Presynaptic Calmodulin targets: lessons from structural proteomics

Introduction: Calmodulin (CaM) is a highly conserved Ca²⁺-binding protein that is exceptionally abundant in the brain. In the presynaptic compartment of neurons, CaM transduces changes in Ca²⁺ concentration into the regulation of synaptic transmission dynamics.

Areas covered: We review selected literature including published CaM interactor screens and outline established and candidate presynaptic CaM targets. We present a workflow of biochemical and structural proteomic methods that were used to identify and characterize the interactions between CaM and Munc13 proteins. Finally, we outline the potential of ion mobility-mass spectrometry (IM-MS) for conformational screening and of protein-protein cross-linking for the structural characterization of CaM complexes.

Expert commentary: Cross-linking/MS and native MS can be applied with considerable throughput to protein mixtures under near-physiological conditions, and thus effectively complement high-resolution structural biology techniques. Experimental distance constraints are applicable best when obtained by combining different cross-linking strategies, i.e. by using cross-linkers with different spacer length and reactivity, and by using the incorporation of unnatural photo-reactive amino acids. Insights from structural proteomics can be used to generate CaM-insensitive mutants of CaM targets for functional studies in vitro or ideally in vivo.     

Role of Prion protein-EGFR multimolecular complex during neuronal differentiation of human dental pulp-derived stem cells

Cellular prion protein (PrP^C) is expressed in a wide variety of stem cells in which regulates their self-renewal as well as differentiation potential. In this study we investigated the presence of PrP^C in human dental pulp-derived stem cells (hDPSCs) and its role in neuronal differentiation process. We show that hDPSCs expresses early PrP^C at low concentration and its expression increases after two weeks of treatment with EGF/bFGF. Then, we analyzed the association of PrP^C with gangliosides and EGF receptor (EGF-R) during neuronal differentiation process. PrP^C associates constitutively with GM2 in control hDPSCs and with GD3 only after neuronal differentiation. Otherwise, EGF-R associates weakly in control hDPSCs and more markedly after neuronal differentiation.

To analyze the functional role of PrP^C in the signal pathway mediated by EGF/EGF-R, a siRNA PrP was applied to ablate PrP^C and its function. The treatment with siRNA PrP significantly prevented Akt and ERK1/2 phosphorylation induced by EGF.

Moreover, siRNA PrP treatment significantly prevented neuronal-specific antigens expression induced by EGF/bFGF, indicating that cellular prion protein is essential for EGF/bFGF-induced hDPSCs differentiation.

These results suggest that PrP^C interact with EGF-R within lipid rafts, playing a role in the multimolecular signaling complexes involved in hDPSCs neuronal differentiation.     

Mass spectrometric analysis of glycosylated viral proteins

Introduction: Viral diseases contribute much to human and animal suffering and enormous efforts are directed at developing appropriate vaccines for protection. Glycoproteins constitute much of the viral surfaces and are obvious targets for such vaccine development. This review describes mass spectrometric methods used for the structural determination of these compounds.

Areas covered: The review describes the structures of the N- and O-linked glycans found on glycoproteins and mass spectrometric methods for their ionization and fragmentation. The steps, such as determination of glycan attachment sites and the structures of the attached glycans following their release from the glycoproteins are described and examples are given of the uses of the various analytical methods using mainly influenza, Ebola and HIV as representative examples. Also included are tables listing work on many other viruses.

Expert commentary: Recent technological advances, such as the introduction of ion mobility techniques, have greatly improved analyses in this area and have enabled larger amounts of information to be gathered in shorter time periods on ever smaller amounts of material. Such techniques should greatly accelerate the discovery of vaccine targets and lead to the production of vaccines for diseases not currently available.     

The landscape of viral proteomics and its potential to impact human health

Introduction: Advances in mass spectrometry-based proteomic technologies are enhancing studies of viral pathogenesis. Identification and quantification of host and viral proteins and modifications in cells and extracellular fluids during infection provides useful information about pathogenesis, and will be critical for directing clinical interventions and diagnostics.

Areas covered: Herein we review and discuss a broad range of global proteomic studies conducted during viral infection, including those of cellular responses, protein modifications, virion packaging, and serum proteomics. We focus on viruses that impact human health and focus on experimental designs that reveal disease processes and surrogate markers.

Expert commentary: Global proteomics is an important component of systems-level studies that aim to define how the interaction of humans and viruses leads to disease. Viral-community resource centers and strategies from other fields (e.g., cancer) will facilitate data sharing and platform-integration for systems-level analyses, and should provide recommended standards and assays for experimental designs and validation.     

