Nascent Folding Research Articles

Response of Foldable Protein Conformations to Non-Physiological Perturbations: Interplay of Thermal Factors and Confinement.

Technological advances frequently interface biomolecules with nanomaterials at non-physiological conditions, necessitating response characterization of key processes. Similar encounters are expected in cellular contexts. We report in silico investigations of the response of diverse protein conformational states to lowering of temperature and imposition of spatial constraints. Conformational states are represented by folded form of the Albumin binding domain (ABD) protein, its compact denatured form, and structurally disordered nascent folding elements. Data from extensive simulations are evaluated to elicit structural, thermodynamic and dynamic responses of the states and their associated environment. Analyses reveal alterations to folding propensity with reduced thermal energy and confinement, with signatures of trend reversal in highly disordered states. Across temperatures, confinement has restrictive effects on volume and energetic fluctuations, leading to narrowing of differences in isothermal compressibility (κ) and heat capacities (Cp). While excess (over ideal gas) entropy of the hydration layer marks dependence on the conformational state at bulk, confinement triggers erasure of differences. These observations are largely consistent with timescales of protein-water hydrogen bonding dynamics. The results implicate multi-factorial associations within a simple bio-nano complex. We expect the current study to motivate investigations of more biologically relevant interfaces towards mechanistic understanding and potential applications.

Prefoldin Subunits and Its Associate Partners: Conservations and Specificities in Plants.

Prefoldins (PFDs) are ubiquitous co-chaperone proteins that originated in archaea during evolution and are present in all eukaryotes, including yeast, mammals, and plants. Typically, prefoldin subunits form hexameric PFD complex (PFDc) that, together with class II chaperonins, mediate the folding of nascent proteins, such as actin and tubulin. In addition to functioning as a co-chaperone in cytoplasm, prefoldin subunits are also localized in the nucleus, which is essential for transcription and post-transcription regulation. However, the specific and critical roles of prefoldins in plants have not been well summarized. In this review, we present an overview of plant prefoldin and its related proteins, summarize the structure of prefoldin/prefoldin-like complex (PFD/PFDLc), and analyze the versatile landscape by prefoldin subunits, from cytoplasm to nucleus regulation. We also focus the specific role of prefoldin-mediated phytohormone response and global plant development. Finally, we overview the emerging prefoldin-like (PFDL) subunits in plants and the novel roles in related processes, and discuss the next direction in further studies.

Peptidyl-tRNA hydrolase is the nascent chain release factor in bacterial ribosome-associated quality control

Rescuing stalled ribosomes often involves their splitting into subunits. In many bacteria, the resultant large subunits bearing peptidyl-tRNAs are processed by the ribosome-associated quality control (RQC) apparatus that extends the C termini of the incomplete nascent polypeptides with polyalanine tails to facilitate their degradation. Although the tailing mechanism is well established, it is unclear how the nascent polypeptides are cleaved off the tRNAs. We show that peptidyl-tRNA hydrolase (Pth), the known role of which has been to hydrolyze ribosome-free peptidyl-tRNA, acts in concert with RQC factors to release nascent polypeptides from large ribosomal subunits. Dislodging from the ribosomal catalytic center is required for peptidyl-tRNA hydrolysis by Pth. Nascent protein folding may prevent peptidyl-tRNA retraction and interfere with the peptide release. However, oligoalanine tailing makes the peptidyl-tRNA ester bond accessible for Pth-catalyzed hydrolysis. Therefore, the oligoalanine tail serves not only as a degron but also as a facilitator of Pth-catalyzed peptidyl-tRNA hydrolysis.

Review of ER stress and unfolded protein response

The cell is the fundamental unit of life, and all of the organelles within it perform essential tasks. Cells must meet their typical requirements for survival and development in order to contribute to the functioning of living things. Protein folding in the rough ER, the major organelle for the synthesis, folding, and modification of secreted and transmembrane proteins, is essential for their proper function in the cytoplasm. Endoplasmic reticulum stress (ERS) occurs when the protein-folding capacity of this organelle is disrupted due to various external causes or intracellular events, resulting to a buildup of misfolded or unfolded proteins. Concerns in this area are possible. Some illnesses, cell death, and metabolic abnormalities are examples. The cell's stress response mechanism is the unfolded protein response (UPR). Reduced translation, degradation of unfolded proteins to reduce damage, and increased production of cell-mate proteins all aid in the folding of nascent proteins. This research uses a literature review approach to learn about the ER, an experiment on ER Stress, the reaction mechanism of unfolded protein response (UPR), diseases induced by ER Stress, and the significance of UPR in these illnesses.

Protein Disulfide Isomerase A2 Is Correlated with Immune Infiltrates and Is a Novel Prognostic Biomarker in Glioma Patients.

