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Introns are excised from pre-messenger RNAs by the spliceosome, which produces mRNAs with continuous protein-coding information. In humans, most pre-mRNAs undergo alternative splicing to expand proteomic diversity. Cryo-electron microscopy (cryo-EM) structures of the yeast spliceosome elucidated how proteins stabilize and remodel an RNA-based active site to effect splicing catalysis. More recent cryo-EM snapshots of the human spliceosome reveal a complex protein scaffold and provide insights into the role of specific human proteins in modulating spliceosome activation, splice site positioning, and the ATPase-mediated dynamics of the active site. The emerging molecular picture highlights how, compared to its yeast counterpart, the human spliceosome has coopted additional protein factors to allow increased plasticity of splice site recognition and remodeling, and potentially to regulate alternative splicing.

This issue of the Biomedical Journal acquaints us with the compelling hypothesis that the vascular glycocalyx lies at the intersection of severe COVID-19 risk factors and damages, and the ways used by artificial intelligence to predict interactions between SARS-CoV-2 and human proteins. Furthermore, we explore the antiviral potential of valinomycin and the long list of COVID-19-related clinical trials, and learn how (not) to fix a broken femoral head. Last but not least, we get to enjoy the tale of the cellular oxygen-sensing system as well as the role of the host complement system during Leptospira infection, and learn that SARS-CoV-2 can sometimes come with a pathogenic plus one.

A cell contains numerous protein molecules. One of the fundamental goals in molecular cell biology is to determine their subcellular locations since this information is extremely important to both basic research and drug development. In this paper, we report a novel and very powerful predictor called "pLoc_bal-mHum" for predicting the subcellular localization of human proteins based on their sequence information alone. Cross-validation tests on exactly the same experiment-confirmed dataset have indicated that the new predictor is remarkably superior to the existing state-of-the-art predictor in identifying the subcellular localization of human proteins. To maximize the convenience for the majority of experimental scientists, a user-friendly web-server for the new predictor has been established at http://www.jci-bioinfo.cn/pLoc_bal-mHum/, by which users can easily get their desired results without the need to go through the detailed mathematics.

Computational identification of human-virus protein-protein interactions (PHIs) is a worthwhile step towards understanding infection mechanisms. Analysis of the PHI networks is important for the determination of path-ogenic diseases. Prediction of these interactions is a popular problem since experimental detection of PHIs is both time-consuming and expensive. The available methods use biological features like amino acid sequences, molecular structure, or biological activities for prediction. Recent studies show that the topological properties of proteins in protein-protein interaction (PPI) networks increase the performance of the predictions. The basic network projections, random-walk-based models, or graph neural networks are used for generating topologically enriched (hybrid) protein embeddings. In this study, we propose a three-stage machine learning pipeline that generates and uses hybrid embeddings for PHI prediction. In the first stage, numerical features are extracted from the amino acid sequences using the Doc2Vec and Byte Pair Encoding method. The amino acid embeddings are used as node features while training a modified GraphSAGE model, which is an improved version of the graph convolutional network. Lastly, the hybrid protein embeddings are used for training a binary interaction classifier model that predicts whether there is an interaction between the given two proteins or not. The proposed method is evaluated with comprehensive experiments to test its functionality and compare it with the state-of-art methods. The experimental results on the benchmark dataset prove the efficiency of the proposed model by having a 3–23% better area under curve (AUC) score than its competitors. Publisher's Version Q2 WOS:000858873400002 PMID: 36037723

Smokeless tobacco (SLT) products are consumed by millions of people in over 130 countries around the world. Consumption of SLT has been estimated to cause a number of diseases accounting to more than 0.65 million deaths per year. There is sufficient epidemiological evidence on the association of SLT products with nicotine addiction, cancers of oral cavity and digestive systems but there is a lack of understanding of the role of toxic chemicals in these diseases. We provide the first comprehensive in-silico analysis of chemical compounds present in different SLT products used worldwide. Many of these compounds are found to have good absorption, solubility and permeability along with mutagenic and toxic properties. They are also found to target more than 350 human proteins involved in a plethora of human biological processes and pathways. Along with all the previously known diseases, the present study has identified the association of compounds of SLT products with a number of unknown diseases like neurodegenerative, immune and cardiac diseases (Left ventricular non compaction, dilated cardiomyopathy etc). These findings indicate far-reaching impact of SLT products on human health than already known which needs further validations using epidemiological, in-vitro and in-vivo methodologies. Thus, this study will provide one stop information for the policy makers in development of regulatory policies on toxic contents of SLT products.

