Affect Protein Folding Research Articles

Interactions of variants of human apolipoprotein A-I with biopolymeric model matrices. Effect of collagen and heparin

BackgroundThe extracellular matrix (ECM) is a complex tridimensional scaffold that actively participates in physiological and pathological events. The objective of this study was to test whether structural proteins of the ECM and glycosaminoglycans (GAGs) may favor the retention of human apolipoprotein A-I (apoA-I) variants associated with amyloidosis and atherosclerosis. MethodsBiopolymeric matrices containing collagen type I (Col, a main macromolecular component of the ECM) with or without heparin (Hep, a model of GAGs) were constructed and characterized, and used to compare the binding of apoA-I having the native sequence (Wt) or Arg173Pro, a natural variant inducing cardiac amyloidosis. Protein binding was observed by fluorescence microscopy and unbound proteins quantified by a colorimetric assay. ResultsBoth, Wt and Arg173Pro bound to the scaffolds containing Col, but the presence of Hep diminished the binding efficiency. Col-Hep matrices retained Arg173Pro more than the Wt. The retained protein was only partially removed from the matrices with saline solutions, indicating that electrostatic interactions may occur but are not the main driving force. Using in addition thermodynamic molecular simulations and size exclusion chromatography approaches, we suggest that the binding of apoA-I variants to the biopolymeric matrices is driven by many low affinity interactions. ConclusionsUnder this scenario Col-Hep scaffolds contribute to the binding of Arg173Pro, as a cooperative platform which could modify the native protein conformation affecting protein folding. General significanceWe show that the composition of the ECM is key to the protein retention, and well characterized biosynthetic matrices offer an invaluable in vitro model to mimic the hallmark of pathologies with interstitial infiltration such as cardiac amyloidosis.

Single-molecule analysis of osmolyte-mediated nanomechanical unfolding behavior of a protein domain

The small organic molecules, known as osmolytes being ubiquitously present in different cell types, affect protein folding, stability and aggregation. However, it is unknown how the osmolytes affect the nanomechanical unfolding behavior of protein domain. Here, we show the osmolyte-dependent mechanical unfolding properties of protein titin immunoglobulin-27 (I27) domain using an atomic force microscopy (AFM)-based single-molecule force spectroscopy. We found that amines and methylamines improved the mechanical stability of I27 domain, whereas polyols had no effect. Interestingly, glycine betaine (GB) or trimethylamine-N-oxide (TMAO) increased the average unfolding force of the protein domain. The kinetic parameters analyzed at single-molecule level reveal that stabilizing effect of osmolytes is due to a decrease in the unfolding rate constant of I27, which was confirmed by molecular dynamics simulations. Our study reveals different effects that diverse osmolytes have on the mechanical properties of the protein, and suggests the potential use of osmolytes in modulating the mechanical stability of proteins required for various nano-biotechnological applications.

Proline Isomerization: From the Chemistry and Biology to Therapeutic Opportunities.

Proline isomerization, the process of interconversion between the cis- and trans-forms of proline, is an important and unique post-translational modification that can affect protein folding and conformations, and ultimately regulate protein functions and biological pathways. Although impactful, the importance and prevalence of proline isomerization as a regulation mechanism in biological systems have not been fully understood or recognized. Aiming to fill gaps and bring new awareness, we attempt to provide a wholistic review on proline isomerization that firstly covers what proline isomerization is and the basic chemistry behind it. In this section, we vividly show that the cause of the unique ability of proline to adopt both cis- and trans-conformations in significant abundance is rooted from the steric hindrance of these two forms being similar, which is different from that in linear residues. We then discuss how proline isomerization was discovered historically followed by an introduction to all three types of proline isomerases and how proline isomerization plays a role in various cellular responses, such as cell cycle regulation, DNA damage repair, T-cell activation, and ion channel gating. We then explore various human diseases that have been linked to the dysregulation of proline isomerization. Finally, we wrap up with the current stage of various inhibitors developed to target proline isomerases as a strategy for therapeutic development.