Soluble CD40 ligand in haemodialysis patients: survival impact and cardiovascular prognostic role

Context: Soluble CD40 ligand (sCD40l) can predict cardiovascular events (CVE) and mortality in haemodialysis (HD) patients (short-, medium-term follow-up studies).

Objective: To evaluate the relationship between sCD40l and survival, CVE and mortality in HD patients on long-term follow-up.

Methods: We registered 46?HD patients’ baseline characteristics, mortality and CVE for 108 months.

Results: SCD40l correlated positively with C-reactive protein, was higher in survivors, but had no impact on survival and was not predictive for CVE or CV mortality.

Conclusion: The levels of sCD40l have no influence on survival or CVE and mortality in HD patients in a long-term follow-up.     

Protein complexes,big data,machine learning and integrative proteomics: lessons learned over a decade of systematic analysis of protein interaction networks

Overview: Elucidation of the networks of physical (functional) interactions present in cells and tissues is fundamental for understanding the molecular organization of biological systems, the mechanistic basis of essential and disease-related processes, and for functional annotation of previously uncharacterized proteins (via guilt-by-association or -correlation). After a decade in the field, we felt it timely to document our own experiences in the systematic analysis of protein interaction networks.

Areas covered: Researchers worldwide have contributed innovative experimental and computational approaches that have driven the rapidly evolving field of ‘functional proteomics’. These include mass spectrometry-based methods to characterize macromolecular complexes on a global-scale and sophisticated data analysis tools – most notably machine learning – that allow for the generation of high-quality protein association maps.

Expert commentary: Here, we recount some key lessons learned, with an emphasis on successful workflows, and challenges, arising from our own and other groups’ ongoing efforts to generate, interpret and report proteome-scale interaction networks in increasingly diverse biological contexts.     

Molecular dynamics and combined docking studies for the identification of Zaire ebola virus inhibitors

Ebola virus (EBOV) is a lethal human pathogen with a risk of global spread of its zoonotic infections, and Ebolavirus Zaire specifically has the highest fatality rate amongst other species. There is a need for continuous effort towards having therapies, as a single licensed treatment to neutralize the EBOV is yet to come into reality. This present study virtually screened the MCULE database containing almost 36 million compounds against the structure of a Zaire Ebola viral protein (VP) 35 and a consensus scoring of both MCULE and CLCDDW docking programs remarked five compounds as potential hits. These compounds, with binding energies ranging from –7.9 to –8.9?kcal/mol, were assessed for predictions of their physicochemical and bioactivity properties, as well as absorption, distribution, metabolism, excretion, and toxicity (ADMET) criteria. The results of the 50?ns molecular dynamics simulations showed the presence of dynamic stability between ligand and protein complexes, and the structures remained significantly unchanged at the ligand-binding site throughout the simulation period. Both docking analysis and molecular dynamics simulation studies suggested strong binding affinity towards the receptor cavity and these selected compounds as potential inhibitors against the Zaire Ebola VP 35. With respect to inhibition constant values, bioavailability radar and other physicochemical properties, compound A (MCULE-1018045960-0-1) appeared to be the most promising hit compound. However, the ligand efficiency and ligand efficiency scale need improvement during optimization, and also validation via in vitro and in vivo studies are necessary to finally make a lead compound in treating Ebola virus diseases.

Communicated by Ramaswamy H. Sarma     

Study on the interaction of fisetholz with BSA/HSA by multi-spectroscopic,cyclic voltammetric,and molecular docking technique

The interaction of fisetholz with bovine serum albumin (BSA) and human serum albumin (HSA) was investigated by multi-spectroscopic, cyclic voltammetric, and molecular docking technique. The results revealed that there was a static quenching of BSA/HSA induced by fisetholz. The binding constants (K_a) and binding sites (n) were calculated at different temperatures (293, 303, and 311?K). The enthalpy change (ΔH) were calculated to be –17.20?kJ mol^?1 (BSA) and –18.28?kJ mol^?1 (HSA) and the entropy change (ΔS) were calculated to be 35.41?J mol^?1 (BSA) and 24.02?J mol^?1 (HSA), respectively, which indicated that the interaction between fisetholz and BSA/HSA was mainly by electrostatic attraction. Based on displacement experiments using site probes, indomethacin and ibuprofen, the binding site of fisetholz to BSA/HSA was identified as sub-domain IIIA, which was further confirmed by molecular docking method. There was little effect of K⁺, Ca²⁺, Cu²⁺, Zn²⁺, and Fe³⁺ on fisetholz-BSA or fisetholz-HSA complex. The spectra of synchronous fluorescence, circular dichroism (CD) and Fourier transform infrared (FT-IR) all showed that fisetholz binding to BSA/HSA leads to secondary structures change of the two serum albumins. According to the Förster non-radiation energy transfer theory, the binding distance between fisetholz and BSA/HSA was 2.94/4.68?nm. The cyclic voltammetry as a supporting tool also indicated that fisetholz interacted with protein.