Pathogens infect host organisms by exploiting host cellular mechanisms and evading host defence mechanisms through molecular pathogen–host interactions (PHIs). Discovering new interactions between pathogen and human proteins is very crucial in understanding the infection mechanisms. By analysing interaction networks, the interactions responsible for infectious diseases can be detected and new drugs disabling these interactions can be delivered. In this paper, we propose a method based on Bayesian matrix factorization for predicting PHIs along with a projection-based technique and combine the results by employing an ensemble method. Furthermore, two features, target similarity and attacker similarity, are utilized for the first time in the literature for PHI prediction. The advantages of the proposed methods are two folds. Firstly, they relieve the need for negative samples which is significant since there is no available dataset providing negative samples for most of the pathogenic systems. Secondly, the experiments demonstrate that the proposed approach outperforms state-of-the-art methods; roughly 20% of top 50 predictions are among recently validated interactions. So, the search space for wet-lab experiments to obtain validated interactions can be considerably narrowed down from a huge number of possible interactions.

Intrinsically disordered proteins are proteins with intrinsically disordered regions (IDRs) that do not adopt a globular structure in their free state. IDRs have unique regions having protein-binding segments of which play pivotal roles in many biological processes. The binding sites in IDRs are heterogeneous in terms of their sequence conservation: some are conserved and others are not. We have been running a database of intrinsically disordered proteins, IDEAL, and collecting such binding segments, which are called protean segments (ProSs), within it. In this study, we compared the sequence conservation of ProSs, structural domains (SDs), and IDRs other than ProSs (non-ProSs) and found that i) functionally constrained residues in ProSs tend to be conserved, ii) the distribution of conservation scores of ProSs is similar to those of SDs but not non-ProSs, and iii) ProSs found in human proteins are mostly conserved only in vertebrates. These results indicate that the conservation patterns in ProSs principally follow the general rules found in SDs. However, we need to consider evolutionary distance when comparing IDR sequences because ProSs can readily emerge and disappear over the course of protein evolution. Moreover, many ProSs may remain to be identified, which may account for the heterogeneity of the sequence conservation of IDRs.

Network-based learning enables the identification of possible undiscovered interactions in biological systems. In this issue of Patterns, Du et al. show that applying these methods to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reveals potential infection targets of the virus and possible interactions between SARS-CoV-2 proteins and human proteins.

S100A6 is a Ca2+-signal transducer that interacts with numerous proteins and regulates their biochemical functions. Here we identified a centrosomal protein, FOR20 (FOP-related protein of 20 kDa) as a novel S100A6 target by screening protein microarrays carrying 19,676 recombinant GST-fused human proteins. Binding experiments revealed that S100A6 interacts with the N-terminal region (residues 1–30) of FOR20 in a Ca2+-dependent manner in vitro and in living cells. Several S100 proteins including S100A1, A2, A4, A11, B also exhibited Ca2+-dependent interactions with FOR20 as well as S100A6. We found that two distantly related centrosomal proteins, FOP and OFD1, also possess N-terminal regions with a significant sequence similarity to the putative S100A6-binding site (residues 1–30) in FOR20 and are capable of binding to S100A6 in a Ca2+-dependent manner. Taken together, these results may indicate that S100A6 interacts with FOR20 and related centrosomal proteins through a conserved N-terminal domain, suggesting a novel Ca2+-dependent regulation of centrosomal function.

A microfluidic platform optimized for bead-based immunoassays is presented. It uses a simple design that incorporates passive valves in the inlet and outlet of a microreactor. The superhydrophobic, passive valves are created by selective plasma nanotexturing and deposition of a hydrophobic layer. The anti-sticking operation of the valves simplify the working principle, reduce the reagents needed, and enhance the fluorescence intensity of the assays performed on chip by reducing non-specific binding compared to assays performed on plate. Two different multi-step immunoassays were tested individually and as a mixture to evaluate the platform performance: IL6 and PDGF-2 human protein assays. The results show that the proposed platform offers faster assay kinetics, allows lower reagent use (5l instead of 50), enhanced fluorescent signals (by a factor of two or more), and reduced incubation time at least by a factor of 2. A possible configuration using the microfluidic platform for a portable biosensing system is also demonstrated. Display Omitted Superhydrophobic valves are incorporated inside a microfluidic.IL6 & PDGF-2 human proteins are detected using an antifouling LOC device.Time of analysis and sensitivity are improved using the LOC device.A portable biosensing system is demonstrated.