Single-cell transcriptome analysis identifies novel biomarkers involved in major liver cancer subtypes.

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the two aggressive subtypes of liver cancer (LC). Immense cellular heterogeneity and cross-talk between cancer and healthy cells make it challenging to treat these cancer subtypes. To address these challenges, the study aims to systematically characterize the tumor heterogeneity of LC subtypes using single-cell RNA sequencing (scRNA-seq) datasets. The study combined 51,927 single cells from HCC, ICC, and healthy scRNA-seq datasets. After integrating the datasets, cell groups with similar gene expression patterns are clustered and cluster annotation has been performed based on gene markers. Cell-cell communication analysis (CCA) was implemented to understand the cross-talk between various cell types. Further, differential gene expression analysis and enrichment analysis were carried out to identify unique molecular drivers associated with HCC and ICC. Our analysis identified T cells, hepatocytes, epithelial cells, and monocyte as the major cell types present in the tumor microenvironment. Among them, abundance of natural killer (NK) cells in HCC, epithelial cells, and hepatocytes in ICC was detected. CCA revealed key interaction between T cells to NK cells in HCC and smooth muscle cells to epithelial cells in the ICC. Additionally, SOX4 and DTHD1 are the top differentially expressed genes (DEGs) in HCC, while keratin and CCL4 are in ICC. Enrichment analysis of DEGs reveals major upregulated genes in HCC affect protein folding mechanism and in ICC alter pathways involved in cell adhesion. The findings suggest potential targets for the development of novel therapeutic strategies for the treatment of these two aggressive subtypes of LC.

Trehalose promotes structural disorderness of intrinsically disordered casein proteins with enhanced chaperone function

Intrinsically disordered proteins (IDPs) are being currently explored for their role in wide range of essential cellular processes especially signalling, cell cycle, transcription, translation and several disease processes like neurodegeneration, cancer, systemic amyloidosis etc. However, as compared to ordered proteins, their high structural dynamics makes them quite sensitive to the changing cellular environment such as changes in the level of cellular metabolites. Since previous studies have shown the ability of trehalose (an important small molecule metabolite) to affect protein folding and formation of high order oligomers, we aim to elucidate the effect of trehalose on the structural–functional elasticity of intrinsically disordered α- and β-casein proteins. We observed that trehalose enhances disorderliness resulting in increased chaperone activity of both the casein proteins. Considering the role of co-solutes in cellular proteostasis, the present study indicates that trehalose acts not only as an osmoprotectant, but also a key regulator of functional activity of various IDPs.

Seasonal influence on expression of heat shock proteins (HSP70 and HSP90) vis-à-vis functional competence of Gir bull semen

The success of assisted reproduction relies on functional competence of frozen-thawed semen. Heat stress affects protein folding leading to aggregation of mis-folded proteins. Hence, a total of 384 (32 ejaculates/bull/season) ejaculates from six matured Gir bulls were used to evaluate physico-morphological parameters, the expression of HSPs (70 and 90) and fertility of frozen-thawed semen. The mean percent individual motility, viability and membrane integrity were significantly (p < 0.01) higher in winter compared to summer. Out of 1200 Gir cows inseminated, 626 confirmed pregnant and the mean conception rate of winter (55.04 ± 0.35) was significantly (p < 0.001) higher than summer (49.33 ± 0.32). A significant (p < 0.01) difference in concentration of HSP70 (ng/mg of protein) but not HSP90was observed between the two seasons. The HSP70 expression in pre-freeze semen of Gir bulls had significant positive correlation with motility (p < 0.01, r = 0.463), viability (p < 0.01, r = 0.565), acrosome integrity (p < 0.05, r = 0.330) and conception rate (p < 0.01, r = 0.431). In conclusion, the season influences physico-morphological parameters and expression of HSP70 but not HSP90 in Gir bull semen. The HSP70 expression is positively correlated with motility, viability, acrosome integrity and fertility of semen. The semen expression of HSP70 may be utilized as biomarker for thermo-tolerance, semen quality and fertilizing capacity of Gir bull semen.

