Proteomes Of Organisms Research Articles

Freestanding bilayer microscope for single-molecule imaging of membrane proteins.

Integral membrane proteins (IMPs) constitute a large fraction of organismal proteomes, playing fundamental roles in physiology and disease. Despite their importance, the mechanisms underlying dynamic features of IMPs, such as anomalous diffusion, protein-protein interactions, and protein clustering, remain largely unknown due to the high complexity of cell membrane environments. Available methods for in vitro studies are insufficient to study IMP dynamics systematically. This publication introduces the freestanding bilayer microscope (FBM), which combines the advantages of freestanding bilayers with single-particle tracking. The FBM, based on planar lipid bilayers, enables the study of IMP dynamics with single-molecule resolution and unconstrained diffusion. This paper benchmarks the FBM against total internal reflection fluorescence imaging on supported bilayers and is used here to estimate ion channel open probability and to examine the diffusion behavior of an ion channel in phase-separated bilayers. The FBM emerges as a powerful tool to examine membrane protein/lipid organization and dynamics to understand cell membrane processes.

FungiRegEx: A Tool for Pattern Identification in Fungal Proteomic Sequences Using Regular Expressions

In the context of proteomic-scale research, it is imperative to automatically analyze numerous species and subspecies to discern distinctive characteristics present in multiple species of the fungi kingdom that contain sequences of interest that could fulfill a specific biological function. To achieve this, complex sequences must be recognized within an organism’s entire set of proteomes. Our study presents FungiRegEx, a piece of software that facilitates the identification of regular expressions of proteomes of fungal organisms and uses real-time data retrieval of the different species from the JGI Mycocosm database without the need to download any file. Integrating a graphical user interface that makes it easy to use, the tool offers regular expression searches on 2402 fungal species from the JGI Mycocosm portal. The tool was validated with the AXSXG sequence and the RXRL effector, demonstrating the effectiveness of FungiRegEx in identifying user-defined patterns in the recovered sequences. This tool allows customization and filtering, and it can save results if required, combining speed, adaptability, and ease of use. It provides an experience without a console and programming, displaying the results in a GUI and making them easier to read. Its architecture guarantees optimized use of resources, time consumption, and implementation flexibility, allowing the customization of specific software parameters for resource management. The tool’s potential for future research and exploration is emphasized, providing a nuanced perspective on its practical use within the fungal genomics community. The tools are available at the addresses mentioned in the text.

The determinants of the rarity of nucleic and peptide short sequences in nature.

The prevalence of nucleic and peptide short sequences across organismal genomes and proteomes has not been thoroughly investigated. We examined 45785 reference genomes and 21871 reference proteomes, spanning archaea, bacteria, eukaryotes and viruses to calculate the rarity of short sequences in them. To capture this, we developed a metric of the rarity of each sequence in nature, the rarity index. We find that the frequency of certain dipeptides in rare oligopeptide sequences is hundreds of times lower than expected, which is not the case for any dinucleotides. We also generate predictive regression models that infer the rarity of nucleic and proteomic sequences across nature or within each domain of life and viruses separately. When examining each of the three domains of life and viruses separately, the R² performance of the model predicting rarity for 5-mer peptides from mono- and dipeptides ranged between 0.814 and 0.932. A separate model predicting rarity for 10-mer oligonucleotides from mono- and dinucleotides achieved R² performance between 0.408 and 0.606. Our results indicate that the mono- and dinucleotide composition of nucleic sequences and the mono- and dipeptide composition of peptide sequences can explain a significant proportion of the variance in their frequencies in nature.

ProNet DB: a proteome-wise database for protein surface property representations and RNA-binding profiles.