Protein disulfide isomerase A2 (PDIA2), a member of the protein disulfide isomerase family, plays a key role in the folding of nascent proteins in the endoplasmic reticulum by forming disulfide bonds, together with enzymes such as thiol isomerase, oxidase, and reductase. This study investigated the clinical significance and potential functions of PDIA2 in glioma. The expression of PDIA2 in gliomas was explored using The Cancer Genome Atlas and Gene Expression Omnibus databases. We analyzed the clinical characteristics of glioma patients and the prognostic and diagnostic value of PDIA2 expression. Kaplan-Meier and Cox regression analyses were used to examine the effect of PDIA2 expression on overall survival, progression-free interval, and disease-specific survival. Furthermore, we performed Gene Set Enrichment Analysis and immune infiltration analysis to investigate the functions of PDIA2. PDIA2 mRNA and protein expression was evaluated in cell lines and glioma tissues. PDIA2 was expressed at low levels in glioma patients. Kaplan-Meier survival analysis showed that glioma patients with low PDIA2 levels had a worse prognosis than those with high PDIA2 levels. Receiver operating characteristic curve analysis indicated the diagnostic and prognostic ability of PDIA2 (area under the curve = 0.918). Pathways associated with PD1, PI3K/AKT, cancer immunotherapy via PD1 blockade, Fceri-mediated NF-kB activation, FOXM1, and DNA repair were enriched in glioma patients with low levels of PDIA2. PDIA2 expression levels were negatively correlated with immune cell infiltrate levels. PDIA2 levels are significantly downregulated in glioma. PDIA2 expression may be a potential biomarker for the diagnosis and prognosis of glioma patients.

SEL1L–HRD1 endoplasmic reticulum-associated degradation controls STING-mediated innate immunity by limiting the size of the activable STING pool

Stimulator of interferon genes (STING) orchestrates the production of proinflammatory cytokines in response to cytosolic double-stranded DNA; however, the pathophysiological significance and molecular mechanism underlying the folding and maturation of nascent STING protein at the endoplasmic reticulum (ER) remain unknown. Here we report that the SEL1L–HRD1 protein complex—the most conserved branch of ER-associated degradation (ERAD)—is a negative regulator of the STING innate immunity by ubiquitinating and targeting nascent STING protein for proteasomal degradation in the basal state. SEL1L or HRD1 deficiency in macrophages specifically amplifies STING signalling and immunity against viral infection and tumour growth. Mechanistically, nascent STING protein is a bona fide substrate of SEL1L–HRD1 in the basal state, uncoupled from ER stress or its sensor inositol-requiring enzyme 1α. Hence, our study not only establishes a key role of SEL1L–HRD1 ERAD in innate immunity by limiting the size of the activable STING pool, but identifies a regulatory mechanism and therapeutic approach to targeting STING.

Structural inventory of cotranslational protein folding by the eukaryotic RAC complex

The challenge of nascent chain folding at the ribosome is met by the conserved ribosome-associated complex (RAC), which forms a chaperone triad with the Hsp70 protein Ssb in fungi, and consists of the non-canonical Hsp70 Ssz1 and the J domain protein Zuotin (Zuo1). Here we determine cryo-EM structures of Chaetomium thermophilum RAC bound to 80S ribosomes. RAC adopts two distinct conformations accommodating continuous ribosomal rotation by a flexible lever arm. It is held together by a tight interaction between the Ssz1 substrate-binding domain and the Zuo1 N terminus, and additional contacts between the Ssz1 nucleotide-binding domain and Zuo1 J- and Zuo1 homology domains, which form a rigid unit. The Zuo1 HPD motif conserved in J-proteins is masked in a non-canonical interaction by the Ssz1 nucleotide-binding domain, and allows the positioning of Ssb for activation by Zuo1. Overall, we provide the basis for understanding how RAC cooperates with Ssb in a dynamic nascent chain interaction and protein folding.

HtpG Is a Metal-Dependent Chaperone Which Assists the DnaK/DnaJ/GrpE Chaperone System of Mycobacterium tuberculosis via Direct Association with DnaJ2.

