Affect Protein Stability Research Articles

Hofmeister effects on protein stability are dependent on the nature of the unfolded state.

The interpretation of a salt's effect on protein stability traditionally discriminates low concentration regimes (<0.3 M), dominated by electrostatic forces, and high concentration regimes, generally described by ion-specific Hofmeister effects. However, increased theoretical and experimental studies have highlighted observations of the Hofmeister phenomena at concentration ranges as low as 0.001 M. Reasonable quantitative predictions of such observations have been successfully achieved throughout the inclusion of ion dispersion forces in classical electrostatic theories. This molecular description is also on the basis of quantitative estimates obtained resorting to surface/bulk solvent partition models developed for ion-specific Hofmeister effects. However, the latter are limited by the availability of reliable structures representative of the unfolded state. Here, we use myoglobin as a model to explore how ion-dependency on the nature of the unfolded state affects protein stability, combining spectroscopic techniques with molecular dynamic simulations. To this end, the thermal and chemical stability of myoglobin was assessed in the presence of three different salts (NaCl, (NH4)2SO4 and Na2SO4), at physiologically relevant concentrations (0-0.3 M). We observed mild destabilization of the native state induced by each ion, attributed to unfavorable neutralization and hydrogen-bonding with the protein side-chains. Both effects, combined with binding of Na+, Cl- and SO42- to the thermally unfolded state, resulted in an overall destabilization of the protein. Contrastingly, ion binding was hindered in the chemically unfolded conformation, due to occupation of the binding sites by urea molecules. Such mechanistic action led to a lower degree of destabilization, promoting surface tension effects that stabilized myoglobin according to the Hofmeister series. Therefore, we demonstrate that Hofmeister effects on protein stability are modulated by the heterogeneous physico-chemical nature of the unfolded state. Altogether, our findings evidence the need to characterize the structure of the unfolded state when attempting to dissect the molecular mechanisms underlying the effects of salts on protein stability.

Quantitative proteomic analysis to capture the role of heat-accumulated proteins in moss plant acquired thermotolerance.

At dawn of a scorching summer day, land plants must anticipate upcoming extreme midday temperatures by timely establishing molecular defences that can keep heat‐labile membranes and proteins functional. A gradual morning pre‐exposure to increasing sub‐damaging temperatures induces heat‐shock proteins (HSPs) that are central to the onset of plant acquired thermotolerance (AT). To gain knowledge on the mechanisms of AT in the model land plant Physcomitrium patens, we used label‐free LC–MS/MS proteomics to quantify the accumulated and depleted proteins before and following a mild heat‐priming treatment. High protein crowding is thought to promote protein aggregation, whereas molecular chaperones prevent and actively revert aggregation. Yet, we found that heat priming (HP) did not accumulate HSP chaperones in chloroplasts, although protein crowding was six times higher than in the cytosol. In contrast, several HSP20s strongly accumulated in the cytosol, yet contributing merely 4% of the net mass increase of heat‐accumulated proteins. This is in poor concordance with their presumed role at preventing the aggregation of heat‐labile proteins. The data suggests that under mild HP unlikely to affect protein stability. Accumulating HSP20s leading to AT, regulate the activity of rare and specific signalling proteins, thereby preventing cell death under noxious heat stress.

Multiple levels of crosstalk in hormone networks regulating plant defense.

SummaryPlant hormones are essential for regulating the interactions between plants and their complex biotic and abiotic environments. Each hormone initiates a specific molecular pathway and these different hormone pathways are integrated in a complex network of synergistic, antagonistic and additive interactions. This inter‐pathway communication is called hormone crosstalk. By influencing the immune network topology, hormone crosstalk is essential for tailoring plant responses to diverse microbes and insects in diverse environmental and internal contexts. Crosstalk provides robustness to the immune system but also drives specificity of induced defense responses against the plethora of biotic interactors. Recent advances in dry‐lab and wet‐lab techniques have greatly enhanced our understanding of the broad‐scale effects of hormone crosstalk on immune network functioning and have revealed underlying principles of crosstalk mechanisms. Molecular studies have demonstrated that hormone crosstalk is modulated at multiple levels of regulation, such as by affecting protein stability, gene transcription and hormone homeostasis. These new insights into hormone crosstalk regulation of plant defense are reviewed here, with a focus on crosstalk acting on the jasmonic acid pathway in Arabidopsis thaliana, highlighting the transcription factors MYC2 and ORA59 as major targets for modulation by other hormones.