NeXtProt is a web-based protein knowledge platform that supports research on human proteins. NeXtProt (release 2015-04-28) lists 20,060 proteins, among them, 3373 canonical proteins (16.8%) lack credible experimental evidence at protein level (PE2:PE5). Therefore, they are considered as "missing proteins". A comprehensive bioinformatic workflow has been proposed to analyze these "missing" proteins. The aims of current study were to analyze physicochemical properties, existence and distribution of the tryptic cleavage sites, and to pinpoint the signature peptides of the missing proteins. Our findings showed that 23.7% of missing proteins were hydrophobic proteins possessing transmembrane domains (TMD). Also, forty missing entries generate tryptic peptides were either out of mass detection range (30aa) or mapped to different proteins (9aa). Additionally, 21% of missing entries didn't generate any unique tryptic peptides. In silico endopeptidase combination strategy increased the possibility of missing proteins identification. Coherently, using both mature protein database and signal peptidome database could be a promising option to identify some missing proteins by targeting their unique N-terminal tryptic peptide from mature protein database and or C-terminus tryptic peptide from signal peptidome database. In conclusion, Identification of missing protein requires additional consideration during sample preparation, extraction, digestion and data analysis to increase its incidence of identification.

Reports of mass spectrometry based assays for peptides and proteins have become increasingly common in the literature. The growing interest of mass spectrometry for use in clinical laboratories has been primarily driven by the inherent selectivity of the platform relative to more traditional platforms such as immunoassays. However, the adoption of mass spectrometry for peptide and protein analysis in the clinic has been relatively slow compared its adoption in non-clinical laboratories such as in biomarker discovery efforts or within laboratories that support pharmaceutical and academic research. Here, we review some of the successful reports of MS based assays for human proteins in multiple stages of assay research, and describe how and why the platform was employed in order to demonstrate where and when mass spectrometry based assays will have value in the future.

Crosstalk between the microbiome and the human host is mediated by specific ligand-receptor interactions involving microbially generated metabolites that can be either agonists or antagonists of human proteins. The evolved co-compatibility of gut microbiota with human systems points to a potentially rich area for discovering new drug-like molecules that are both highly specific modulators of human pathways and derisked for adverse effects. In this review, we discuss the rapidly growing research into the role of microbial metabolites in human health and suggest potential strategies for developing these molecules into therapeutic agents.

The bow-and-arrow was a widely used weapon in the Postclassic and Contact periods in the Maya lowlands. A sample of 108 arrow points from varied archaeological contexts in the lakes region of central Peten, northern Guatemala, was submitted for cross-over immunoelectrophoresis (CIEP) analysis. Analysis resulted in 25 positive matches to available antisera for a wide range of local and introduced fauna, from small and large land mammals to avians. These findings indicate possible uses in subsistence and ritual, as well as the first immunological identification of human proteins from projectile weaponry in Mesoamerica. This study did not reveal strong correlations between targeted fauna and point morphology, although larger points were likely used for larger game.

Many eukaryotic proteins exert their physiological function in specific cellular compartments. Proteins of the inter-membrane space (IMS) of mitochondria, for example, are synthesized in the cytoplasm and translocate to the IMS, where they are further processed to their mature form. In-cell Nuclear Magnetic Resonance (NMR) has proven to be an ideal approach to investigate eukaryotic proteins at the atomic level, inside the cytoplasm. Here we show that proteins inside intact mitochondria isolated from human cells can be structurally characterized by NMR (in-mitochondria NMR). By this approach, we characterized the folding and maturation state of two human proteins in the IMS, SOD1 and Mia40. Both observed proteins were in the folded state. Mia40 was in the oxidized, functional state, while SOD1 disulfide bond formation was promoted by increasing the level of the SOD1 chaperone, CCS, in the IMS.

