is crucial for male fertility. Our previous study demonstrated that the loss of the B-lymphoma Mo-MLV insertion region 1 (BMI1) could cause male infertility and found a potential interaction between BMI1 and proline-rich protein 11 (PRR11); however, the specific co-regulatory effects of BMI1/PRR11 on spermatogonia maintenance remain unclear.The expression of PRR11 was downregulated in a mouse spermatogonia cell line (GC-1) via transfection with PRR11-siRNAs, and PRR11 knockdown was verified by real-time reverse transcriptase polymerase chain reaction (RT-qPCR). The proliferative activity of GC-1 cells was determined using the cell counting kit (CCK-8), colony formation, and 5-ethynyl-2-deoxyuridine (EdU) incorporation assay. A Transwell assay was performed to evaluate the effects of PRR11 on GC-1 cell migration. A terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to measure GC-1 cell apoptosis. Furthermore, co-immunoprecipitation, RT-qPCR, and western blot analyses were used for investigating the regulatory mechanisms involved in this regulation. It was found that downregulation of PRR11 could cause a marked inhibition of proliferation and migration and induced apoptosis in GC-1 cells. Moreover, silencing of PRR11 obviously led to a reduction in the BMI1 protein level. PRR11 was found to interact with BMII at the endogenous protein level. PRR11 knockdown produced a decrease in BMI1 protein stability via an increase in BMI1 ubiquitination after which derepression in the transcription of protein tyrosine phosphatase receptor type M (Ptprm) occurred. Importantly, knockdown of Ptprm in PRR11-deficient GC-1 cells led to a reversal of proliferation and migration of GC-1 cells.This study uncovered a novel mechanism by which PRR11 cooperated with BMI1 to facilitate GC-1 maintenance through targeting Ptprm. Our findings may provide a better understanding of the regulatory network in spermatogonia maintenance.

An essential step in engineering proteins and understanding disease-causing missense mutations is to accurately model protein stability changes when such mutations occur. Here, we developed a new sequence-based predictor for the

Extraordinarily stable protein and peptide structures are critically demanded in many applications. Typical approaches to enhance protein and peptide stability are strengthening certain interactions. Here, we develop a very different approach: stabilizing peptide structures through side-chain-locked knots. More specifically, a peptide core consists of a knot, which is prevented from unknotting and unfolding by large side chains of amino acids at knot boundaries. These side chains impose free energy barriers for unknotting. The free energy barriers are quantified using all-atom and coarse-grained simulations. The barriers become infinitely high for large side chains and tight knot cores, resulting in stable peptide structures, which never unfold unless one chemical bond is broken. The extraordinary stability is essentially kinetic stability. Our new approach lifts the thermodynamic restriction in designing peptide structures, provides extra freedom in selecting sequence and structural motifs that are thermodynamically unstable, and should expand the functionality of peptides. This work also provides a bottom-up understanding of how knotting enhances protein stability.

Leptin is a polypeptide hormone produced in the adipose tissue and governs many processes in the body. Recently, polymorphisms in the LEP gene revealed a significant change in body weight regulation, energy balance, food intake, and reproductive hormone secretion. This study considers its crucial role in the regulation of the economically important traits of sheep. Several computational tools, including SIFT, Predict SNP2, SNAP2, and PROVEAN, have been used to screen out the deleterious nsSNPs. Following the screening of 11 nsSNPs in the sheep genome, 5 nsSNPs, T86M (C → T), D98N (G → A), N136T (A → C), R142Q (G → A), and P157Q (C → A), were predicted to have a significant deleterious effect on the LEP protein function, leading to phenotypic difference. The analysis of proteins’ stability change due to amino acid substitution using the I-stable, SDM, and DynaMut consistently confirmed that three nsSNPs (T86M (C → T), D98N (G → A), and P157Q (C → A)) increased protein stability. It is suggested that these three nsSNPs may enhance the evolvability of LEP protein, which is vital for the evolutionary adaptation of sheep. Our findings demonstrate that the five nsSNPs reported in this study might be responsible for sheep’s structural and functional modifications of LEP protein. This is the first comprehensive report on the sheep LEP gene. It narrow downs the candidate nsSNPs for in vitro experiments to facilitate the development of reliable molecular markers for associated traits.

Increasing the temperature by just a few degrees may lead to structural perturbation or unfolding of the protein and consequent loss of function. The concepts of flexibility and rigidity are fundamental for understanding the relationships between function, structure and stability. Protein unfolding can often be triggered by thermal fluctuations with flexible residues usually on the protein surface. Therefore, identification and knowledge of the effect of modification to flexible regions in protein structures are required for efficient protein engineering and the rational design of thermally stable proteins. The most flexible regions in protein are loops, hence their rigidification is one of the effective strategies for increasing thermal stability. Directed evolution or rational design by computational prediction can also lead to the generation of thermally stable proteins. Computational protein design has been improved significantly in recent years and has successfully produced de novo stable backbone structures with optimized sequences and functions. This review discusses intramolecular and intermolecular interactions that determine the protein structure, and the strategies utilized in the mutagenesis of mesophilic proteins to stabilize and improve the functional characteristics of biocatalysts by describing efficient techniques and strategies to rigidify flexible loops at appropriate positions in the structure of the protein.

Nano/microparticles are widely used as vaccine adjuvants to improve antigen stability and enhance immune response. Conformational stability of a given protein was normally assessed using differential scanning calorimetry (DSC) for the optimization of formulation and for ensuring antigen stability in vaccine products. Here, a higher throughput version, namely the microtiter plate-based differential scanning fluorimetry (DSF) method was developed and optimized for assessing the protein thermal stability in the particulate adjuvant-adsorbed form. Using recombinant human papillomavirus (HPV) vaccine antigens, along with several model proteins, enhanced sensitivity and correlation to the well-established differential scanning calorimetry were demonstrated. Higher throughput and much smaller sample consumption (1/10 ∼ 1/20 of the amount needed as compared to DSC) make the plate-based DSF a method of choice for formulation development, particularly during the early developmental phase of a project where the sample amount is usually quite limited.