Effect of urea and glycine betaine on the hydration sphere of model molecules for the surface features of proteins

Water properties may significantly affect protein stability. Osmolytes are compounds that intrinsically affect water in many different ways and thus can influence proteins with this type of indirect mechanism. In this study, we characterize water properties in ternary solutions: model–water–osmolyte, with two model molecules: N-methylacetamide (NMA) and dimethyl sulfoxide (DMSO) and two osmolytes: glycine betaine (TMG) and urea. We focus primarily on the water affected simultaneously by two solutes and propose a new FTIR-based experimental approach to study their properties. Our findings, supported by DFT and AIMD calculations, indicate that TMG promotes hydration cage enhancement around both model molecules. However, direct model–TMG interactions are rare. Simultaneously, urea interacts with them directly, displacing water molecules and weakening the hydrogen bonds in overlapped hydration spheres. The latter behavior results from the cooperativity loss between hydrophobic and hydrophilic-type hydration exhibited by the model molecules in the absence of urea. Relating the obtained results to protein systems, in the case of both osmolytes, the potential mechanism of stabilization–destabilization of these biomolecules is enthalpy-driven.

Spectroscopic Evidence of the Salt-Induced Conformational Change around the Localized Electric Charges on the Protein Surface of Fibronectin Type III

The effect of salt on the electrostatic interaction of a protein is an important issue, because addition of salt affects protein stability and association/aggregation. Although adding salt is a generally recognized strategy to improve protein stability, this improvement does not necessarily occur. The lack of an effect upon the addition of salt was previously confirmed for the tenth fibronectin type III domain from human fibronectin (FN3) by thermal stability analysis. However, the detailed molecular mechanism is unknown. In the present study, by employing the negatively charged carboxyl triad on the surface of FN3 as a case study, the molecular mechanism of the inefficient NaCl effect on protein stability was experimentally addressed using spectroscopic methods. Complementary analysis using Raman spectroscopy and 8-anilino-1-naphthalenesulfonic acid fluorescence revealed the three-phase behavior of the salt-protein interaction between NaCl and FN3 over a wide salt concentration range from 100 mM to 4.0 M, suggesting that the Na+-specific binding to the negatively charged carboxyl triad causes a local conformational change around the binding site with an accompanying structural change in the overall protein, which contributes to the protein's structural destabilization. This spectroscopic evidence clarifies the molecular understanding of the inefficiency of salt to improve protein stability. The findings will inform the optimization of formulation conditions.

Endocrine Resistance in Breast Cancer: The Role of Estrogen Receptor Stability.

Therapy of hormone receptor positive breast cancer (BCa) generally targets estrogen receptor (ER) function and signaling by reducing estrogen production or by blocking its interaction with the ER. Despite good long-term responses, resistance to treatment remains a significant issue, with approximately 40% of BCa patients developing resistance to ET. Mutations in the gene encoding ERα, ESR1, have been identified in BCa patients and are implicated as drivers of resistance and disease recurrence. Understanding the molecular consequences of these mutations on ER protein levels and its activity, which is tightly regulated, is vital. ER activity is in part controlled via its short protein half-life and therefore changes to its stability, either through mutations or alterations in pathways involved in protein stability, may play a role in therapy resistance. Understanding these connections and how ESR1 alterations could affect protein stability may identify novel biomarkers of resistance. This review explores the current reported data regarding posttranslational modifications (PTMs) of the ER and the potential impact of known resistance associated ESR1 mutations on ER regulation by affecting these PTMs in the context of ET resistance.

A pan-cancer assessment of alterations of the kinase domain of ULK1, an upstream regulator of autophagy

Autophagy is a key clearance process to recycle damaged cellular components. One important upstream regulator of autophagy is ULK1 kinase. Several three-dimensional structures of the ULK1 catalytic domain are available, but a comprehensive study, including molecular dynamics, is missing. Also, an exhaustive description of ULK1 alterations found in cancer samples is presently lacking. We here applied a framework which links -omics data to structural protein ensembles to study ULK1 alterations from genomics data available for more than 30 cancer types. We predicted the effects of mutations on ULK1 function and structural stability, accounting for protein dynamics, and the different layers of changes that a mutation can induce in a protein at the functional and structural level. ULK1 is down-regulated in gynecological tumors. In other cancer types, ULK2 could compensate for ULK1 downregulation and, in the majority of the cases, no marked changes in expression have been found. 36 missense mutations of ULK1, not limited to the catalytic domain, are co-occurring with mutations in a large number of ULK1 interactors or substrates, suggesting a pronounced effect of the upstream steps of autophagy in many cancer types. Moreover, our results pinpoint that more than 50% of the mutations in the kinase domain of ULK1, here investigated, are predicted to affect protein stability. Three mutations (S184F, D102N, and A28V) are predicted with only impact on kinase activity, either modifying the functional dynamics or the capability to exert effects from distal sites to the functional and catalytic regions. The framework here applied could be extended to other protein targets to aid the classification of missense mutations from cancer genomics studies, as well as to prioritize variants for experimental validation, or to select the appropriate biological readouts for experiments.