Proteins are responsible for performing the vast majority of cellular functions which are critical to a cell's survival. The knowledge of the subcellular localization of proteins can provide valuable information about their molecular functions. Therefore, one of the fundamental goals in cell biology and proteomics is to analyze the subcellular localizations and functions of these proteins. Recent large-scale human genomics and proteomics studies have made it possible to characterize human proteins at a subcellular localization level. In this study, according to the annotation in Swiss-Prot, 8842 human proteins were classified into seven subcellular localizations. Human proteins in the seven subcellular localizations were compared by using topological properties, biological properties, codon usage indices, mRNA expression levels, protein complexity and physicochemical properties. All these properties were found to be significantly different in the seven categories. In addition, based on these properties and pseudo-amino acid compositions, a machine learning classifier was built for the prediction of protein subcellular localization. The study presented here was an attempt to address the aforementioned properties for comparing human proteins of different subcellular localizations. We hope our findings presented in this study may provide important help for the prediction of protein subcellular localization and for understanding the general function of human proteins in cells.

Graphical abstract, Highlights ⿢ Optimized protocol to collect the secretome of human BM-MSC. ⿢ Truly secreted proteins may be difficult to detect for fetal bovine serum presence. ⿢ Cells should be transferred into a serum-free medium prior to secretome collection., The analysis of mesenchymal stromal cells secretome is fundamental to identify key players of processes involving these cells. Truly secreted proteins may be difficult to detect in MS based analysis of conditioned media (CM) due to proteins supplemented with fetal bovine serum (FBS). We compared different growth conditions to determine the effect of varying FBS concentration on the number and quantity of truly secreted human proteins vs contaminating bovine proteins. The results suggest that to minimize interference cells should be grown in presence of FBS until confluence and transferred into a serum-free medium prior to secretome collection.

The task of recognizing and normalizing protein name mentions in biomedical literature is a challenging task and important for text mining applications such as protein–protein interactions, pathway reconstruction and many more. In this paper, we present ProNormz, an integrated approach for human proteins (HPs) tagging and normalization. In Homo sapiens, a greater number of biological processes are regulated by a large human gene family called protein kinases by post translational phosphorylation. Recognition and normalization of human protein kinases (HPKs) is considered to be important for the extraction of the underlying information on its regulatory mechanism from biomedical literature. ProNormz distinguishes HPKs from other HPs besides tagging and normalization. To our knowledge, ProNormz is the first normalization system available to distinguish HPKs from other HPs in addition to gene normalization task. ProNormz incorporates a specialized synonyms dictionary for human proteins and protein kinases, a set of 15 string matching rules and a disambiguation module to achieve the normalization. Experimental results on benchmark BioCreative II training and test datasets show that our integrated approach achieve a fairly good performance and outperforms more sophisticated semantic similarity and disambiguation systems presented in BioCreative II GN task. As a freely available web tool, ProNormz is useful to developers as extensible gene normalization implementation, to researchers as a standard for comparing their innovative techniques, and to biologists for normalization and categorization of HPs and HPKs mentions in biomedical literature. URL: http://www.biominingbu.org/pronormz.

The veterinary vaccine market is forecast to grow consistently over the next several years [12xVeterinary Vaccines Market to 2017. GBI Research. See all References[12]. Moreover, the vaccine market for companion animals is growing more rapidly than any other animal health sector [13xSee all References[13]. From a technological standpoint, research and innovation will increasingly focus on knowledge-based engineered products and the development of more combination vaccines and marker vaccines.An alternative approach is the application of approved human drugs for veterinary purposes. The Animal Medicinal Drug Use Clarification Act of 1994 allows veterinarians in the USA to prescribe approved human drugs for animals under certain conditions. This approach is constrained, however, by product costs and by likely immunological reaction of animals to human proteins. Overall, therefore, the field of veterinary biopharmaceuticals is likely to remain distinct from the human biopharmaceutical sector into the future.