Genome-wide association studies in adults have identified variants in hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) and mitochondrial amidoxime reducing component 1 (MTARC1) as protective against nonalcoholic fatty liver disease (NAFLD). We aimed to test their association with pediatric NAFLD liver histology and investigate their function using metabolomics. A total of 1450 children (729 with NAFLD, 399 with liver histology) were genotyped for rs72613567T>TA in HSD17B13, rs2642438G>A in MTARC1, and rs738409C>G in patatin-like phospholipase domain-containing protein 3 (PNPLA3). Genotype-histology associations were tested using ordinal regression. Untargeted hepatic proteomics and plasma lipidomics were performed in a subset of children. We found rs72613567T>TA in HSD17B13 to be associated with lower odds of NAFLD diagnosis (odds ratio, 0.7; 95% confidence interval, 0.6-0.9) and a lower grade of portal inflammation (p < 0.001). rs2642438G>A in MTARC1 was associated with a lower grade of hepatic steatosis (p = 0.02). Proteomics found reduced expression of HSD17B13 in carriers of the protective -TA allele. MTARC1 levels were unaffected by genotype. Both variants were associated with down-regulation of fibrogenic pathways. HSD17B13 perturbs plasma phosphatidylcholines and triglycerides. In silico modeling suggested p.Ala165Thr disrupts the stability and metal binding of MTARC1. Conclusion: Both HSD17B13 and MTARC1 variants are associated with less severe pediatric NAFLD. These results provide further evidence for shared genetic mechanisms between pediatric and adult NAFLD.

Motivation Prediction of protein stability change upon mutation (ΔΔG) is crucial for facilitating protein engineering and understanding of protein folding principles. Robust prediction of protein folding free energy change requires the knowledge of protein three-dimensional (3D) structure. In case, protein 3D structure is not available, one can predict the structure from protein sequence; however, the perspectives of ΔΔG predictions for predicted protein structures are unknown. The accuracy of using 3D structures of the best templates for the ΔΔG prediction is also unclear. Results To investigate these questions, we used a representative set of seven diverse and accurate publicly available tools (FoldX, Eris, Rosetta, DDGun, ACDC-NN, ThermoNet and DynaMut) for stability change prediction combined with AlphaFold or I-Tasser for protein 3D structure prediction. We found that best templates perform consistently better than (or similar to) homology models for all ΔΔG predictors. Our findings imply using the best template structure for the prediction of protein stability change upon mutation if the protein 3D structure is not available. Availability and implementation The data are available at https://github.com/ivankovlab/template-vs-model. Supplementary information Supplementary data are available at Bioinformatics online.

Enzymes play a powerful role as catalysts with high specificity and activity under mild environmental conditions. Significant hurdles, such as reduced solubility, reduced shelf-life, aggregate formation, and toxicity, are still ongoing struggles that scientists come across when purifying recombinant proteins. Over the past three decades, PEGylation techniques have been utilized to significantly overcome low solubility; increased protein stability, shelf-life, and bioactivity; and prevented protein aggregate formation. This review seeks to highlight the impact of PEG-based formulations that are significantly utilized to obtain favourable protein physiochemical properties. The authors further discuss other techniques that can be employed such as coexpression studies and nanotechnology-based skills to obtaining favourable protein physiochemical properties.

Background: The analysis of biofluid samples with low protein content (e.g., urine or saliva) can be challenging for downstream analysis methods with limited sensitivity. To circumvent this problem, sample processing methods are employed to increase the protein concentration in analyzed samples. However, for some techniques, like differential scanning calorimetry (DSC) that characterizes thermally-induced unfolding of biomolecules, sample processing must not affect native protein structure and stability. Methods: We evaluated centrifugal concentration and stirred cell ultrafiltration, two common methods of sample concentration characterized by a low risk of protein denaturation, with the goal of establishing a protocol for DSC analysis of low concentration biospecimens. Results: Our studies indicate that both methods can affect protein stability assessed by DSC and, even after optimization of several parameters, the obtained DSC profile (thermogram) suggested that sample processing affects the structure or intermolecular interactions of component proteins contributing to altered thermal stability detectable by DSC. We also found a relationship between changes in thermograms and low protein concentration, indicating that diluting biospecimens to concentrations below 0.1 mg/mL can perturb the intermolecular environment and affect the structure of proteins present in the solution. Conclusions: Dilution of samples below 0.1 mg/mL, as well as concentration of samples with low protein content, resulted in affected thermogram shapes suggesting changes in protein stability. This should be taken into account when concentrating dilute samples or employing techniques that lower the protein concentration (e.g., fractionation), when downstream applications include techniques, such as DSC, that require the preservation of native protein forms.

The extracellular-signal-regulated kinase 2 (ERK2), a mitogen-activated protein kinase (MAPK) located downstream of the Ras-Raf-MEK-ERK signal transduction cascade, is involved in the regulation of a large variety of cellular processes. The ERK2, activated by phosphorylation, is the principal effector of a central signaling cascade that converts extracellular stimuli into cells. Deregulation of the ERK2 signaling pathway is related to many human diseases, including cancer. This study reports a comprehensive biophysical analysis of structural, function, and stability data of pure, recombinant human non-phosphorylated (NP-) and phosphorylated (P-) ERK2 wild-type and missense variants in the common docking site (CD-site) found in cancer tissues. Because the CD-site is involved in interaction with protein substrates and regulators, a biophysical characterization of missense variants adds information about the impact of point mutations on the ERK2 structure–function relationship. Most of the P-ERK2 variants in the CD-site display a reduced catalytic efficiency, and for the P-ERK2 D321E, D321N, D321V and E322K, changes in thermodynamic stability are observed. The thermal stability of NP-ERK2 and P-ERK2 D321E, D321G, and E322K is decreased with respect to the wild-type. In general, a single residue mutation in the CD-site may lead to structural local changes that reflects in alterations in the global ERK2 stability and catalysis.