Preparation of the protein corona: How washing shapes the proteome and influences cellular uptake of nanocarriers

A protein coat, termed the protein corona, assembles around the nanocarriers´ surface once it gets in contact with a biological environment. We show that the media used for the washing of protein corona can be crucial. This is true for the downstream analysis as well as for the pre-coating used in in vitro or in vivo. This has been widely overlooked so far. In this paper we focus on eight different washing media and analyze how they influence the composition of the hard protein corona of several nanocarriers incubated with human blood plasma and serum. SDS-PAGE and LC-MS analysis showed major differences in protein corona profiles when using diverse washing media. While plasma and serum proteins already have different complexities, each washing media changes the composition of proteins detected by downstream methods with different key proteins bound to the nanocarriers´ surface. Furthermore, the protein structure of the most abundant blood proteins incubated in the different media was analyzed with nanoDSF. This also emphasized the importance of the washing media, which had a significant influence on the protein adsorption stability. Lastly, cell uptake experiments for HeLa and RAW 264.7 macrophages also indicated an influence of the washing media. In conclusion, picking a specific washing media is on the one hand an important factor for downstream detection of protein compositions and may on the other hand be used to deliberately tune the protein corona for pre-adsorbed proteins from complex protein compositions. This might further support a guided delivery of the nanocarrier to a desired location within a physiological environment. Statement of SignificanceThe successfully application of nanocarriers as drug delivery vehicles is currently hampered by a limited understanding of the nanocarriers´ behavior in a complex biological environment. Once the nanocarrier comes into contact with blood plasma or serum, biomolecules rapidly adsorb onto their surface, covering the nanocarriers and forming a protein corona, which then dictates their biological identity. Analyzing the composition of this dynamic network of bound molecules, has already been shown to be influenced by various factors. However, the impact of the washing media used for the protein corona preparation has so far been neglected. In the present study, we demonstrate a quantitative influence of the washing media on the composition of the hard corona of different nanocarrier systems, which additionally affects protein stability and cellular uptake behavior.

An allosteric pathway explains beneficial fitness in yeast for long-range mutations in an essential TIM barrel enzyme.

Protein evolution proceeds by a complex response of organismal fitness to mutations that can simultaneously affect protein stability, structure, and enzymatic activity. To probe the relationship between genotype and phenotype, we chose a fundamental paradigm for protein evolution, folding, and design, the (βα)8 TIM barrel fold. Here, we demonstrate the role of long‐range allosteric interactions in the adaptation of an essential hyperthermophilic TIM barrel enzyme to mesophilic conditions in a yeast host. Beneficial fitness effects observed with single and double mutations of the canonical βα‐hairpin clamps and the α‐helical shell distal to the active site revealed an underlying energy network between opposite faces of the cylindrical β‐barrel. We experimentally determined the fitness of multiple mutants in the energetic phase plane, contrasting the energy barrier of the chemical reaction and the folding free energy of the protein. For the system studied, the reaction energy barrier was the primary determinant of organism fitness. Our observations of long‐range epistatic interactions uncovered an allosteric pathway in an ancient and ubiquitous enzyme that may provide a novel way of designing proteins with a desired activity and stability profile.