Communicated by Ramaswamy H. Sarma     

Glycosylation profiling to evaluate glycoprotein immunogens against HIV-1

Introduction: Much of the efforts to develop a vaccine against the human immunodeficiency virus (HIV) have focused on the design of recombinant mimics of the viral attachment glycoprotein (Env). The leading immunogens exhibit native-like antigenic properties and are being investigated for their ability to induce broadly neutralizing antibodies (bNAbs). Understanding the relative abundance of glycans at particular glycosylation sites on these immunogens is important as most bNAbs have evolved to recognize or evade the dense coat of glycans that masks much of the protein surface. Understanding the glycan structures on candidate immunogens enables triaging between native-like conformations and immunogens lacking key structural features as steric constraints limit glycan processing. The sensitivity of the processing state of a particular glycan to its structural environment has led to the need for quantitative glycan profiling and site-specific analysis to probe the structural integrity of immunogens.

Areas covered: We review analytical methodologies for HIV immunogen evaluation and discuss how these studies have led to a greater understanding of the structural constraints that control the glycosylation state of the HIV attachment and fusion spike.

Expert commentary: Total composition and site-specific glycosylation profiling are emerging as standard methods in the evaluation of Env-based immunogen candidates.     

The influence of Cu+ binding to hypoxanthine on stabilization of mismatches involving hypoxanthine and DNA bases: a DFT study

In the present work, the influence of Cu⁺ binding to N3- and N7-positions of hypoxanthine on energetic, geometrical and topological properties of hypoxanthine–guanine, hypoxanthine–adenine, hypoxanthine–cytosine, hypoxanthine–thymine and hypoxanthine–hypoxanthine mismatches is theoretically investigated. The calculations, in gas phase, are performed at B3LYP/6-311++G(3df,3pd) level of theory. Unlike the other mispairs, Cu⁺ binding to N3-position of hypoxanthine causes the proton transfer process from enol form of hypoxanthine to imino forms of adenine and cytosine. This process also occurs in all mismatches having enol form of hypoxanthine when Cu⁺ binds to N7-position of hypoxanthine. The mismatches are stabilized by hydrogen bonds. The influence of Cu⁺ on hydrogen bonds is also examined by atoms in molecules (AIM) and natural bond orbital (NBO) analyses.

Communicated by Ramaswamy H. Sarma     

Effect of confounding factors on a phospho-flow assay of ribosomal S6 protein for therapeutic drug monitoring of the mTOR-inhibitor everolimus in heart transplanted patients

Context: Several assays of monitoring immune cell function have been developed to enhance therapeutic drug monitoring.

Objective: An in vitro-validated whole-blood assay of phosphorylated ribosomal protein S6 (pS6RP) was evaluated for confounders to monitor the mTOR-inhibitor everolimus (ERL).

Materials and methods: Whole blood samples from 87 heart transplant recipients were analyzed for pS6RP-expression in CD3-positive T-cells by phospho-flow analysis.

Results: ERL blood concentration, laboratory parameters, co-medications, demographic and clinical data were reviewed.

Conclusion: Evaluating the pS6RP-assay revealed that pS6RP is influenced by cyclosporine A (CsA) blood concentration, duration of ERL treatment, co-medication with thiazide diuretics and different metabolic parameters.     

Hepatitis C virus core or NS3/4A protein expression preconditions hepatocytes against oxidative stress and endoplasmic reticulum stress

Objectives: The occurrence of oxidative stress and endoplasmic reticulum (ER) stress in hepatitis C virus (HCV) infection has been demonstrated and play an important role in liver injury. During viral infection, hepatocytes must handle not only the replication of the virus, but also inflammatory signals generating oxidative stress and damage. Although several mechanisms exist to overcome cellular stress, little attention has been given to the adaptive response of hepatocytes during exposure to multiple noxious triggers.

Methods: In the present study, Huh-7 cells and hepatocytes expressing HCV Core or NS3/4A proteins, both inducers of oxidative and ER stress, were additionally challenged with the superoxide anion generator menadione to mimic external oxidative stress. The production of reactive oxygen species (ROS) as well as the response to oxidative stress and ER stress were investigated.