Background: Radiotherapy constitutes an important therapeutic option for prostate cancer. However, prostate cancer cells often acquire resistance during cancer progression, limiting the cytotoxic effects of radiotherapy. Among factors regulating sensitivity to radiotherapy are members of the Bcl-2 protein family, known to regulate apoptosis at the mitochondrial level. Here, we analyzed the role of anti-apoptotic Mcl-1 and USP9x, a deubiquitinase stabilizing Mcl-1 protein levels, in prostate cancer progression and response to radiotherapy. Methods: Changes in Mcl-1 and USP9x levels during prostate cancer progression were determined by immunohistochemistry. Neutralization of Mcl-1 and USP9x was achieved by siRNA-mediated knockdown. We analyzed Mcl-1 stability after translational inhibition by cycloheximide. Cell death was determined by flow cytometry using an exclusion assay of mitochondrial membrane potential-sensitive dye. Changes in the clonogenic potential were examined by colony formation assay. Results: Protein levels of Mcl-1 and USP9x increased during prostate cancer progression, and high protein levels correlated with advanced prostate cancer stages. The stability of Mcl-1 reflected Mcl-1 protein levels in LNCaP and PC3 prostate cancer cells. Moreover, radiotherapy itself affected Mcl-1 protein turnover in prostate cancer cells. Particularly in LNCaP cells, the knockdown of USP9x expression reduced Mcl-1 protein levels and increased sensitivity to radiotherapy. Conclusion: Posttranslational regulation of protein stability was often responsible for high protein levels of Mcl-1. Moreover, we demonstrated that deubiquitinase USP9x as a factor regulating Mcl-1 levels in prostate cancer cells, thus limiting cytotoxic response to radiotherapy.

The evolutionary and functional studies suggested that the emergence of the Omicron variants can be determined by multiple fitness trade-offs including the immune escape, binding affinity, conformational plasticity, protein stability and allosteric modulation. In this study, we embarked on a systematic comparative analysis of the conformational dynamics, electrostatics, protein stability and allostery in the different functional states of spike trimers for BA.1, BA.2, and BA.2.75 variants. Using efficient and accurate coarse-grained simulations and atomistic reconstruction of the ensembles, we examined conformational dynamics of the spike trimers that agrees with the recent functional studies, suggesting that BA.2.75 trimers are the most stable among these variants. A systematic mutational scanning of the inter-protomer interfaces in the spike trimers revealed a group of conserved structural stability hotspots that play a key role in modulation of functional dynamics and are also involved in the inter-protomer couplings through local contacts and interaction networks with the Omicron mutational sites. The results of mutational scanning provided evidence that BA.2.75 trimers are more stable than BA.2 and comparable in stability to BA.1 variant. Using dynamic network modeling of the S Omicron BA.1, BA.2 and BA.2.75 trimers we showed that the key network positions driving long-range signaling are associated with the major stability hotspots that are inter-connected along potential communication pathways, while sites of Omicron mutations may often correspond to weak spots of stability and allostery but are coupled to the major stability hotspots through interaction networks. The presented analysis of the BA.1, BA.2 and BA.2.75 trimers suggested that thermodynamic stability of BA.1 and BA.2.75 variants may be intimately linked with the residue interaction network organization that allows for a broad ensemble of allosteric communications in which signaling between structural stability hotspots may be modulated by the Omicron mutational sites. The findings provided plausible rationale for mechanisms in which Omicron mutations can evolve to balance thermodynamic stability and conformational adaptability in order to ensure proper tradeoff between stability, binding and immune escape.

Most cases of hepatocellular carcinoma (HCC) arise with the fibrotic microenvironment where hepatic stellate cells (HSCs) and carcinoma-associated fibroblasts (CAFs) are critical components in HCC progression. Therefore, CAF normalization could be a feasible therapy for HCC. Galectin-1 (Gal-1), a β-galactoside-binding lectin, is critical for HSC activation and liver fibrosis. However, few studies has evaluated the pathological role of Gal-1 in HCC stroma and its role in hepatic CAF is unclear. Here we showed that Gal-1 mainly expressed in HCC stroma, but not cancer cells. High expression of Gal-1 is correlated with CAF markers and poor prognoses of HCC patients. In co-culture systems, targeting Gal-1 in CAFs or HSCs, using small hairpin (sh)RNAs or an therapeutic inhibitor (LLS30), downregulated plasminogen activator inhibitor-2 (PAI-2) production which suppressed cancer stem-like cell properties and invasion ability of HCC in a paracrine manner. The Gal-1-targeting effect was mediated by increased a disintegrin and metalloprotease 17 (ADAM17)-dependent TNF-receptor 1 (TNFR1) shedding/cleavage which inhibited the TNF-α → JNK → c-Jun/ATF2 signaling axis of pro-inflammatory gene transcription. Silencing Gal-1 in CAFs inhibited CAF-augmented HCC progression and reprogrammed the CAF-mediated inflammatory responses in a co-injection xenograft model. Taken together, the findings uncover a crucial role of Gal-1 in CAFs that orchestrates an inflammatory CSC niche supporting HCC progression and demonstrate that targeting Gal-1 could be a potential therapy for fibrosis-related HCC.