The rapid growth in the number of experimental and predicted protein structures and more complicated protein structures poses a significant challenge for computational biology in leveraging structural information and accurate representation of protein surface properties. Recently, AlphaFold2 released the comprehensive proteomes of various species, and protein surface property representation plays a crucial role in protein-molecule interaction predictions, including those involving proteins, nucleic acids and compounds. Here, we proposed the first extensive database, namely ProNet DB, that integrates multiple protein surface representations and RNA-binding landscape for 326 175 protein structures. This collection encompasses the 16 model organism proteomes from the AlphaFold Protein Structure Database and experimentally validated structures from the Protein Data Bank. For each protein, ProNet DB provides access to the original protein structures along with the detailed surface property representations encompassing hydrophobicity, charge distribution and hydrogen bonding potential as well as interactive features such as the interacting face and RNA-binding sites and preferences. To facilitate an intuitive interpretation of these properties and the RNA-binding landscape, ProNet DB incorporates visualization tools like Mol* and an Online 3D Viewer, allowing for the direct observation and analysis of these representations on protein surfaces. The availability of pre-computed features enables instantaneous access for users, significantly advancing computational biology research in areas such as molecular mechanism elucidation, geometry-based drug discovery and the development of novel therapeutic approaches. Database URL: https://proj.cse.cuhk.edu.hk/aihlab/pronet/.

Comparison of cysteine content in whole proteomes across the three domains of life

An empirical observation suggests that Giardia lamblia proteins have larger cysteine content than their counterparts in other organisms. As this parasite lacks conventional antioxidant stress systems, it is generally accepted that high cysteine content helps G. lamblia cope with oxygen toxicity, a strategy apparently shared by other organisms. Here, we question whether the high cysteine content in some organisms is genuine or just a simple assumption based on singular observations. To this end, we analyzed the cysteine content in 78 proteomes of organisms spanning the three domains of life. The results indicate that the cysteine content in eukaryota is approximately double that in archaea and bacteria, with G. lamblia among the highest. Atypical cysteine contents were found in a few organisms correlating with specific environmental conditions, supporting the evolutionary amino acid-level selection of amino acid composition.

Visiting Molecular Mimicry Once More: Pathogenicity, Virulence, and Autoimmunity.

The concept of molecular mimicry describes situations in which antigen sharing between parasites and hosts could benefit pathogen evasion from host immune responses. However, antigen sharing can generate host responses to parasite-derived self-like peptides, triggering autoimmunity. Since its conception, molecular mimicry and the consequent potential cross-reactivity following infections have been repeatedly described in humans, raising increasing interest among immunologists. Here, we reviewed this concept focusing on the challenge of maintaining host immune tolerance to self-components in parasitic diseases. We focused on the studies that used genomics and bioinformatics to estimate the extent of antigen sharing between proteomes of different organisms. In addition, we comparatively analyzed human and murine proteomes for peptide sharing with proteomes of pathogenic and non-pathogenic organisms. We conclude that, although the amount of antigenic sharing between hosts and both pathogenic and non-pathogenic parasites and bacteria is massive, the degree of this antigen sharing is not related to pathogenicity or virulence. In addition, because the development of autoimmunity in response to infections by microorganisms endowed with cross-reacting antigens is rare, we conclude that molecular mimicry by itself is not a sufficient factor to disrupt intact self-tolerance mechanisms.

αCharges: partial atomic charges for AlphaFold structures in high quality.

The AlphaFold2 prediction algorithm opened up the possibility of exploring proteins' structural space at an unprecedented scale. Currently, >200 million protein structures predicted by this approach are deposited in AlphaFoldDB, covering entire proteomes of multiple organisms, including humans. Predicted structures are, however, stored without detailed functional annotations describing their chemical behaviour. Partial atomic charges, which map electron distribution over a molecule and provide a clue to its chemical reactivity, are an important example of such data. We introduce the web application αCharges: a tool for the quick calculation of partial atomic charges for protein structures from AlphaFoldDB. The charges are calculated by the recent empirical method SQE+qp, parameterised for this class of molecules using robust quantum mechanics charges (B3LYP/6-31G*/NPA) on PROPKA3 protonated structures. The computed partial atomic charges can be downloaded in common data formats or visualised via the powerful Mol* viewer.The αCharges application is freely available at https://alphacharges.ncbr.muni.cz with no login requirement.

Quantification of snake venom proteomes by mass spectrometry-considerations and perspectives.