Results: We demonstrate that hepatocytes diminish oxidative stress through a reduction in ROS production, ER-stress markers (HSPA5 [GRP78], sXBP1) and apoptosis (caspase-3 activity) despite external oxidative stress. Interestingly, the level of the autophagy substrate protein p62 was downregulated together with HCV Core degradation, suggesting that hepatocytes can overcome excess oxidative stress through autophagic degradation of one of the stressors, thereby increasing cell survival.

Duscussion: In conclusion, hepatocytes exposed to direct and indirect oxidative stress inducers are able to cope with cellular stress associated with viral hepatitis and thus promote cell survival.     

Proteomics for cancer drug design

Introduction: Signal transduction cascades drive cellular proliferation, apoptosis, immune, and survival pathways. Proteins have emerged as actionable drug targets because they are often dysregulated in cancer, due to underlying genetic mutations, or dysregulated signaling pathways. Cancer drug development relies on proteomic technologies to identify potential biomarkers, mechanisms-of-action, and to identify protein binding hot spots.

Areas covered: Brief summaries of proteomic technologies for drug discovery include mass spectrometry, reverse phase protein arrays, chemoproteomics, and fragment based screening. Protein-protein interface mapping is presented as a promising method for peptide therapeutic development. The topic of biosimilar therapeutics is presented as an opportunity to apply proteomic technologies to this new class of cancer drug.

Expert opinion: Proteomic technologies are indispensable for drug discovery. A suite of technologies including mass spectrometry, reverse phase protein arrays, and protein-protein interaction mapping provide complimentary information for drug development. These assays have matured into well controlled, robust technologies. Recent regulatory approval of biosimilar therapeutics provides another opportunity to decipher the molecular nuances of their unique mechanisms of action. The ability to identify previously hidden protein hot spots is expanding the gamut of potential drug targets. Proteomic profiling permits lead compound evaluation beyond the one drug, one target paradigm.     

Elevated serum YKL-40 levels in patients with Kawasaki disease

Context: YKL-40 is an inflammatory biomarker for endothelial dysfunction that may have a role in Kawasaki disease (KD).

Objectives: We investigated the association of serum YKL-40 levels with KD and established laboratory parameters for YKL-40 levels and other inflammatory markers.

Methods: YKL-40 levels and other inflammatory markers of 23 KD patients, 9 disease control patients and 11 age-matched healthy controls.

Results: YKL-40 concentration in the serum of KD patients significantly increased during the acute disease phase compared with those of disease controls and healthy controls.

Conclusions: Increased YKL-40 levels may provide a useful inflammatory marker for patients with KD.     

Nutritional status,energy expenditure,and protein oxidative stress after kidney transplantation

Objectives: To evaluate the association between nutritional status, resting energy expenditure (REE), and protein oxidative stress in patients after kidney transplantation (KT).

Methodology: The study evaluated 35 patients transplanted at the time of hospital discharge and 3 months after regarding: body composition, REE (by indirect calorimetry), and injury factor (IF); serum urea, creatinine, glucose, albumin, total protein, advanced oxidation protein products (AOPP), vitamin C.

Results: Three months after discharge, there was an improvement in renal function, nutritional status, and oxidative stress, with a standardization in the REE/kg. There was an increase in body weight, mainly in fat mass. The correlations showed that a greater cold ischemia time resulted in a deeper decline in vitamin C; a longer hospital length stay resulted in a greater reduction in AOPP; the higher preoperative body weight showed greater increases in body fat and glucose after transplantation. For decreases in REE and IF, there were increases in total protein. Finally, at hospital discharge there was a greater gain in weight, lower albumin, and total protein among individuals who had rejection episodes.

Discussion: The KT improves many of metabolic abnormalities, with the improvement of nutritional status, oxidative stress, and normalization of REE.     

Mass spectrometry protein tests: ready for prime time (or not)

Introduction: Mass spectrometry has played an important role in protein biomarker discovery. Yet, very few of the candidate biomarkers have been validated, and mass spectrometry-based protein tests have not made a significant inroad into clinical laboratories.

Areas covered: Offered here is a unique perspective on the future of mass spectrometry protein tests, in view of the following determinants: the true demand for such clinical tests, end-users requirements, platforms and systems design, sample preparation bottlenecks, analytical and clinical validation, and regulatory approval.