Point mutations in the catalytic domain disrupt cellulose synthase (CESA6) vesicle trafficking and protein dynamics.

Cellulose, the main component of the plant cell wall, is synthesized by the multimeric cellulose synthase (CESA) complex (CSC). In plant cells, CSCs are assembled in the endoplasmic reticulum or Golgi and transported through the endomembrane system to the plasma membrane (PM). However, how CESA catalytic activity or conserved motifs around the catalytic core influence vesicle trafficking or protein dynamics is not well understood. Here, we used yellow fluorescent protein (YFP)-tagged AtCESA6 and created 18 mutants in key motifs of the catalytic domain to analyze how they affected seedling growth, cellulose biosynthesis, complex formation, and CSC dynamics and trafficking in Arabidopsis thaliana. Seedling growth and cellulose content were reduced by nearly all mutations. Moreover, mutations in most conserved motifs slowed CSC movement in the PM as well as delivery of CSCs to the PM. Interestingly, mutations in the DDG and QXXRW motifs affected YFP-CESA6 abundance in the Golgi. These mutations also perturbed post-Golgi trafficking of CSCs. The 18 mutations were divided into two groups based on their phenotypes; we propose that Group I mutations cause CSC trafficking defects whereas Group II mutations, especially in the QXXRW motif, affect protein folding and/or CSC rosette formation. Collectively, our results demonstrate that the CESA6 catalytic domain is essential for cellulose biosynthesis as well as CSC formation, protein folding and dynamics, and vesicle trafficking.

Teicoplanin aglycone media and carboxypeptidase Y: Tools for finding low-abundance D-amino acidsandepimericpeptides.

D-amino acids and epimeric peptides/proteins can play crucial biological roles and adversely affect protein folding and oligopeptide aggregation in age-related pathologies in humans. This has ignited interest in free D-amino acids as well as those incorporated in peptides/proteins and their effects in humans. However, such stereoisomeric analytes are often elusive and in low abundance with few existing methodologies capable of scouting for and identifying them. In this work, we examine the feasibility of using teicoplanin aglycone, a macrocyclic antibiotic, which has been reported to strongly retain D-amino acids and peptides with a D-amino acid on the C-terminus, for use as asolidphaseextraction(SPE)medium. The HPLC retention factors of L-/D-amino acids and C-terminus modified D-amino acid-containing peptides and their L-amino acid exclusive counterparts on teicoplanin aglycone are presented. Retention curve differences between amino acids and peptides highlight regions of solvent composition that can be utilized for their separation. This approach is particularly useful when coupled with enzymatic hydrolysis via carboxypeptidase Y to eliminate all L-amino acid exclusive peptides. The remaining peptides with carboxy-terminal D-amino acids are then more easily concentrated and identified.

Oxidation state of SNAP-25 and formation of adducts.

SNARE proteins drive neuronal exocytosis by forming a 4-helix coiled-coil. This mechanism requires SNARE proteins to undergo a disordered-to-ordered state transition. SNAP-25 is unique among the SNARE proteins because it contributes two alpha helices to the 4-helix bundle, compared to Synaptobrevin and Syntaxin which only contribute one alpha helix each. Furthermore, neurons express SNAP-25 in two different isoforms, SNAP-25a and SNAP-25b, depending on the brain region and the developmental stage of the neuron, but the benefit of having two isoforms is unknown. A key difference between these two isoforms is in the location of the four cysteines in the cysteine-rich region of the linker connecting the two SNARE domains. We hypothesize that cysteine location and redox state combine to alter the structure (and function) of each protein. Using circular dichroism (CD) spectrometry, we observe structural similarities and differences between SNAP-25a and SNAP-25b. We demonstrate, using mass spectrometry, that sulfur-containing reducing agents form adducts with the cysteines. Through CD spectrometry, we demonstrate differences in protein folding with temperature, oxidation state, and presence of adducts on SNAP-25. By understanding how oxidation affects protein folding, we hope to better understand differences between the two isoforms of SNAP-25 and how oxidative stress may alter SNARE-driven neurotransmitter release.