Estimation of varicocele associated human ARG2 and NOS1 proteins and computational analysis on the effect of its nsSNPs

One of the major causes of varicocele is nitrosative stress. Two genes namely arginase 2 (ARG2) and nitric oxide synthase 1 (NOS1) are important in the mechanism of nitric oxide (NO) in the body. Semen samples from three different categories were taken, fertile (n = 20), Infertile (n = 20), and unilateral varicocele (n = 15). Quantitative estimation of ARG2 and NOS1 was carried out through the ELISA kit method. t-Test and ANOVA were calculated using Graphpad Prism. For the in silico analysis, the sequence of the proteins obtained from dbSNP was used in online tools such as nsSNPAnalyzer, PolyPhen-2, Fathmm, I-Mutant 2.0, SNPs&GO, PhD-SNP, PANTHER, SNAP2, PROVEAN, SIFT, and SNPeffect for screening deleterious mutants. These mutants were further evaluated with Swiss PDB and PyMOL for recording energy minimization, Root mean square deviation (RMSD) value, TM score, Hydrogen bonding, and Comparative modeling. Further, ConSurf, Netsurf, and STRING tools were used for evaluating conserved regions, stability, and protein-protein interactions respectively. The results of ARG2 protein were, fertile = 0.168 ± 0.007 U/ml, infertile = 0.201 ± 0.004 U/ml and, varicocele = 0.092 ± 0.002 U/ml. Results of NOS1 protein were fertile = 32.61 ± 2.8 nM/mg, infertile = 19.33 ± 3.7 nM/mg and, varicocele = 54.74 ± 4.8 nM/mg. From statistical analysis, the parameters were highly significant. From the in silico retrieved data, there were 130 and 499 nsSNPs (non-synonymous SNP) in ARG2 and NOS1 respectively. After screening with online tools, 6 deleterious nsSNPs each of ARG2 and NOS1 were considered for analysis through Swiss PDB. FoldX result of A52P mutant (ConSurf score = 9) of ARG2 was found to severely affect protein stability and that of A363T mutant (ConSurf score = 9) of NOS1 revealed a structural change. A52P had a higher RMSD value and A363T of NOS1 developed a new H-bond with 580th position. In varicocele cases, the ARG2 protein is found in lower quantity. The A52P variant of this protein can cause dysfunction and induce nitrosative stress. However, in infertile cases, NOS1 protein is found in lower quantity and the A363T variant can result in a decrease in NO leading to other forms of infertility.

An Angelman syndrome substitution in the HECT E3 ubiquitin ligase C-terminal Lobe of E6AP affects protein stability and activity.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by speech impairment, intellectual disability, ataxia, and epilepsy. AS is caused by mutations in the maternal copy of UBE3A located on chromosome 15q11-13. UBE3A codes for E6AP (E6 Associated Protein), a prominent member of the HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family. E6AP catalyzes the posttranslational attachment of ubiquitin via its HECT domain onto various intracellular target proteins to regulate DNA repair and cell cycle progression. The HECT domain consists of an N-lobe, required for E2~ubiquitin recruitment, while the C-lobe contains the conserved catalytic cysteine required for ubiquitin transfer. Previous genetic studies of AS patients have identified point mutations in UBE3A that result in amino acid substitutions or premature termination during translation. An AS transversion mutation (codon change from ATA to AAA) within the region of the gene that codes for the catalytic HECT domain of E6AP has been annotated (I827K), but the molecular basis for this loss of function substitution remained elusive. Here, we demonstrate that the I827K substitution destabilizes the 3D fold causing protein aggregation of the C-terminal lobe of E6AP using a combination of spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy. Our fluorescent ubiquitin activity assays with E6AP-I827K show decreased ubiquitin thiolester formation and ubiquitin discharge. Using 3D models in combination with our biochemical and biophysical results, we rationalize why the I827K disrupts E6AP-dependent ubiquitylation. This work provides new insight into the E6AP mechanism and how its malfunction can be linked to the AS phenotype.

Probing the Effects of Heterogeneous Oxidative Modifications on the Stability of Cytochrome c in Solution and in the Gas Phase.

Covalent modifications by reactive oxygen species can modulate the function and stability of proteins. Thermal unfolding experiments in solution are a standard tool for probing oxidation-induced stability changes. Complementary to such solution investigations, the stability of electrosprayed protein ions can be assessed in the gas phase by collision-induced unfolding (CIU) and ion-mobility spectrometry. A question that remains to be explored is whether oxidation-induced stability alterations in solution are mirrored by the CIU behavior of gaseous protein ions. Here, we address this question using chloramine-T-oxidized cytochrome c (CT-cyt c) as a model system. CT-cyt c comprises various proteoforms that have undergone MetO formation (+16 Da) and Lys carbonylation (LysCH2-NH2 → LysCHO, -1 Da). We found that CT-cyt c in solution was destabilized, with a ∼5 °C reduced melting temperature compared to unmodified controls. Surprisingly, CIU experiments revealed the opposite trend, i.e., a stabilization of CT-cyt c in the gas phase. To pinpoint the source of this effect, we performed proteoform-resolved CIU on CT-cyt c fractions that had been separated by cation exchange chromatography. In this way, it was possible to identify MetO formation at residue 80 as the key modification responsible for stabilization in the gas phase. Possibly, this effect is caused by newly formed contacts of the sulfoxide with aromatic residues in the protein core. Overall, our results demonstrate that oxidative modifications can affect protein stability in solution and in the gas phase very differently.