Submitted: 23.01.2022, Revision Requested: 17.02.2022, Last Revision Received: 19.02.2022, Accepted: 21.03.2022, Published Online: 13.04.2022. Content of this journal is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. Objective: Coronavirus Disease 2019 (COVID19) is a viral disease caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2). The high mutation propensity of the SARS CoV-2 genome is one of the biggest threats to the long-term validity of treatment options. Helicases are anti-viral targets because of the vital role they play in the viral life cycle. In this study, changes in the protein structure caused by SARS CoV-2 nsp13 mutations were investigated to contribute to the development of effective antiviral drugs. Materials and Methods: Genome data of 298 individuals located in the China location were examined. The mutant model was built using deep learning algorithms. Model quality assessment was done with QMEAN. Protein stability analyses were performed with DynaMut2 and Cutoff Scanning Matrix stability. Changes in substrate affinity were performed with Haddock v2.4. Results: In this study, twenty-eight mutations in nsp13 were identified (23 sense, 5 missense). The changes in protein structure caused by the five missense mutations (Leu14Phe, Arg15Ser, Arg21Ser, Leu235Phe, Ala454Thr) were modeled. The mutations caused a decrease in the stability of SARS CoV-2 helicase (-0.99, -1.66, -1.15, -0.54, and -0.73 for Leu14Phe, Arg15Ser, Arg21Ser, Leu235Phe, Ala454Thr, respectively). The mutations reduced the helicase's affinity to the substrate. The docking scores for wild-type and mutant helicase were -84.4±1.4 kcal.mol-1 and -71.1±6.7 kcal.mol-1, respectively. Conclusion: Helicase mutations caused a decrease in the protein stability and nucleic acid affinity of the SARS CoV-2 helicase. The results provide important data on the development of potential antivirals and the effect of mutation on the functions of viral proteins.

Changes in protein stability due to side-chain mutations are evaluated by alchemical free-energy calculations based on classical molecular dynamics (MD) simulations in explicit solvent using the GROMOS force field. Three proteins of different complexity with a total number of 93 single-point mutations are analyzed, and the relative free-energy differences are discussed with respect to configurational sampling and (dis)agreement with experimental data. For the smallest protein studied, a 34-residue WW domain, the starting structure dependence of the alchemical free-energy changes, is discussed in detail. Deviations from previous simulations for the two other proteins are shown to result from insufficient sampling in the earlier studies. Hamiltonian replica exchange in combination with multiple starting structures and sufficient sampling time of more than 100 ns per intermediate alchemical state is required in some cases to reach convergence.

Background OTULIN-related autoinflammatory syndrome (ORAS) is an autosomal recessive disease characterized by systemic inflammation, recurrent fever. Due to limited knowledge about the OTULIN DNA variants that cause ORAS, the diagnosis and treatment of this disease is difficult. In this study, we aim to identify OTULIN DNA variants responsible for the genetic pathology of ORAS and observe the effects of these variants on the OTULIN protein structure and the function with different bioinformatics approaches. Methods The present study included 3230 individuals with the suspicion of an autoinflammatory disease who were referred to Ege University Children's Hospital Molecular Medicine Laboratory. OTULIN variants were detected using a panel consisting of 37 different autoinflammatory diseases (AID) genes via targeted Next-Generation Sequencing. Results As a result of the study, DNA variants associated with various AID were detected in 65% of the individuals to whom the panel was applied. Among these variants, only three different OTULIN variants (p.Val82Ile, p.Gln115His and p.Leu131_Arg132insLeuCysThrGlu) were detected. The pathogenic effects of the variants detected in the OTULIN gene were determined by using Polyphen2 as Probably Pathogenic for the p.Val82Ile and benign for the p.Gln115His. At the same time, the effects of these variants on the structure and function of the OTULIN protein were investigated by in silico approaches. Both variants reduce protein stability and binding affinity. Conclusion The results of the current study suggest that the evaluation of OTULIN variants with in silico approaches will contribute to the development of personalized treatments by diagnosing the disease specific to the variant.

Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs), superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between partially folded and unfolded conformations. We rationalize the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the center toward both termini simultaneously. Most strikingly, we also directly observe the protein's superhelical tertiary structure in the force signal. Using protein engineering, crystallography, and single-molecule experiments, we show that the superhelical geometry can be altered by carefully placed amino acid substitutions, and we examine how these sequence changes affect intrinsic repeat stability and inter-repeat coupling. Our findings provide the means to dissect and modulate repeat-protein stability and dynamics, which will be essential for researchers to understand the function of natural repeat proteins and to exploit artificial repeats proteins in nanotechnology and biomedical applications.

Ras family proteins are membrane-bound GTPases that control proliferation, survival, and motility. Many forms of cancers are driven by the acquisition of somatic mutations in a RAS gene. In pancreatic cancer (PC), more than 90% of tumors carry an activating mutation in KRAS. Mutations in components of the Ras signaling pathway can also be the cause of RASopathies, a group of developmental disorders. In a subset of RASopathies, the causal mutations are in the LZTR1 protein, a substrate adaptor for E3 ubiquitin ligases that promote the degradation of Ras proteins. Here, we show that the function of LZTR1 is regulated by the glycogen synthase kinase 3 (GSK3). In PC cells, inhibiting or silencing GSK3 led to a decline in the level of Ras proteins, including both wild type Ras proteins and the oncogenic Kras protein. This decline was accompanied by a 3-fold decrease in the half-life of Ras proteins and was blocked by the inhibition of the proteasome or the knockdown of LZTR1. Irrespective of the mutational status of KRAS, the decline in Ras proteins was observed and accompanied by a loss of cell proliferation. This loss of proliferation was blocked by the knockdown of LZTR1 and could be recapitulated by the silencing of either KRAS or GSK3. These results reveal a novel GSK3-regulated LZTR1-dependent mechanism that controls the stability of Ras proteins and proliferation of PC cells. The significance of this novel pathway to Ras signaling and its contribution to the therapeutic properties of GSK3 inhibitors are both discussed.