The advent of soft ionization mass spectrometry-based proteomics in the 1990s led to the development of a new dimension in biology that conceptually allows for the integral analysis of whole proteomes. This transition from a reductionist to a global-integrative approach is conditioned to the capability of proteomic platforms to generate and analyze complete qualitative and quantitative proteomics data. Paradoxically, the underlying analytical technique, molecular mass spectrometry, is inherently nonquantitative. The turn of the century witnessed the development of analytical strategies to endow proteomics with the ability to quantify proteomes of model organisms in the sense of "an organism for which comprehensive molecular (genomic and/or transcriptomic) resources are available." This essay presents an overview of the strategies and the lights and shadows of the most popular quantification methods highlighting the common misuse of label-free approaches developed for model species' when applied to quantify the individual components of proteomes of nonmodel species (In this essay we use the term "non-model" organisms for species lacking comprehensive molecular (genomic and/or transcriptomic) resources, a circumstance that, as we detail in this review-essay, conditions the quantification of their proteomes.). We also point out the opportunity of combining elemental and molecular mass spectrometry systems into a hybrid instrumental configuration for the parallel identification and absolute quantification of venom proteomes. The successful application of this novel mass spectrometry configuration in snake venomics represents a proof-of-concept for a broader and more routine application of hybrid elemental/molecular mass spectrometry setups in other areas of the proteomics field, such as phosphoproteomics, metallomics, and in general in any biological process where a heteroatom (i.e., any atom other than C, H, O, N) forms integral part of its mechanism.

Assessment Of Acute Toxicity Of Chlorpyrifos And Its Sub-Lethal Effects On Protein Patterns Of Cyprinus Carpio(L.) By Using SDS-PAGE

The present study was focused on investigating the acute toxicity of chlorpyrifos and its impact on electrophoretic protein patterns in the tissues of Cyprinus carpio, an acute toxicity bioassay was conducted in the semi-static system and the fingerlings of C. carpio were exposed to various chlorpyrifos(50%EC)concentrations ranging from 0.1mg/L to 0.7mg/L for 96 h, and acute toxicity was determined as 0.318mg/L. Based on the LC50 value, the Cyprinus carpio were exposed to two sub-lethal concentrations viz., SL-1 (1/5th of LC50, 0.0636mg/l) and SL-2 (1/10th of LC50,0.0318 mg/l)  of chlorpyrifos. Proteins have a unique role in cell metabolism and proteomic analysis provides valuable information when variations that occur within the proteome of organisms are compared as a consequence of biological perturbations or external stimuli. The study's goal was to investigate the influence of chlorpyrifos on electrophoretic protein patterns by performing SDS-PAGE on muscle, liver, gill, and brain tissues after exposing fish to two sublethal concentrations  in a semi-static system for 3 weeks. The intensity of protein subunits in exposed tissues decreased, and some subunits disappeared, CPF influences both high-intensity protein bands and low intensity as well in both the sub-lethal concentrations. The study has suggested that the changes in the protein banding are more prominent in the SL-1 concentration of chlorpyrifos.The variations in protein subunits band patterns are due to protein deprivation of various proteins, proteolysis, or maybe insufficient amino acid incorporation into the polypeptide chain due to induced stress imposed by the toxicant.   &nbsp

Search and Identification of Amyloid Proteins

Amyloids are fibrillar proteins with a cross-β structure. Pathological amyloids are associated with the development of a number of incurable diseases, while functional amyloids regulate vital processes. The detection of unknown amyloids in living objects is a difficult task, and therefore the question of the prevalence and biological significance of amyloids remains open. We present a description of two methods, the combination of which makes it possible to find and identify amyloid proteins in the proteome of various organisms. The method of proteomic screening for amyloids allows the detection of the proteins that form SDS-resistant aggregates. SDS resistance is a general feature of amyloid fibrils. Protein aggregates resistant to SDS treatment can be collected by ultracentrifugation and further identified by mass spectrometry. However, in addition to amyloids, SDS-resistant aggregates contain some non-amyloid proteins. To test the amyloid properties of proteins identified by proteomic screening, we developed the method of fibril immunoprecipitation followed by Congo red staining and birefringence analysis. The methods of proteomic screening and immunoprecipitation of fibrillar proteins have been successfully tested and applied for the identification of amyloid proteins in yeast and vertebrates.

General features of transmembrane beta barrels from a large database.