Involvement of testicular N-glycoproteome heterogeneity in seasonal spermatogenesis of the American mink (Neovison vison).

Spermatogenesis in the American mink is characterized by an annual cycle of transition involving completely inactive and fully activated stages. N-glycosylation of proteins has emerged as an important regulator as it affects protein folding, secretion, degradation, and activity. However, the function of protein N-glycosylation in seasonal spermatogenesis of the American mink remains unclear. In the present study, we established a proteome-wide stoichiometry of N-glycosylation in mink testes at various phases of spermatogenesis using N-linked glycosylated-peptide enrichment in combination with liquid chromatography-tandem mass spectrometry analysis. A total of 532 N-glycosylated sites matching the canonical Asn-X-Ser/Thr motif were identified in 357 testicular proteins. Both the number of glycoproteins and the sites of N-glycosylated proteins in mink testes were highly dynamic at different stages. Functional analyses showed that testicular proteins with different N-glycosylation might play a vital role in spermatogenesis by affecting their folding, distribution, stability, and activity. Overall, our data suggest that the dynamics of N-glycosylation of testicular proteins are involved in seasonal spermatogenesis in the American mink.

Emerging roles for G-quadruplexes in proteostasis.

How nucleic acids interact with proteins, and how they affect protein folding, aggregation, and misfolding is a still-evolving area of research. Considerable effort is now focusing on a particular structure of RNA and DNA, G-quadruplexes, and their role in protein homeostasis and disease. In this state-of-the-art review, we track recent reports on how G-quadruplexes influence protein aggregation, proteolysis, phase separation, and protein misfolding diseases, and pose currently unanswered questions in the advance of this scientific field.

The Effects of Temperature on Cellular Physiology.

Temperature impacts biological systems across all length and timescales. Cells and the enzymes that comprise them respond to temperature fluctuations on short timescales, and temperature can affect protein folding, the molecular composition of cells, and volume expansion. Entire ecosystems exhibit temperature-dependent behaviors, and global warming threatens to disrupt thermal homeostasis in microbes that are important for human and planetary health. Intriguingly, the growth rate of most species follows the Arrhenius law of equilibrium thermodynamics, with an activation energy similar to that of individual enzymes but with maximal growth rates and over temperature ranges that are species specific. In this review, we discuss how the temperature dependence of critical cellular processes, such as the central dogma and membrane fluidity, contributes to the temperature dependence of growth. We conclude with a discussion of adaptation to temperature shifts and the effects of temperature on evolution and on the properties of microbial ecosystems.

S-Nitrosylation of OTUB1 Alters Its Stability and Ubc13 Binding.

S-Nitrosylation is a reversible post-translational modification that regulates protein function involving the covalent attachment of the nitric oxide (NO) moiety to sulfhydryl residues of the protein. It is an important regulator in the cell signaling process under physiological conditions. However, the release of an excess amount of NO due to dysregulated NOS machinery causes aberrant S-nitrosylation of proteins, which affects protein folding, localization, and activity. Here, we have shown that OTUB1, a deubiquitinating enzyme, undergoes S-nitrosylation under redox stress conditions in vivo and in vitro. Previously, we have shown that OTUB1 forms an amyloid-like structure that promotes phosphorylation of α-synuclein and neuronal toxicity. However, the mechanistic insight into OTUB1 aggregation remains elusive. Here, we identified that OTUB1 undergoes S-nitrosylation in SH-SY5Y neuroblastoma cells under rotenone-induced stress, as well as excitotoxic conditions, and in rotenone-treated mouse brains. The in vitro S-nitrosylation of OTUB1 followed by mass-spectrometry analysis has identified cysteine-23 and cysteine-91 as S-nitrosylation sites. S-Nitrosylated OTUB1 (SNO-OTUB1) diminished its catalytic activity, impaired its native structure, promoted amyloid-like aggregation, and compromised its binding with Ubc13. Thus, our results demonstrated that nitrosylation of OTUB1 might play a crucial role in regulating the ubiquitin signaling and Parkinson's disease pathology.