A total of 26 toxicology studies performed with biological drugs (monoclonal antibodies and related immunoglobulins as well as recombinant human proteins) in the primate have been evaluated to give an insight into the types of study designs used and results. The evaluation involved examination of pivotal repeat dose studies which ranged from 2 to 13 weeks in duration, used to support early clinical investigation. Either no findings were seen or restricted to those which could largely be explained by the drug's pharmacology. Based on these results and supporting findings from a literature review of other similar drugs in development or approved for marketed use, a case has been made to revisit aspects of the standard design approach of toxicology studies for biological drugs. Thus, consideration should be given to a move away from the use of three drug-treated and numerous non-dose recovery groups currently used to support initial clinical entry to a robust toxicological assessment that could be achieved in many cases with one control and one or two drug-treated groups and with non-dose recovery groups only for the control and highest level used. An argument for not routinely measuring for anti-drug antibodies unless there is a specific drug-related reason is also made.

The effect of two Escherichiacoli expression strains on the production of recombinant human protein fragments was evaluated. High-throughput protein production projects, such as the Swedish Human Protein Atlas project, are dependent on high protein yield and purity. By changing strain from E. coli BL21(DE3) to E. coli Rosetta(DE3) the overall success rate of the protein production has increased dramatically. The Rosetta(DE3) strain compensates for a number of rare codons. Here, we describe how the protein expression of human gene fragments in E. coli strains BL21(DE3) and Rosetta(DE3) was evaluated in two stages. Initially a test set of 68 recombinant proteins that previously had been expressed in BL21(DE3) was retransformed and expressed in Rosetta(DE3). The test set generated very positive results with an improved expression yield and a significantly better purity of the protein product which prompted us to implement the Rosetta(DE3) strain in the high-throughput protein production. Except for analysis of protein yield and purity the sequences were also analyzed regarding number of rare codons and rare codon clusters. The content of rare codons showed to have a significant effect on the protein purity. Based on the results of this study the atlas project permanently changed expression strain to Rosetta(DE3).

A number of diseases are treated by passive administration of human proteins. Human coagulation factor VIII (FVIII) is one such protein which is administered to hemophilia A patients in order to manage and treat hemorrhagic incidences. This mode of therapy suffers from the side effect of generating anti-FVIII antibodies (inhibitors) which neutralizes the function of the infused FVIII. At a time when efficient viral screening procedures are at place, development of inhibitors poses the greatest threat to such a therapy. Various predisposing factors, both patient and product-related, are responsible for the development of inhibitory antibodies. A proper understanding of these "risk-factors" would eventually help to better design therapeutic regimen to tackle hemophilia A.

The ability to identify classically secreted proteins is an important component of targeted therapeutic studies and the discovery of circulating biomarkers. Here, we review some of the most recent programs available for the in silico prediction of secretory proteins, the performance of which is benchmarked with an independent set of annotated human proteins. The description of these programs and the results of this benchmarking provide insights into the most recently developed prediction programs, which will enable investigators to make more informed decisions about which program best addresses their research needs.

Gene Ontology (GO) is a common language for the functional annotation of gene products. We have developed a computational tool, GOKey, to predict the GO function of proteins based on their sequence features and the support vector machine (SVM) method. Several measures, including improved handling of the problem caused by unbalanced positive and negative training data and postprocessing strategies to evaluate the posterior probability and statistical significance of SVM outputs, have been adopted to improve the prediction performance of GOKey. The GOKey has been trained to predict the 36 GO categories of the 'molecular function' of GO slims, and could be easily extended to other GO categories. The results of 5-fold cross validation with 10,603 GO-mapped proteins demonstrate that the performance of GOKey is better than that of standard SVMs. Comparisons with other computational tools for GO function prediction also show that the performance of GOKey is satisfactory. Further, GOKey has been applied to predict the GO functions for 5381 novel human proteins in the Ensembl database. The results show that 93% of the novel proteins can be assigned one or more GO terms, and some evidences supporting the predictions have been found. GOKey can be accessed at http://infosci.hust.edu.cn.

This review outlines the concept of population proteomics and its implication in the discovery and validation of cancer-specific protein modulations. Population proteomics is an applied subdiscipline of proteomics engaging in the investigation of human proteins across and within populations to define and better understand protein diversity. Population proteomics focuses on interrogation of specific proteins from large number of individuals, utilizing top-down, targeted affinity mass spectrometry approaches to probe protein modifications. Deglycosylation, sequence truncations, side-chain residue modifications, and other modifications have been reported for myriad of proteins, yet little is know about their incidence rate in the general population. Such information can be gathered via population proteomics and would greatly aid the biomarker discovery efforts. Discovery of novel protein modifications is also expected from such large scale population proteomics, expanding the protein knowledge database. In regard to cancer protein biomarkers, their validation via population proteomics-based approaches is advantageous as mass spectrometry detection is used both in the discovery and validation process, which is essential for the detection of those structurally modified protein biomarkers.