Large datasets for particular classes of proteins can contribute a birds-eye view that is more accurate than that gathered from studying particular exemplars. We used co-evolution data in the form of predicted contact maps to create a large, high-quality database of transmembrane β-barrels (TMBB). We classify sequences for our database using IsItABarrel, an approach combining topology prediction and rule-based TMBB classification. By applying simple feature detection on generated contact maps, our method achieves 96% balanced accuracy when discriminating this structural fold from other protein classes. Our database contains almost two million bacterial TMBB proteins--significantly larger and more accurate than previous datasets. We assessed the TMBB-content of 2,959 proteomes of bacterial organisms across several taxonomic categories and find tremendous variance among TMBB-containing organisms with some having as many as 300 TMBBs (6.79% of proteome) and others as few as 10 (0.27% of proteome). The distribution of the lengths of the TMBBs is suggestive of previously hypothesized duplication events. In addition, we find that the C-terminal β-signal varies among different classes of bacteria and type of protein though it is most commonly GHyGHyGφ+φ. The database is available through our IsItABarrel webapp and we anticipate that its quality and size will serve as a useful resource where high quality TMBB sequence data is required.

Estimating Total Quantitative Protein Content in Escherichia coli, Saccharomyces cerevisiae, and HeLa Cells.

The continuous improvement of proteomic techniques, most notably mass spectrometry, has generated quantified proteomes of many organisms with unprecedented depth and accuracy. However, there is still a significant discrepancy in the reported numbers of total protein molecules per specific cell type. In this article, we explore the results of proteomic studies of Escherichia coli, Saccharomyces cerevisiae, and HeLa cells in terms of total protein copy numbers per cell. We observe up to a ten-fold difference between reported values. Investigating possible reasons for this discrepancy, we conclude that neither an unmeasured fraction of the proteome nor biases in the quantification of individual proteins can explain the observed discrepancy. We normalize protein copy numbers in each study using a total protein amount per cell as reported in the literature and create integrated proteome maps of the selected model organisms. Our results indicate that cells contain from one to three million protein molecules per µm3 and that protein copy density decreases with increasing organism complexity.

Detection of MicroRNAs by Northern Blot.

Small RNAs (sRNAs) are key regulators of transcriptomes and proteomes of organisms through their sequence-specific interaction with complementary RNA targets. sRNAs can be classified according to their origin and mode of action into different classes such as: microRNAs (miRNAs), small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). The abundance and specific spatio-temporal expression of many sRNAs, especially miRNAs, is relevant for their biological function. Northern blotting is a widely used technique to study sRNAs because it is quantitative, relatively inexpensive, and readily available for most laboratories. This chapter describes the protocols for radioactive and non-radioactive sRNA Northern blot analysis, which includes RNA extraction, polyacrylamide gel electrophoresis, membrane transfer, hybridisation and detection of sRNA using oligonucleotide probes. The protocol is described to prepare most of the reagents needed in the lab, but also timesaving commercial reagent alternatives are included. Suggestions and nuances obtained from experience are included as Notes.

Application of genetically modified microorganisms for potential human amyloids search

Amyloids are fibrous protein structures often found in patients with severe diseases, such as Alzheimers, Parkinsons diseases etc. A number of studies have shown that the production of heterologous amyloidogenic proteins in Saccharomyces cerevisiae strains results in formation of amyloid aggregates with properties similar to those found in mammals.
 Amyloid aggregates formed in yeasts usually do not have their own phenotypic manifestation. To assess amyloidogenic potential of individual proteins a yeast test-system was developed under supervision of Prof. Y.O. Chernoff. The system is based on usage of genetically modified S. cerevisiae cells auxotrophic for certain growth factors, allowing effective phenotypic selection to search for amyloidogenic proteins within proteomes of various organisms [1]. Using this test-system, our laboratory evaluated amyloid potential of a spectrum of human proteins, the amyloidogenicity of which was previously predicted by bioinformatics algorithms. The proteins that have shown amyloidogenic potential in yeast-based model are being currently tested in vitro and in vivo. Some mutant Escherichia coli strains can be applied for studying propensity of heterologous proteins to form amyloids in vitro. Thus, application of genetically modified microorganisms makes it possible to identify new human amyloidogenic proteins and to improve predictive ability of bioinformatics algorithms.
 The research is supported by RSF grant №20-14-00148 and by St. Petersburg State University (project No. 93025998). Authors acknowledge SPbSU Resource Centers Chromas, Molecular and Cell Technologies and Biobank.