How physical forces drive the process of helical membrane protein folding.

Protein folding is a fundamental process of life with important implications throughout biology. Indeed, tens of thousands of mutations have been associated with diseases, and most of these mutations are believed to affect protein folding rather than function. Correct folding is also a key element of design. These factors have motivated decades of research on protein folding. Unfortunately, knowledge of membrane protein folding lags that of soluble proteins. This gap is partly caused by the greater technical challenges associated with membrane protein studies, but also because of additional complexities. While soluble proteins fold in a homogenous water environment, membrane proteins fold in a setting that ranges from bulk water to highly charged to apolar. Thus, the forces that drive folding vary in different regions of the protein, and this complexity needs to be incorporated into our understanding of the folding process. Here, we review our understanding of membrane protein folding biophysics. Despite the greater challenge, better model systems and new experimental techniques are starting to unravel the forces and pathways in membrane protein folding.

Going through changes: effect of phosphoserine on secondary structure preference and dipole-dipole interactions in model peptides

Intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids and lack a single, well-defined tertiary structure. IDPs are frequently the targets of post-translational modifications (PTMs) which can affect protein folding or stability, activity, susceptibility to degradation, and function. Phosphorylation is one of the most common PTMs and occurs on serine, threonine, and tyrosine residues. Hyperphosphorylation of some IDPs is associated with disease states, such as the tau protein in Alzheimer’s disease and α-synuclein in Parkinson’s disease.

Biological factors in the synthetic construction of overlapping genes

BackgroundOverlapping genes (OLGs) with long protein-coding overlapping sequences are disallowed by standard genome annotation programs, outside of viruses. Recently however they have been discovered in Archaea, diverse Bacteria, and Mammals. The biological factors underlying life’s ability to create overlapping genes require more study, and may have important applications in understanding evolution and in biotechnology. A previous study claimed that protein domains from viruses were much better suited to forming overlaps than those from other cellular organisms - in this study we assessed this claim, in order to discover what might underlie taxonomic differences in the creation of gene overlaps.ResultsAfter overlapping arbitrary Pfam domain pairs and evaluating them with Hidden Markov Models we find OLG construction to be much less constrained than expected. For instance, close to 10% of the constructed sequences cannot be distinguished from typical sequences in their protein family. Most are also indistinguishable from natural protein sequences regarding identity and secondary structure. Surprisingly, contrary to a previous study, virus domains were much less suitable for designing OLGs than bacterial or eukaryotic domains were. In general, the amount of amino acid change required to force a domain to overlap is approximately equal to the variation observed within a typical domain family. The resulting high similarity between natural sequences and those altered so as to overlap is mostly due to the combination of high redundancy in the genetic code and the evolutionary exchangeability of many amino acids.ConclusionsSynthetic overlapping genes which closely resemble natural gene sequences, as measured by HMM profiles, are remarkably easy to construct, and most arbitrary domain pairs can be altered so as to overlap while retaining high similarity to the original sequences. Future work however will need to assess important factors not considered such as intragenic interactions which affect protein folding. While the analysis here is not sufficient to guarantee functional folding proteins, further analysis of constructed OLGs will improve our understanding of the origin of these remarkable genetic elements across life and opens up exciting possibilities for synthetic biology.