Much attention has been paid recently to proteins with partially or fully disordered structures, which are found to exist mostly in eukaryotes and are involved mainly in pivotal cellular processes such as transcriptional regulation, translation and cellular signal transduction. Long disordered sequences are sometimes inserted within the single structural domains of proteins, forming loops from the molecular surface. Such intrinsically disordered loops (IDLs) either are invisible in X-ray crystallography, or hamper protein crystallization itself due to great flexibility. Perhaps because of this, such long disordered sequences have not been characterized adequately. Here, we propose an informational method that stringently identifies IDLs in the structural domains of proteins using the amino acid sequence alone. A genome-wide survey of human proteins conducted with the method identified 50 IDL-containing proteins, several of which have experimentally determined 3D structures. Similar searches in other entirely sequenced organisms revealed that IDLs are prevalent in eukaryotes, while they are much less so in prokaryotes. As there is a statistically significant coincidence between the boundaries of IDLs and those of exons, we suggest that IDLs were produced mainly by exon addition in eukaryotes. IDLs are almost always located at the surface of proteins and are enriched with hydrophilic residues, and IDL-containing proteins tend to be intracellular. Some of the well-characterized proteins with IDLs illustrate that IDLs play pivotal roles in the switching of intracellular signaling or regulatory functions, suggesting that IDL insertion is an effective way to create functionally different domain variants.

This paper reports reconstitution of 5'-nick-directed mismatch repair using purified human proteins. The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. In this system, MutSbeta plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSalpha, which efficiently corrects both types of heteroduplexes. MutLalpha reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLalpha, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch. Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutSalpha-MutLalpha complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision.

The instrument-cleaning step is recognised as critical in achieving sterility or high-level disinfection in the medical instrument reprocessing cycle. Proteins in the residual bioburden on instruments after their use present a serious challenge to successful reprocessing, as they can adversely affect both chemical and physical sterilisation. The commercial products used in the cleaning step have a variety of functionalities, one of the most important of which is the elimination of human proteins from the instrument prior to the sterilisation/high level disinfection step. The methodology employed in this study is SDS-PAGE, a gel electrophoresis technique, which is a standard for determining protein molecular weights. Some proteins have low water-solubility and can be reliably eliminated only when broken down into small water-soluble fragments. The study involves the comparison of protein molecular weights before and after treatment with various cleaning products to give a clear indication of protein digestion efficacy. The study concludes that many products claiming protein digestion properties have virtually no efficacy in this regard, whilst others tested are highly effective. This is alarming since most of the products tested claim 'removal of all proteins' within two minutes.

The short consensus repeat domain (SCR, complement control protein module, sushi-domain) is a structural unit found in multiple adjacent copies in more than 40 human proteins. Each bead-like domain is composed of approximately 60 residues and the adjacent domains are connected in a head-to-tail fashion with linkers that consist of two to 12 amino acid residues. Based on experimentally determined structures the neighbouring SCR domains interact with each other at the so-called hinge or interdomain contact region. The functions mediated by the SCR domains have been studied using mutagenesis but the possible effects of the mutations on the hinge regions and interdomain angles have not been analysed. In this study, the linker and three loops in conserved locations were found to be responsible for the interdomain contact regions of all the solved experimental structures. The interdomain contact regions were identified in sequences of 140 human SCR domain pairs, and distinct hydrophobic and charge features were found in different subsets of SCR proteins and functional domains. To compare the possible associations of the interdomain contact region characteristics to the interdomain orientations all the experimentally solved SCR structures were subjected to a uniform calculation of tilt, twist, and skew angles that define the interdomain orientation. The twist and skew angles were found to have a linear correlation and the spatial location of one loop of the N-terminal domain (N#1) was found to have an effect on the skew angle. Thus, we describe location of the interdomain contact regions in primary structures of SCR domains and report that the orientation of adjacent SCR domains is not random and depends partially on the interdomain contact regions. On the basis of these results, mutations within the interdomain contact regions and subsequent loss-of-function effects caused by changes in the interdomain orientation can be avoided in mutagenesis studies.