Specific Features of the Proteomic Response of Thermophilic Bacterium Geobacillus icigianus to Terahertz Irradiation

Studying the effects of terahertz (THz) radiation on the proteome of temperature-sensitive organisms is limited by a number of significant technical difficulties, one of which is maintaining an optimal temperature range to avoid thermal shock as much as possible. In the case of extremophilic species with an increased temperature tolerance, it is easier to isolate the effects of THz radiation directly. We studied the proteomic response to terahertz radiation of the thermophilic Geobacillus icigianus, persisting under wide temperature fluctuations with a 60 °C optimum. The experiments were performed with a terahertz free-electron laser (FEL) from the Siberian Center for Synchrotron and Terahertz Radiation, designed and employed by the Institute of Nuclear Physics of the SB of the RAS. A G. icigianus culture in LB medium was THz-irradiated for 15 min with 0.23 W/cm2 and 130 μm, using a specially designed cuvette. The life cycle of this bacterium proceeds under conditions of wide temperature and osmotic fluctuations, which makes its enzyme systems stress-resistant. The expression of several proteins was shown to change immediately after fifteen minutes of irradiation and after ten minutes of incubation at the end of exposure. The metabolic systems of electron transport, regulation of transcription and translation, cell growth and chemotaxis, synthesis of peptidoglycan, riboflavin, NADH, FAD and pyridoxal phosphate cofactors, Krebs cycle, ATP synthesis, chaperone and protease activity, and DNA repair, including methylated DNA, take part in the fast response to THz radiation. When the response developed after incubation, the systems of the cell's anti-stress defense, chemotaxis, and, partially, cell growth were restored, but the respiration and energy metabolism, biosynthesis of riboflavin, cofactors, peptidoglycan, and translation system components remained affected and the amino acid metabolism system was involved.

Targeting plant UBX proteins: AI-enhanced lessons from distant cousins.

Across all eukaryotic kingdoms, ubiquitin regulatory X (UBX) domain-containing adaptor proteins control the segregase cell division control protein 48 (CDC48), and thereby also control cellular proteostasis and adaptation. The structures and biological roles of UBX proteins in animals and fungi have garnered considerable attention. However, their counterparts in plants remain markedly understudied. Since 2021, the artificial intelligence (AI)-based algorithm AlphaFold has provided predictions of protein structural features that can be highly accurate. Predictions of the proteomes of all major model organisms are now freely accessible to the entire research community through user-friendly web interfaces. We propose that the combination of cross-kingdom comparison with AF analysis produces a wealth of testable hypotheses to inspire and guide experimental research on plant UBX domain-containing (PUX) proteins.

Evaluation of acute toxicity of carbosulfan and its sub-lethal effects on protein patterns of Cyprinus carpio (L.) using SDS-PAGE

Among all types of water pollutants, pesticides are of the greatest concern. Carbosulfan is one such carbamate pesticide extensively employed in rural communities, that enters the aquatic environment by the proximity of agricultural lands to water bodies or through direct application in such environments. The consequences of these sublethal pollutants/toxicants exposures can be quantified in terms of biochemical, physiological, or the fish organism's histological reactions. Proteins have a unique role in cell metabolism and proteomic analysis provides valuable information when variations that occur within the proteome of organisms are compared as a consequence of biological perturbations or external stimuli. The study's goal was to investigate the acute toxicity of carbosulfan and its influence on electrophoretic protein patterns in the tissues of Cyprinus carpio. Fingerlings of C.carpio were subjected to various carbosulfan concentrations ranging from 2.6 mg/L to 4.6 mg/L for 96 h, and acute toxicity was determined as 3.41 mg/L. The comparison of the 96 h LC50 values between fish species shows that C.carpio is more resistant to carbosulfan. SDS-PAGE was performed on muscle, liver, gill, and brain tissues after exposing fish to two sublethal concentrations (1/10th and 1/5th of the 96 h LC50) in a semi-static system for 3 weeks. The intensity of protein subunits in carbosulfan-exposed tissues decreased, and some protein subunits disappeared. Carbosulfan attacks proteins having two or more domains easily in low concentrations, and as concentration increases, it begins to attack single domain proteins as well. Variations in protein subunits band patterns due to protein deprivation, proteolysis, or maybe insufficient amino acid incorporation into the polypeptide chain. The study concludes that carbosulfan provides a larger risk to the animal and alters the entire physiology of the fish. Given the importance of fish in human nutrition, biological monitoring of water and fish meat should be done frequently to ensure the continued safety of freshwater food. To prevent these pesticides and other toxins from entering the environment, safe disposal and recycling of domestic sewage and industrial wastes should be practiced and precautions should be taken while using even low concentrations of carbosulfan, and prohibiting or restricting carbosulfan usage is preferable.