A novel COLEC10 mutation in a child with 3MC syndrome

3MC syndrome is an autosomal recessive disorder encompassing four rare disorders previously known as the Malpuech, Michels, Mingarelli and Carnevale syndromes. They are characterized by a variable spectrum of abnormalities, including facial dysmorphisms, along with genital, limb and vesico-renal anomalies. The syndrome was originally attributed to mutations in MASP1 and COLEC11, which code for proteins involved in the lectin complement pathway. More recently, mutations in COLEC10, a third gene coding for collectin CL-L1, were identified in a limited number of patients with 3MC syndrome. Here we describe a 4-years-old patient with typical 3MC phenotypic characteristics, including blepharophimosis, telecanthus, high arched eyebrows, fifth finger clinodactyly, sacral dimple and horseshoe kidney. Initial genetic analysis was based on clinical exome sequencing, where only MASP1 and COLEC11 genes are present, without evidence of pathogenic variants. Sanger sequencing of COLEC10 identified the homozygous frameshift variant c.807_810delCTGT; p.Cys270Serfs*33, which results in the loss of the natural stop codon. The resulting protein is 24 amino acids longer and lacks a conserved cysteine residue (Cys270), which could affect protein folding. Segregation studies confirmed that both parents were carriers for the variant: interestingly they originate from the same area of Apulia in southern Italy. Plasma levels of CL-L1 in the patient and her parents were within normal range, suggesting that this variant does not modify transcription or secretion. However, the variant affects the chemo-attractive feature of CL-L1, as HeLa cells migrate significantly less in response to the mutant protein compared to the wild-type one.

Recombinant and endogenous ways to produce methylated phospholipids in Escherichia coli

Escherichia coli is the daily workhorse in molecular biology research labs and an important platform microorganism in white biotechnology. Its cytoplasmic membrane is primarily composed of the phospholipids phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL). As in most other bacteria, the typical eukaryotic phosphatidylcholine (PC) is not a regular component of the E. coli membrane. PC is known to act as a substrate in various metabolic or catabolic reactions, to affect protein folding and membrane insertion, and to activate proteins that originate from eukaryotic environments. Options to manipulate the E. coli membrane to include non-native lipids such as PC might make it an even more powerful and versatile tool for biotechnology and protein biochemistry. This article outlines different strategies how E. coli can be engineered to produce PC and other methylated PE derivatives. Several of these approaches rely on the ectopic expression of genes from natural PC-producing organisms. These include PC synthases, lysolipid acyltransferases, and several phospholipid N-methyltransferases with diverse substrate and product preferences. In addition, we show that E. coli has the capacity to produce PC by its own enzyme repertoire provided that appropriate precursors are supplied. Screening of the E. coli Keio knockout collection revealed the lysophospholipid transporter LplT to be responsible for the uptake of lyso-PC, which is then further acylated to PC by the acyltransferase-acyl carrier protein synthetase Aas. Overall, our study shows that the membrane composition of the most routinely used model bacterium can readily be tailored on demand.Key points• Escherichia coli can be engineered to produce non-native methylated PE derivatives.• These lipids can be produced by foreign and endogenous proteins.• Modification of E. coli membrane offers potential for biotechnology and research.Graphical abstract

Role of Glycoproteins during Fruit Ripening and Seed Development.

Approximately thirty percent of the proteins synthesized in animal or plant cells travel through the secretory pathway. Seventy to eighty percent of those proteins are glycosylated. Thus, glycosylation is an important protein modification that is related to many cellular processes, such as differentiation, recognition, development, signal transduction, and immune response. Additionally, glycosylation affects protein folding, solubility, stability, biogenesis, and activity. Specifically, in plants, glycosylation has recently been related to the fruit ripening process. This review aims to provide valuable information and discuss the available literature focused on three principal topics: (I) glycosylations as a key posttranslational modification in development in plants, (II) experimental and bioinformatics tools to analyze glycosylations, and (III) a literature review related to glycosylations in fruit ripening. Based on these three topics, we propose that it is necessary to increase the number of studies related to posttranslational modifications, specifically protein glycosylation because the specific role of glycosylation in the posttranslational process and how this process affects normal fruit development and ripening remain unclear to date.

Chaperonin Abundance Enhances Bacterial Fitness.

The ability of chaperonins to buffer mutations that affect protein folding pathways suggests that their abundance should be evolutionarily advantageous. Here, we investigate the effect of chaperonin overproduction on cellular fitness in Escherichia coli. We demonstrate that chaperonin abundance confers 1) an ability to tolerate higher temperatures, 2) improved cellular fitness, and 3) enhanced folding of metabolic enzymes, which is expected to lead to enhanced energy harvesting potential.