Mismatch-provoked excision directed by a strand break located 3′ or 5′ to the mispair has been reconstituted using purified human proteins. While MutSα, EXOI, and RPA are sufficient to support hydrolysis directed by a 5′ strand break, 3′ directed excision also requires MutLα, PCNA, and RFC. EXOI interacts with PCNA. RFC and PCNA suppress EXOI-mediated 5′ to 3′ hydrolysis when the nick that directs excision is located 3′ to the mispair and activate 3′ to 5′ excision, which is dependent on loaded PCNA and apparently mediated by a cryptic EXOI 3′ to 5′ hydrolytic function. By contrast, RFC and PCNA have only a limited effect on 5′ to 3′ excision directed by a 5′ strand break.

Since the early 1980s when gonadotrophins were first routinely used in ovarian stimulation regimens, there have been important advances in pharmaceutical product development, leading to the availability today of highly purified and consistent human gonadotrophins produced by recombinant technology, that have benefited cycle management and treatment outcomes. This 'evolution' has introduced scientifically robust and 'state of the art' processes and equipment to ensure that the product is consistent, pure and safe, and is the optimal way to reduce risk of any viral, protein, and bacterial contamination. Production of human proteins by recombinant technology has resulted in improvements in product purity, consistency and delivery as well as enabling the design of future generations of gonadotrophins (e.g. longer-acting forms and orally active mimetics).

Background Protein cross-linking and fluorescence are widely recognized markers of oxidative aging in human proteins. Oxidative protein aging is a combinatorial process in which diversity arises from the heterogeneity of the targets and is amplified by the nonselective nature of the reactants. The cross-links themselves defy analysis because they are generally embedded in a covalent matrix. Arthropods rely upon oxidative cross-linking in the hardening of the cuticle — a process known as sclerotization. Among arthropods, scorpions are noteworthy in that the process of sclerotization is accompanied by the buildup of strong visible fluorescence. To date, the nature of the fluorescent species has remained a mystery. Results We have identified one of the soluble fluorescent components of the scorpions Centuroides vittatus and Pandinus Imperator as β-carboline — a tryptophan derivative that has previously been identified by hydrolysis and oxidation of lens protein. We have also shown that β-carboline-3-carboxylic acid is released from both scorpion exuvia (the shed cuticle) and human cataracts upon hydrolysis, suggesting that the protein-bound β-carboline and free β-carboline have common chemical origins. Conclusions Cataractogenesis and cuticular sclerotization are disparate oxidative processes — the former is collateral and the latter is constitutive. The common formation of β-carbolines shows that similar patterns of reactivity are operative. These fundamental mechanisms provide predictive insight into the consequences of human protein aging.

Immunoadhesins are chimeric, antibody-like molecules that combine the functional domain of a binding protein, usually a receptor, ligand, or cell-adhesion molecule, with immunoglobulin constant domains, usually including the hinge and Fc regions. Immunoadhesins have many applications as research tools, for example, in studies of receptor–ligand interactions. In addition, human immunoadhesins have potential applications in the clinic and may provide an alternative to human monoclonal antibodies as agents that recognize and neutralize foreign or human proteins that possess pathologic functions.

Cytokines are key mediators of the immune system, dictating the quality of the host response to infection. The importance of such immune mediators to the development of immune and inflammatory responses has emerged from work in mouse and man, however it has now become necessary to produce the equivalent (and novel) cytokines in ruminants. Over the past three years recombinant DNA techniques have allowed the cloning of numerous ovine cytokines. These include interleukins -1, -2 and -3 (IL-1, -2 and -3), interferon-gamma (IFN-gamma), ovine trophoblast protein (oTP-1), tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The predicted amino-acid sequences of these ovine proteins show varying degrees of similarity with the equivalent human proteins thus explaining why some of the cytokines are not biologically cross-reactive between species. Recombinant ovine proteins have been produced for IFN-gamma, oTP-1, IL-1, IL-3 and GM-CSF. Their biological activities are very similar to those of their human counterparts. Although it is too early to tell whether the recombinant ovine proteins will be of use in the treatment or prophylaxis of infectious disease, work in cattle and pigs has indicated the potential usefulness of cytokines in this role.