In the process of structure-function studies on the class II molecule expressed in autoimmunity prone SJL mice, I-A(s), we discovered a disparity from the reported sequence of the MHC class II beta chain. The variant is localized at a highly conserved site of the beta chain, at residue 58. Our studies revealed that this single amino acid substitution of Pro for Ala at this residue, found in I-A(s), changes the structure of the MHC class II molecule, as evidenced by a loss of recognition by two monoclonal antibodies, and elements of MHC class II conformational stability identified through molecular dynamics simulation. Two other rare polymorphisms in I-A(s) involved in hydrogen bonding potential between the alpha chain and the peptide main chain are located at the same end of the MHC class II binding pocket, studied in parallel may impact the consequences of the β chain variant. Despite striking changes in MHC class II structure, CD4 T cell recognition of influenza-derived peptides was preserved. These disparate findings were reconciled by discovering, through monoclonal antibody blocking approaches, that CD4 T cell recognition by I-A(s) restricted CD4 T cells focused more on the region of MHC class II at the peptide’s amino terminus. These studies argue that the conformational variability or flexibility of the MHC class II molecule in that region of I-A(s) select a CD4 T cell repertoire that deviates from the prototypical docking mode onto MHC class II peptide complexes. Overall, our results are consistent with the view that naturally occurring MHC class II molecules can possess polymorphisms that destabilize prototypical features of the MHC class II molecule but that can maintain T cell recognition of the MHC class II:peptide ligand via alternate docking modes

Many types of cancer feature TP53 mutations with oncogenic properties. However, whether the oncogenic activity of mutant p53 is affected by the cellular metabolic state is unknown. Here we show that cancer-associated mutant p53 protein is stabilized by 2-hydroxyglutarate generated by malic enzyme 2. Mechanistically, malic enzyme 2 promotes the production of 2-hydroxyglutarate by adjusting glutaminolysis, as well as through a reaction that requires pyruvate and NADPH. Malic enzyme 2 depletion decreases cellular 2-hydroxyglutarate levels in vitro and in vivo, whereas elevated malic enzyme 2 expression increases 2-hydroxyglutarate production. We further show that 2-hydroxyglutarate binds directly to mutant p53, which reduces Mdm2-mediated mutant p53 ubiquitination and degradation. 2-Hydroxyglutarate supplementation is sufficient for maintaining mutant p53 protein stability in malic enzyme 2-depleted cells, and restores tumour growth of malic enzyme 2-ablated cells, but not of cells that lack mutant p53. Our findings reveal the previously unrecognized versatility of malic enzyme 2 catalytic functions, and uncover a role for mutant p53 in sensing cellular 2-hydroxyglutarate levels, which contribute to the stabilization of mutant p53 and tumour growth.

The intermolecular interactions of noble gases in biological systems are associated with numerous biochemical responses, including apoptosis, inflammation, anesthesia, analgesia, and neuroprotection. The molecular modes of action underlying these responses are largely unknown. This is in large part due to the limited experimental techniques to study protein-gas interactions. The few techniques that are amenable to such studies are relatively low throughput and require large amounts of purified proteins. Thus, they do not enable the large-scale analyses that are useful for protein-target discovery. Here we report the application of Stability of Proteins from Rates of Oxidation (SPROX) and limited proteolysis (LiP) methodologies to detect protein-xenon interactions on the proteomic scale using protein folding stability measurements. Over 5,000 methionine-containing peptides and over 5,000 semi-tryptic peptides, mapping to ~1,500 and ~950 proteins, respectively, in the yeast proteome, were assayed for Xe-interacting activity using the SPROX and LiP techniques. The SPROX and LiP analyses identified 31 and 60 Xe-interacting proteins, respectively, none of which were previously known to bind Xe. A bioinformatics analysis of the proteomic results revealed that these Xe-interacting proteins were enriched in those involved in ATP-driven processes. A fraction of the protein targets that were identified is tied to previously established modes of action related to xenon’s anesthetic and organoprotective properties. These results enrich our knowledge and understanding of biologically relevant xenon interactions. The sample preparation protocols and analytical methodologies developed here for xenon are also generally applicable to the discovery of a wide range of other protein-gas interactions in complex biological mixtures, such as cell lysates.

In Arabidopsis, the differentiation of epidermal cells into stomata is regulated by endogenous and environmental signals. Sugar is required for plant epidermal cell proliferation and differentiation. However, it is unclear how epidermal cells maintain division and differentiation to generate proper amounts of stomata in response to different sugar availability. Here, we show that two evolutionarily conserved kinase Snf1-related protein kinase 1 (SnRK1) and Target of rapamycin (TOR) play critical roles in the regulation of stomatal development under different sugar availability. When plants are grown on a medium containing 1% sucrose, sucrose-activated TOR promotes the stomatal development by inducing the expression of SPEECHLESS (SPCH), a master regulator of stomatal development. SnRK1 promotes stomatal development through phosphorylating and stabilizing SPCH. However, under the high sucrose conditions, the highly accumulated trehalose-6-phosphate (Tre6P) represses the activity of KIN10, the catalytic α-subunit of SnRK1, by reducing the interaction between KIN10 and its upstream kinase, consequently promoting SPCH degradation and inhibiting stomatal development. Our findings revealed that TOR and SnRK1 finely regulate SPCH expression and protein stability to optimize the stomatal development in response to exogenously supplied sugar.

Monoclonal antibodies are the fastest growing class of therapeutics. However, aggregation limits their shelf life and can lead to adverse immune responses. Assessment and optimization of the long-term antibody stability are therefore key challenges in the biologic drug development. Here, we present a platform based on the analysis of temperature-dependent aggregation data that can dramatically shorten the assessment of the long-term aggregation stability and thus accelerate the optimization of antibody formulations. For a set of antibodies used in the therapeutic areas from oncology to rheumatology and osteoporosis, we obtain an accurate prediction of aggregate fractions for up to three years using the data obtained on a much shorter time scale. Significantly, the strategy combining kinetic and thermodynamic analysis not only contributes to a better understanding of the molecular mechanisms of antibody aggregation but has already proven to be very effective in the development and production of biological therapeutics.