Protein Stability and Photostability under In Vitro Vitreal Conditions - Implications for Long Acting Delivery of Protein Therapeutics for Ocular Disease.

To evaluate the stability of a model Mab1 and Fab1 under in vitro vitreal conditions in the presence of various metabolites and in the presence of light at λ> 400nM. A full length IgG1 monoclonal antibody (Mab1) and a fab fragment (Fab1) were formulated with various metabolites typically found in the vitreous humor and subjected to visible light treatment. Both proteins were analyzed using a variety of biochemical techniques. Furthermore, Fab1 was also tested for antigen binding ability using surface plasmon resonance. Our data shows that some vitreal metabolites such as riboflavin and ascorbic acid affect protein stability, via formation of reactive oxygen species (ROS) and advanced glycation end products (AGE) s respectively. In contrast, metabolites such as glutathione may protect these proteins from light-induced degradation to some extent. Ascorbic acid and riboflavin were found to photosensitize therapeutic proteins especially when exposed to light. Ascorbic acid reacted with proteins even in the absence of light. Antioxidants such as glutathione helped limit photooxidation under ambient or blue light exposure. Since antioxidant capacity in the eye decreases with age we recommend understanding long term stability, including photooxidation and photosensitization, of new candidate proteins in the context of controlled or sustained release technologies for ocular diseases.

Meltome atlas-thermal proteome stability across the tree of life.

We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30-90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org.

Insights into the Effect of Genetic Variation in CYP2C9 (*11) on Drug Binding.

Cytochrome P450s (CYP) are a family of membrane associated heme‐containing enzymes involved in xenobiotic metabolism with recent relevance in pharmacogenomics. The human CYP2C9 enzyme metabolizes up to 15–20% of drugs that include warfarin, losartan, ibuprofen, and tolbutamide. It is highly polymorphic with several genetic variations associated with significantly altered activity of the enzyme. The CYP2C9*11 allele occurs predominantly in African Americans but also in Caucasian and other ethnic populations with a frequency of around 0.05. The *11 genetic variation represents a substitution of amino acid residue at 335 from arginine to tryptophan (R335W) that has demonstrated decreased catalytic activity towards various substrates including S‐warfarin. The R335W was created in CYP2C9 wild‐type (WT) construct using site‐directed mutagenesis and co‐expressed in E. Coli alongside chaperone proteins (GroEL/ES). Generation of purified CYP2C9*11 protein enabled catalytic studies and crystallization in complex with the drug substrate losartan. The enzymatic assays revealed marked reduction in losartan turn‐over by CYP2C9*11 compared to the WT enzyme. Additionally, comparison of the amino acid sequence revealed that R335 is conserved across the CYP2C subfamily of enzymes. The R335 is located on the distal J‐J’ loop and interacts with multiple amino acid side chains on the J and J’ helices. The computational analysis suggests that the substitution with hydrophobic and bulkier tryptophan located on the surface region may result in destabilizing interactions with several helices including those located near the active site heme, affecting protein stability and function. Overall, these observations establish the effect of distal variation prompting further evaluation by structural and biophysical studies.Support or Funding InformationThe research was funded by the start‐up funds from the Albany College of Pharmacy and Health Sciences.

Revealing the sequence determinants of SMYD1 lysine methyltransferase substrate selectivity with lysine‐oriented peptide libraries

Lysine methylation is a protein post‐translational modification (PTMs) that can affect protein stability, activity, subcellular localization, and molecular interactions. Revealing protein substrates for lysine methyltransferases (KMTs) will help connect enzymatic activity to cellular functions and pathologies associated with these enzymes. Toward this goal, we developed a high‐throughput screening assay to define the sequence determinants of KMT substrate selectivity. This assay queries a highly degenerate lysine‐oriented peptide library (K‐OPL) to identify amino acid preferences at three resides N‐ and C‐terminal to a fixed central lysine. Here, we demonstrate the utility of this approach to facilitate identification of several novel substrates of KMT activity, including for SMYD1 (SET and MYND domain‐containing protein 1), a KMT expressed in cardiac and skeletal muscle that is essential for heart development and organ homeostasis. We further demonstrate the utility of this tool, in conjunction with in silico and biophysical approaches, for revealing the impact of a cardiac disease‐associated mutation of SMYD1 on its KMT function. Collectively, our studies reveal new hypotheses toward a better understanding of SMYD1 function in cardiac and skeletal muscle development and implicate its aberrant KMT activity in heart disease.Support or Funding InformationNIH R356M14736