The field of protein therapy has flourished in the past few years, mainly due to the advent of recombinant DNA techniques and large-scale manufacturing processes. It was believed that human-derived proteins would eliminate the problems of short circulating lives, low stability and immunogenicity associated with the use of non-human proteins as drugs. Unfortunately, it has been demonstrated that many human proteins still suffer from short circulating lives and low stability, and often require the use of large doses to maintain therapeutic efficacy. This in turn increases the chances for the development of an adverse immune response, or at least for the development of clearing antibodies. The discovery that the modification of proteins with poly(ethylene glycol) (PEGNOLOGYSM) results in an adduct with decreased to non-existent immunogenicity increased circulating life, increased stability and opened the way for the development of protein pharmaceuticals that would realize the promise of protein therapy. In this paper, we will review the clinical value of PEGNOLOGYSM as researched by Enzon Inc. and discuss the future for this technology.

Human proteins, including enzymes, manufactured with recombinant-DNA yeasts can be used to treat a variety of medical conditions; some redesign of molecular structure (and production techniques) should increase their specificity, efficacy and immunotolerance. Prevention and treatment of diseases due to reactive oxygen species (ROS) and reactive nitrogen species (RNS) may become possible. Therapeutic vaccines for some diseases may be produced by design-specification for particular desirable protein features, on an individual patient basis.

We employed inside-out patch-clamp technique to investigate the mechanism of CFTR inhibition by a thiazolidinone derivative, CFTRinh-172. We found that application of CFTRinh-172 results in an increase of the mean closed time, and a decrease of the mean open time of WT-CFTR channels. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. We have shown that though CFTRinh-172 can bind to both open and closed state of the channel, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance following binding of the inhibitor to the channel. Our data also indicate that stabilization of the open state leads to more potent inhibition: K1/2 is reduced to nanomolar range for locked-open CFTR channels. Interestingly, we found that although there is >95% homology between pig and human proteins, pig CFTR is significantly less sensitive to CFTRinh-172 (K1/2 = 8.05 ± 3.38 μM for pig CFTR vs K1/2 = 1.24 ± 0.14 μM for human CFTR). This result is especially puzzling since the open time for pig CFTR is ∼5 times longer than that of human CFTR. Like its human counterpart, pig CFTR, locked open with PPi, becomes more sensitive to CFTRinh-172 (K1/2 = 2.21 ± 0.41 μM). However, compared to human CFTR locked open with PPi, pig channels are >100 fold less sensitive to the inhibitor. We also found that dog CFTR behaves very similarly to pig CFTR, including a longer open time and a lower sensitivity to CFTRinh-172. Sequence analysis and chimera approach allowed us to reveal the region between the R- and NBD2- domains as largely responsible for this difference in sensitivity. Experiments further identifying responsible amino acid residue(s) are under way.

Protein-DNA interactions (PDIs) mediate a broad range of functions essential for cellular differentiation, function, and survival. However, it is still a daunting task to comprehensively identify and profile sequence-specific PDIs in complex genomes. Here, we have used a combined bioinformatics and protein microarray-based strategy to systematically characterize the human protein-DNA interactome. We identified 17,718 PDIs between 460 DNA motifs predicted to regulate transcription and 4,191 human proteins of various functional classes. Among them, we recovered many known PDIs for transcription factors (TFs). We also identified a large number of new PDIs for known TFs, as well as for previously uncharacterized TFs. Remarkably, we found that over three hundred proteins not previously annotated as TFs also showed sequence-specific PDIs, including RNA binding proteins, mitochondrial proteins, and protein kinases. One of such unconventional DNA-binding proteins, MAPK1, acts as a transcriptional repressor for interferon gamma-induced genes.

PPL Therapeutics (Edinburgh, UK) have announced the birth of two lambs (Cupid and Diana) carrying a gene that was inserted at a specific chromosomal site. It has been possible to create transgenic livestock animals for some time, but the novelty of the new technique lies in the combination of two techniques: (1) replacing a specific gene in vitro, followed by (2) nuclear transfer (a la Dolly). PPL are intending to use this technology to produce large amounts of human proteins, uncontaminated by their ovine or bovine equivalents, in the milk of farm animals. One potential application is the production of human serum albumin, used to treat burn victims. This is currently produced from human serum.