The androgen receptor (AR) plays a central role in prostate, muscle, bone and adipose tissue. Moreover, dysregulated AR activity is a driving force in prostate cancer (PCa) initiation and progression. Consequently, antagonizing AR signalling cascades via antiandrogenic therapy is a crucial treatment option in PCa management. Besides, very high androgen levels also inhibit PCa cells’ growth, so this effect could also be applied in PCa therapy. However, on the molecular and cellular level, these mechanisms have hardly been investigated so far. Therefore, the present study describes the effects of varying androgen concentrations on the viability of PCa cells as well as localization, transactivation, and protein stability of the AR. For this purpose, cell viability was determined via WST1 assay. Alterations in AR transactivity were detected by qPCR analysis of AR target genes. A fluorescent AR fusion protein was used to analyse AR localization microscopically. Changes in AR protein expression were detected by Western blot. Our results showed that high androgen concentrations reduce the cell viability in LNCaP and C4-2 cell lines. In addition, androgens have been reported to increase AR transactivity, AR localization, and AR protein expression levels. However, high androgen levels did not reduce these parameters. Furthermore, this study revealed an androgen-induced increase in AR protein synthesis. In conclusion, inhibitory effects on cell viability by high androgen levels are due to AR downstream signalling or non-genomic AR activity. Moreover, hormonal activation of the AR leads to a self-induced stabilization of the receptor, resulting in increased AR activity. Therefore, in clinical use, a therapeutic reduction in androgen levels represents a clinical target and would lead to a decrease in AR activity and, thus, AR-driven PCa progression.

ATP-binding cassette (ABC) proteins play important roles in cells as importers and exporters but as membrane proteins they are subject to well-known challenges of isolating pure and stable samples for study. One solution to this problem is to use styrene-maleic acid lipid particles (SMALPs). Styrene-maleic acid (SMA) can be added directly to membranes, forming stable nanoparticles incorporating membrane proteins and lipids. Here we use Sav1866, a well-characterised bacterial protein, as a proxy for ABC proteins in general. We show that stable and monodispersed Sav1866 can be purified at high yield using SMA. This protein can be used for biophysical characterisations showing that its overall structure is consistent with existing evidence. However, like other ABC proteins in SMALPs it does not hydrolyse ATP. The lack of ATPase activity in ABC–SMALPs may result from conformational trapping of the proteins in SMALPs. Undertaken in a controlled manner, conformational trapping is a useful tool to stabilise protein samples into a single conformation for structural studies. Due to their inability to hydrolyse ATP, the conformation of Sav1866–SMALPs cannot be altered using ATP and vanadate after purification. To achieve controlled trapping of Sav1866–SMALPs we show that Sav1866 in crude membranes can be incubated with ATP, magnesium and sodium orthovanadate. Subsequent solubilisation and purification with SMA produces a sample of Sav1866–SMALPs with enhanced stability, and in a single conformational state. This method may be generally applicable to vanadate-sensitive ABC proteins and overcomes a limitation of the SMALP system for the study of this protein family.

Accurate prediction of protein stability changes upon mutation (ΔΔG) is increasingly important to evolution studies, protein engineering, and screening of disease-causing gene variants but is challenged by biases in training data. We investigated 45 linear regression models trained on data sets that account systematically for destabilization bias and mutation-type bias BM. The models were externally validated on three test data sets probing different pathologies and for internal consistency (symmetry and neutrality). Model structure and performance substantially depended on training data and even fitting method. We developed two final models: SimBa-IB for typical natural mutations and SimBa-SYM for situations where stabilizing and destabilizing mutations occur to a similar extent. SimBa-SYM, despite is simplicity, is essentially non-biased (vs. the Ssym data set) while still performing well for all data sets (R ~ 0.46-0.54, MAE = 1.16-1.24 kcal/mol). The simple models provide advantage in terms of interpretability, use and future improvement, and are freely available on GitHub.

Human glutathione peroxidase1 (hGPx1) is a good antioxidant and potential drug, but the limited availability and poor stability of hGPx1 have affected its development and application. To solve this problem, we prepared a hGPx1 mutant (GPx1M) with high activity in an Escherichia coli BL21(DE3)cys auxotrophic strain using a single protein production (SPP) system. In this study, the GPx1M was conjugated with methoxypolyethylene glycol-succinimidyl succinate (SS-mPEG, Mw = 5 kDa) chains to enhance its stability. SS-mPEG-GPx1M and GPx1M exhibited similar enzymatic activity and stability toward pH and temperature change, and in a few cases, SS-mPEG-GPx1M was discovered to widen the range of pH stability and increase the temperature stability. Lys 38 was confirmed as PEGylated site by liquid-mass spectrometry. H9c2 cardiomyoblast cells and Sprague-Dawley (SD) rats were used to evaluate the effects of GPx1M and SS-mPEG-GPx1M on preventing or alleviating adriamycin (ADR)-mediated cardiotoxicity, respectively. The results indicated that GPx1M and SS-mPEG-GPx1M had good antioxidant effects in vitro and in vivo, and the effect of SS-mPEG-GPx1M is more prominent than GPx1M in vivo. Thus, PEGylation might be a promising method for the application of GPx1M as an important antioxidant and potential drug.

BRCA1 is frequently down-regulated in breast cancer, the underlying mechanism is unclear. Here we identified DCAF8L1, an X-linked gene product, as a DDB1-Cullin associated Factor (DCAF) for CUL4 E3 ligases to target BRCA1 and BARD1 for proteasomal degradation. Forced expression of DCAF8L1 caused reduction of BRCA1 and BARD1, and impaired DNA damage repair function, conferring increased sensitivity to irradiation and DNA damaging agents, as well as Olaparib, a PARPi anticancer drug; while depletion of DCAF8L1 restored BRCA1 and suppressed the growth of its xenograft tumors. Furthermore, the expression of DCAF8L1 was induced in human H9 ES cells during transition from primed to naïve state when Xi chromosome was reactivated. Aberrant expression of DCAF8L1 was observed in human breast fibroadenoma and breast cancer. These findings suggest that CRL4

Target residence time plays a crucial role in the pharmacological activity of small molecule inhibitors. Little is known, however, about the underlying causes of inhibitor residence time at the molecular level, which complicates drug optimization processes. Here, we employ all-atom molecular dynamics simulations (~400 μs in total) to gain insight into the binding modes of two structurally similar p38α MAPK inhibitors (type I and type I½) with short and long residence times that otherwise show nearly identical inhibitory activities in the low nanomolar IC50 range. Our results highlight the importance of protein conformational stability and solvent exposure, buried surface area of the ligand and binding site resolvation energy for residence time. These findings are further confirmed by simulations with a structurally diverse short residence time inhibitor SB203580. In summary, our data provide guidance in compound design when aiming for inhibitors with improved target residence time., The molecular determinants of the residence time of a small molecule inhibitor at its target protein are not well understood. Here, Pantsar et al. show that the target protein’s conformational stability and solvent exposure are key factors governing the target residence time of kinase inhibitors.