Potential of Single Pulse and Multiplexed Drift-Tube Ion Mobility Spectrometry Coupled to Micropillar Array Column for Proteomics Studies.

Proteomics is one of the most significant methodologies to better understand the molecular pathways involved in diseases and to improve their diagnosis, treatment and follow-up. The investigation of the proteome of complex organisms is challenging from an analytical point of view, because of the large number of proteins present in a wide range of concentrations. In this study, nanofluidic chromatography, using a micropillar array column, was coupled to drift-tube ion mobility and time-of-flight mass spectrometry to identify as many proteins as possible in a protein digest standard of HeLa cells. Several chromatographic parameters were optimized. The high interest of drift-tube ion mobility to increase the number of identifications and to separate isobaric coeluting peptides was demonstrated. Multiplexed drift-tube ion mobility spectrometry was also investigated, to increase the sensitivity in proteomics studies. This innovative proteomics platform will be useful for analyzing patient samples to better understand unresolved disorders.

Proteoform Identification and Quantification Using Intact Protein Database Search Engine ProteinGoggle.

Proteomics studies the proteome of organisms, especially proteins that are differentially expressed under certain physiological or pathological conditions; qualitative identification of protein sequences and posttranslational modifications (PTMs) and their positions can help us systematically understand the structure and function of proteoforms. With the development and relative popularity of soft ionization technology (such as electrospray ionization technology) and high mass measurement accuracy and high-resolution mass spectrometers (such as orbitrap), the mass spectrometry (MS) characterization of complete proteins (the so-called top-down proteomics) has become possible and has gradually become popular. Corresponding database search engines and protein identification bioinformatics tools have also been greatly developed. This chapter provides a brief overview of intact protein database search algorithm "isotopic mass-to-charge ratio and envelope fingerprinting" and search engine ProteinGoggle.

The Road Less Traveled? Unconventional Protein Secretion at Parasite-Host Interfaces.

Protein secretion in eukaryotic cells is a well-studied process, which has been known for decades and is dealt with by any standard cell biology textbook. However, over the past 20 years, several studies led to the realization that protein secretion as a process might not be as uniform among different cargos as once thought. While in classic canonical secretion proteins carry a signal sequence, the secretory or surface proteome of several organisms demonstrated a lack of such signals in several secreted proteins. Other proteins were found to indeed carry a leader sequence, but simply circumvent the Golgi apparatus, which in canonical secretion is generally responsible for the modification and sorting of secretory proteins after their passage through the endoplasmic reticulum (ER). These alternative mechanisms of protein translocation to, or across, the plasma membrane were collectively termed “unconventional protein secretion” (UPS). To date, many research groups have studied UPS in their respective model organism of choice, with surprising reports on the proportion of unconventionally secreted proteins and their crucial roles for the cell and survival of the organism. Involved in processes such as immune responses and cell proliferation, and including far more different cargo proteins in different organisms than anyone had expected, unconventional secretion does not seem so unconventional after all. Alongside mammalian cells, much work on this topic has been done on protist parasites, including genera Leishmania, Trypanosoma, Plasmodium, Trichomonas, Giardia, and Entamoeba. Studies on protein secretion have mainly focused on parasite-derived virulence factors as a main source of pathogenicity for hosts. Given their need to secrete a variety of substrates, which may not be compatible with canonical secretion pathways, the study of mechanisms for alternative secretion pathways is particularly interesting in protist parasites. In this review, we provide an overview on the current status of knowledge on UPS in parasitic protists preceded by a brief overview of UPS in the mammalian cell model with a focus on IL-1β and FGF-2 as paradigmatic UPS substrates.