Expert commentary: Fresh thoughts and attitudes toward MS protein tests are required in order to move them toward clinical utilization and diagnostic use en masse, with critical emphasis on content, simplicity and cost. In its current format and state of the art, they are simply not ready for prime time.     

Inflammatory signature in lung tissues in patients with combined pulmonary fibrosis and emphysema

Background: The aetiology and inflammatory profile of combined pulmonary fibrosis and emphysema (CPFE) remain uncertain currently.

Objective: We aimed to examine the levels of inflammatory proteins in lung tissue in a cohort of patients with emphysema, interstitial pulmonary fibrosis (IPF), and CPFE.

Materials and methods: Explanted lungs were obtained from subjects with emphysema, IPF, CPFE, (or normal subjects), and tissue extracts were prepared. Thirty-four inflammatory proteins were measured in each tissue section.

Results: The levels of all 34 proteins were virtually indistinguishable in IPF compared with CPFE tissues, and collectively, the inflammatory profile in the emphysematous tissues were distinct from IPF and CPFE. Moreover, inflammatory protein levels were independent of the severity of the level of diseased tissue.

Conclusions: We find that emphysematous lung tissues have a distinct inflammatory profile compared with either IPF or CPFE. However, the inflammatory profile in CPFE lungs is essentially identical to lungs from patients with IPF. These data suggest that distinct inflammatory processes collectively contribute to the disease processes in patients with emphysema, when compared to IPF and CPFE.     

Advances in cell-free protein array methods

Introduction: Cell-free protein microarrays represent a special form of protein microarray which display proteins made fresh at the time of the experiment, avoiding storage and denaturation. They have been used increasingly in basic and translational research over the past decade to study protein-protein interactions, the pathogen-host relationship, post-translational modifications, and antibody biomarkers of different human diseases. Their role in the first blood-based diagnostic test for early stage breast cancer highlights their value in managing human health. Cell-free protein microarrays will continue to evolve to become widespread tools for research and clinical management.

Areas covered: We review the advantages and disadvantages of different cell-free protein arrays, with an emphasis on the methods that have been studied in the last five years. We also discuss the applications of each microarray method.

Expert commentary: Given the growing roles and impact of cell-free protein microarrays in research and medicine, we discuss: 1) the current technical and practical limitations of cell-free protein microarrays; 2) the biomarker discovery and verification pipeline using protein microarrays; and 3) how cell-free protein microarrays will advance over the next five years, both in their technology and applications.     

Phytoremediation and absorption isotherms of heavy metal ions by Convolvulus tricolor (CTC)

In recent years, use of plants for remediation of contaminated soils, especially removal of heavy metals, is considered. The current study tends to investigate the removal of lead and nickel ions by the Convolvulus tricolor (CTC), was grown for 30 days in different concentrations of lead and nickel ions. Then concentration of them in soil and different organs of the plant was measured by atomic absorption spectrometry.

The highest absorbed of them occurred in concentration 0.001N, which highest Pb²⁺ accumulation is in the aerial parts of the plant: leaf > stem > root and for Ni²+: root > stem > leaf. No ion was observed into the flowers and nickel ion absorption is more of lead ion in different plant organs of CTC.

The experimental isotherm data were investigated using isotherms of Langmuir, Freundlich, BET, Temkin and Dubinin–Radushkevich (DRK). The correlation coefficient for all of them is calculated that show the best correlation coefficient for Ni²⁺ adsorption is obtained BET model, whereas for Pb²⁺ adsorption in root is Freundlich model but for its leaf and stem is BET model. The results show, CTC is suitable for Pb²⁺ and Ni²⁺ and this technique is in-situ method, simple, and low cost.     

The role of mass spectrometry in the characterization of biologic protein products

Introduction: Mass spectrometry (MS) is widely used in the characterization of biomolecules including peptide and protein therapeutics. These biotechnology products have seen rapid growth over the past few decades and continue to dominate the global pharmaceutical market. Advances in MS instrumentation and techniques have enhanced protein characterization capabilities and supported an increased development of biopharmaceutical products.

Areas covered: This review describes recent developments in MS-based biotherapeutic analysis including sequence determination, post-translational modifications (PTMs) and higher order structure (HOS) analysis along with improvements in ionization and dissociation methods. An outlook of emerging applications of MS in the lifecycle of product development such as comparability, biosimilarity and quality control practices is also presented.

Expert commentary: MS-based methods have established their utility in the analysis of new biotechnology products and their lifecycle appropriate implementation. In the future, MS will likely continue to grow as one of the leading protein identification and characterization techniques in the biopharmaceutical industry landscape.