Congenital cataract, a common disease with lens opacification, causes blindness in the newborn worldwide and is mainly caused by abnormal aggregation of crystallin. As the main structural protein in the mammalian lens, βB1-crystallin has an important role in the maintenance of lens transparency. Recently, the L116P mutation in βB1-CRY was found in a Chinese family with congenital nuclear cataracts, while its underlying pathogenic mechanism remains unclear. In the current study, the βB1 wild-type protein was purified, and the mutated form, βB1-L116P, was examined for examining the effect on structural stability and susceptibility against environmental stresses. Our results reveal low solubility and structural stability of βB1-L116P at physiological temperature, which markedly impaired the protein structure and the oligomerization of βB1-crystallin. Under guanidine hydrochloride-induced denaturing conditions, βB1-L116P mutation perturbed the protein unfolding process, making it prone to amyloid fibrils aggregation. More importantly, the L116P mutation increased susceptibility of βB1-crystallin against UV radiation. βB1-L116P overexpression led to the formation of more serious intracellular aggresomes under UV radiation or oxidative stress. Furthermore, the βB1-L116P mutation increased the sensitivity to the proteolysis process. These results indicate that the low structural stability, susceptibility to amyloid fibrils aggregation, and protease degradation of βB1-L116P may contribute to cataract development and associated symptoms.

The mitogen-activated protein kinase OsMPK1 is involved in abscisic acid (ABA) biosynthesis in rice (Oryza sativa L.). However, the underlying molecular mechanisms of OsMPK1 in regulating ABA biosynthesis are poorly understood. Here, by using yeast two-hybrid assay and firefly luciferase complementary imaging assay, we show that OsMPK1 physically interact with a short-chain dehydrogenase protein OsABA2. However, OsMPK5, a homolog of OsMPK1, does not interact with OsABA2. Further, OsMPK1 can phosphorylate OsABA2S197 in vitro. Phosphorylation at the position of OsABA2S197 does not affect its subcellular localization, but enhances the stability of OsABA2 protein. We also found that OsABA2 has feedback regulation on OsMPK1 kinase activity. Further research reveals that OsMPK1 and OsABA2 coordinately regulate the biosynthesis of ABA, and phosphorylation of OsABA2 at Ser197 by OsMPK1 plays a crucial role in regulating the biosynthesis of ABA. Finally, genetic analysis showed that OsABA2 can enhance the sensitivity of rice to ABA and the tolerance of rice to drought and salt stress.

Hyaluronic acid (HA) is now widely known for its ability to bind water and impart texture. The combined effects of HA and kappa-carrageenan (KC) have not yet been investigated, though. In this study, we looked at the synergistic effects of HA and KC (concentrations of 0.1 and 0.25%, and ratios of 85:15, 70:30, and 50:50 for each concentration) on the rheological properties, heat stability, protein phase separation, water-holding capacity, emulsification properties, and foaming properties of skim milk. When HA and KC were combined in various ratios with a skim milk sample, this resulted in lesser protein phase separation and a higher water-holding capacity than when HA and KC were utilized separately. Similarly, for the sample with a 0.1% concentration, the combination of HA + KC blends demonstrated a synergistic impact with greater emulsifying activity and stability. The samples with a concentration of 0.25% did not exhibit this synergistic effect, and the emulsifying activity and stability were mostly due to the HA’s higher emulsifying activity and stability at 0.25% concentration. Similarly, for rheological (apparent viscosity, consistency coefficient K, and flow behavior index n) and foaming properties, the synergistic effect of the HA + KC blend was not readily apparent; rather, these values were mostly due to an increase in the amount of KC in the HA + KC blend ratios. When HC-control and KC-control samples were compared to various HA + KC mix ratios, there was no discernible difference in the heat stability. With the added benefits of protein stability (reduced phase separation), increased water-holding capacity, improved emulsification capabilities, and foaming abilities, the combination of HA + KC would be highly helpful in many texture-modifying applications.

The circadian clock is a timekeeping, homeostatic system that temporally coordinates all major cellular processes. The function of the circadian clock is compensated in the face of variable environmental conditions ranging from normal to stress-inducing conditions. Salinity is a critical environmental factor affecting plant growth, and plants have evolved the SALT OVERLY SENSITIVE (SOS) pathway to acquire halotolerance. However, the regulatory systems for clock compensation under salinity are unclear. Here, we show that the plasma membrane Na

Alphaviruses can cause severe human arthritis and encephalitis. During virus infection, structural changes of viral glycoproteins in the acidified endosome trigger virus–host membrane fusion for delivery of the capsid core and RNA genome into the cytosol to initiate virus translation and replication. However, mechanisms by which E1 and E2 glycoproteins rearrange in this process remain unknown. Here, we investigate prefusion cryoelectron microscopy (cryo-EM) structures of eastern equine encephalitis virus (EEEV) under acidic conditions. With models fitted into the low-pH cryo-EM maps, we suggest that E2 dissociates from E1, accompanied by a rotation (∼60°) of the E2-B domain (E2-B) to expose E1 fusion loops. Cryo-EM reconstructions of EEEV bound to a protective antibody at acidic and neutral pH suggest that stabilization of E2-B prevents dissociation of E2 from E1. These findings reveal conformational changes of the glycoprotein spikes in the acidified host endosome. Stabilization of E2-B may provide a strategy for antiviral agent development.