The nuclear receptor NR4A1 is regulated by SUMO modification to induce autophagic cell death.

NR4A is a nuclear receptor protein family whose members act as sensors of cellular environment and regulate multiple processes such as metabolism, proliferation, migration, apoptosis, and autophagy. Since the ligand binding domains of these receptors have no cavity for ligand interaction, their function is most likely regulated by protein abundance and post-translational modifications. In particular, NR4A1 is regulated by protein abundance, phosphorylation, and subcellular distribution (nuclear-cytoplasmic translocation), and acts both as a transcription factor and as a regulator of other interacting proteins. SUMOylation is a post-translational modification that can affect protein stability, transcriptional activity, alter protein-protein interactions and modify intracellular localization of target proteins. In the present study we evaluated the role of SUMOylation as a posttranslational modification that can regulate the activity of NR4A1 to induce autophagy-dependent cell death. We focused on a model potentially relevant for neuronal cell death and demonstrated that NR4A1 needs to be SUMOylated to induce autophagic cell death. We observed that a triple mutant in SUMOylation sites has reduced SUMOylation, increased transcriptional activity, altered intracellular distribution, and more importantly, its ability to induce autophagic cell death is impaired.

Deciphering the impact of missense mutations on structure and dynamics of SMAD4 protein involved in pathogenesis of gall bladder cancer

Gall bladder cancer (GBC) is the most common malignancy of biliary tract cancer associated with high mortality rate and poor prognosis due to lack of suitable biomarkers. In this study, we explored the structural and functional effects of different missense mutations occurs in SMAD4 that was associated with the development of GBC. We utilized in silico methods to predict the harmful effects of nonsynonymous missense mutations and monitored the stability of protein. We found that all mutations (D351N, G352E, R361C, R361H, E526Q) associated with SMAD4 were deleterious in nature resulting in the formation of deformed or unstable protein structure. Molecular dynamics simulation studies revealed how these mutations affect protein stability, structure, conformation and function. We observed, different mutants increase the compactness and rigidity of SMAD4 protein, alter secondary structure composition, decrease the surface area and protein-ligand interaction and affect its conformation. Findings of current work indicated that the analyzed mutations might affect the structure of protein and its caliber to interact with other molecules, which probably related to functional impairment of SMAD4 upon D351N, G352E, R361C, R361H, E526Q mutations and their involvement in cancer. Hence, the present study has significance of rational drug design and further increase our understanding of GBC development. Communicated by Ramaswamy H. Sarma

N-Terminal Amino Acids Determine KLF4 Protein Stability in 2A Peptide-Linked Polycistronic Reprogramming Constructs

SummaryA common strategy for multi-protein expression is to link genes by self-cleaving 2A peptide sequences. Yet, little is known how the 2A peptide-derived N-terminal proline or adjacent non-native residues introduced during cDNA cloning affects protein stoichiometry. Polycistronic reprogramming constructs with altered KLF4 protein stoichiometry can influence induced pluripotent stem cell (iPSC) generation. We studied the impact of N-terminal 2A peptide-adjacent residues on the protein stability of two KLF4 isoforms, and assayed their capacity to generate iPSCs. Here, we show that the N-terminal proline remnant of the 2A peptide, alone or in combination with leucine, introduced during polycistronic cloning, destabilizes KLF4 resulting in increased protein degradation, which hinders reprogramming. Interestingly, the addition of charged and hydrophilic amino acids, such as glutamate or lysine stabilizes KLF4, enhancing reprogramming phenotypes. These findings raise awareness that N-terminal modification with 2A peptide-derived proline or additional cloning conventions may affect protein stability within polycistronic constructs.

Massively parallel variant characterization identifies NUDT15 alleles associated with thiopurine toxicity

As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction was quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine the variants' effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in patients treated with thiopurines with far superior sensitivity and specificity compared to bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,152 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.