The TIM barrel is one of the oldest and most ubiquitous protein folds and is often referred to as the most successful in nature, due to its capability to host a diverse set of functions. This specific protein fold is characterized by a highly ordered topology with a central, eight-stranded, parallel β-barrel surrounded by eight α-helices. Interestingly, sequence analysis of natural TIM barrels revealed a low sequence conservation in this protein family despite a high structural similarity. Due to this high versatility in sequence and function the TIM barrel is a highly interesting target for protein design. The first validated de novo TIM barrel sTIM11 features a minimalistic structure of this protein fold. Due to its idealized topology it is an excellent system to study the folding determinants of the TIM-barrel fold and can also be used to create tailormade enzymes. This work aims to improve this designed protein and create a set of diverse de novo TIM barrels. In the first part of this thesis, the original sTIM11 was stabilized by two different strategies. In a first approach, the hydrophobic packing of sTIM11 was improved by applying a fixed-backbone design strategy. In a modular approach initial stabilizing mutations in different regions of the barrel were identified and subsequently combined. This led to the construction of a large set of DeNovoTIMs with significantly improved stabilities and where crystal structures verified the formation of improved hydrophobic clusters. In a second approach, inspired by natural TIM barrels, a salt bridge network was installed in the β-barrel of three different de novo TIM barrels. Analysis of these salt bridge cluster variants revealed highly stabilizing, but also destabilizing effects. Structural analysis verified the formation of salt bridges but with various geometries ranging from single pair interactions to completely formed salt bridge networks. This highlights the challenges of designing salt bridges and especially salt bridge clusters. Even though all three analysed proteins have a highly similar fold, the influence on stability as well as the geometric formation of the salt bridges can vary significantly. In the second part of this work, the de novo TIM barrel is further diversified by the introduction of coiled coils into its βα-loops. Due to its minimalistic design principle, sTIM11 lacks any larger surface areas or cavities which can be utilized for installation of binding or catalytic sites. Therefore, the introduction of additional structural elements is a first step towards the creation of functional de novo TIM barrels. Using a multistep design approach, a de novo designed antiparallel coiled coil was introduced into one and subsequently into a second βα-loop of the de novo TIM barrel creating a set of eight ccTIMs. Biochemical and biophysical analysis demonstrate the formation of additional α-helical elements with stabilizing interactions, which indicates a successful design. The research shown in this work created a large and diverse set of de novo TIM barrels which can be further utilized to build functional proteins and also to investigate folding determinants of TIM barrels.

Fibrillar aggregation is a promising route to enhance protein functionality for food, while prefibrillar oligomers in vivo are suspected to be pathogenic. Here, the role of intramolecular disulfide bonds in fibrillation (80 °C, pH 2) of the major whey protein β-lactoglobulin (BLG) was studied. Different cysteine-modified variants were used for this investigation, removing one or both disulfide bonds in BLG. Denaturation occurred upon heating at pH 2.0 up to 1 h, but residual structure elements prevented aggregation of intact bovine BLG. Heating for longer caused acidic hydrolysis, which resulted in the release of peptides with enhanced aggregation tendency. Partial destabilisation by removal of C66–C160 (by recombinant substitution) or both disulfide bonds (by chemical cleavage) was found to affect this hydrolysis rate, but only cleavage of both disulfide bonds accelerate the aggregation kinetics. No major impact on the fibrillar morphology was observed. In contrast, recombinant removal of both disulfide bonds led to complete structural disruption and instantaneous aggregation of intact BLG at pH 2 prior to heating. These small aggregates (10–50 nm) led to the formation of worm-like aggregates within 1 h of heating, which slowed down the acidic hydrolysis and inhibited further aggregation into fibrils. We concluded that fibrillation can be accelerated by structural destabilisation through disulfide bond cleavage, while complete destabilisation can actually hinder it. These insights can be used for the production of functional protein aggregates, and possibly for the avoidance of pathogenic fibrillation of proteins.

Posttranslational modifications of proteins can impact their therapeutic efficacy, stability, and potential for pharmaceutical development. The Group AStreptococcus pyogenesC5a peptidase (ScpA) is a multi-domain protein composed of an N-terminal signal peptide, a catalytic domain (including propeptide), three fibronectin domains, and cell membrane-associated domains. It is one of several proteins produced by Group AS. pyogenesknown to cleave components of the human complement system. After signal peptide removal, ScpA undergoes autoproteolysis and cleaves its propeptide for full maturation. The exact location and mechanism of the propeptide cleavage, and the impact of this cleavage on stability and activity, are not clearly understood, and the exact primary sequence of the final enzyme is not known. A form of ScpA with no autoproteolysis fragments of propeptide present may be more desirable for pharmaceutical development from a regulatory and a biocompatibility in the body perspective. The current study describes an in-depth structural and functional characterization of propeptide truncated variants of ScpA expressed inEscherichia colicells. All three purified ScpA variants, ScpA, 79ΔPro, and 92ΔPro, starting with N32, D79, and A92 positions, respectively, showed similar activity against C5a, which suggests a propeptide-independent activity profile of ScpA. CE-SDS and MALDI top-down sequencing analyses highlight a time-dependent propeptide autoproteolysis of ScpA at 37 °C with a distinct end point at A92 and/or D93. In comparison, all three variants of ScpA exhibit similar stability, melting temperatures, and secondary structure orientation. In summary, this work not only highlights propeptide localization but also provides a strategy to recombinantly produce a final mature and active form of ScpA without any propeptide-related fragments.

The use of transcriptomes as reliable proxies for cellular proteomes is controversial. In the small intestine, enterocytes operate for 4 days as they migrate along villi, which are highly graded microenvironments. Spatial transcriptomics have demonstrated profound zonation in enterocyte gene expression, but how this variability translates to protein content is unclear. Here we show that enterocyte proteins and messenger RNAs along the villus axis are zonated, yet often spatially discordant. Using spatial sorting with zonated surface markers, together with a Bayesian approach to infer protein translation and degradation rates from the combined spatial profiles, we find that, while many genes exhibit proteins zonated toward the villus tip, mRNA is zonated toward the villus bottom. Finally, we demonstrate that space-independent protein synthesis delays can explain many of the mRNA-protein discordances. Our work provides a proteomic spatial blueprint of the intestinal epithelium, highlighting the importance of protein measurements for inferring cell states in tissues that operate outside of steady state.