Protein folding is a crucial process in maintaining protein homeostasis, also known as proteostasis, in the cell. The requirement for the assistance of molecular chaperones in the appropriate folding of several proteins has already called into question the previously held view of spontaneous protein folding. These chaperones are highly ubiquitous cellular proteins, which not only help in mediating the proper folding of other nascent polypeptides but are also involved in refolding of the misfolded or the aggregated proteins. Hsp90 family proteins such as high-temperature protein G (HtpG) are abundant and ubiquitously expressed in both eukaryotic and prokaryotic cells. Although HtpG is known as an ATP-dependent chaperone protein in most organisms, function of this protein remains obscured in mycobacterial pathogens. Here, we aim to investigate significance of HtpG as a chaperone in the physiology of Mycobacterium tuberculosis. We report that M. tuberculosis HtpG (mHtpG) is a metal-dependent ATPase which exhibits chaperonin activity towards denatured proteins in coordination with the DnaK/DnaJ/GrpE chaperone system via direct association with DnaJ2. Increased expression of DnaJ1, DnaJ2, ClpX, and ClpC1 in a ΔhtpG mutant strain further suggests cooperativity of mHtpG with various chaperones and proteostasis machinery in M. tuberculosis. IMPORTANCE M. tuberculosis is exposed to variety of extracellular stressful conditions and has evolved mechanisms to endure and adapt to the adverse conditions for survival. mHtpG, despite being dispensable for M. tuberculosis growth under in vitro conditions, exhibits a strong and direct association with DnaJ2 cochaperone and assists the mycobacterial DnaK/DnaJ/GrpE (KJE) chaperone system. These findings suggest the potential role of mHtpG in stress management of the pathogen. Mycobacterial chaperones are responsible for folding of nascent protein as well as reactivation of protein aggregates. M. tuberculosis shows differential adaptive response subject to the availability of mHtpG. While its presence facilitates improved protein refolding via stimulation of the KJE chaperone activity, in the absence of mHtpG, M. tuberculosis enhances expression of DnaJ1/J2 cochaperones as well as Clp protease machinery for maintenance of proteostasis. Overall, this study provides a framework for future investigation to better decipher the mycobacterial proteostasis network in the light of stress adaptability and/or survival.

Integrated transcriptome reveals that d-limonene inhibits Candida tropicalis by disrupting metabolism

d-limonene is a common natural flavor additive in food industry. Meanwhile, it can also inhibit the opportunistic pathogens Candida tropicalis by disrupting the relative balance of cell phenotypic parameters, but the molecular mechanism is still unclear. Therefore, this study attempted to reveal the molecular mechanism by integrated transcriptome and phenotypic parameters. The results suggested that d-limonene disrupted the biosynthesis of cell wall and membrane, the transcription of genes encoding key enzymes in glycolysis pathway, tricarboxylic acid cycle and oxidative phosphorylation pathway, and the proper folding of nascent protein in endoplasmic reticulum of Candida tropicalis, and thus damaged the integrity of cell wall and membrane, reduced the supply of NADH + H+ and FADH2 substrate-level for oxidative phosphorylation and ATP production, and increased the endoplasmic reticulum stress and unfolded protein response, which resulted in metabolic disorder of Candida tropicalis, including shrinkage and adhesiveness of cell morphology, permeabilization and depolarization of cellular and mitochondrial membrane, decrease of mitochondrial ATP, and increase of endogenous reactive oxygen species. These findings will facilitate a better understanding of anti-C. tropicalis mechanisms of d-limonene, as well as provide further insights into its potential applications in food preservation and preventive medicine.

Visualization of translation and protein biogenesis at the ER membrane

The dynamic ribosome–translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome1,2. It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation3–7, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.

Luminal H2 O2 promotes ER Ca2+ dysregulation and toxicity of palmitate in insulin-secreting INS-1E cells.

The endoplasmic reticulum (ER) lumen is not only the major site for the assembly and folding of newly synthesized proteins but also the main intracellular Ca2+ store. Ca2+ ions are involved in versatile biochemical processes, including posttranslational processing and folding of nascent proteins. Disruption of ER Ca2+ homeostasis is usually accompanied by an ER stress response that can ultimately lead to apoptosis if unresolved. Abnormal ER Ca2+ depletion has been linked to pancreatic β-cell dysfunction and death under lipotoxic conditions. However, the underlying mechanisms how the β-cell toxic saturated free fatty acid palmitate perturbs ER Ca2+ homeostasis and its interplay with other organelles are not fully understood. In the present study, we demonstrate that treatment of insulin-secreting INS-1E cells with palmitate diminished ER Ca2+ levels, elevated cytosolic/mitochondrial Ca2+ content, lowered the mitochondrial membrane potential, and ATP content. In addition, palmitate-pretreated β-cells contained significantly less luminal Ca2+ , revealed a severely impaired ER Ca2+ reuptake rate, and substantially lower insulin content. Importantly, detoxification of luminal H2 O2 by expression of the ER-resident glutathione peroxidase 8 (GPx8) abrogated the lipotoxic effects of palmitate. Moreover, GPx8 supported oxidative protein folding and preserved insulin content under lipotoxic conditions. A direct involvement of luminal H2 O2 in palmitate-mediated ER Ca2+ depletion could be corroborated by the ectopic expression of an ER-luminal active catalase. Our data point to the critical role of luminal H2 O2 in palmitate-mediated depletion of ER Ca2+ through redox-dependent impairment of Ca2+ ATPase pump activity upstream of mitochondrial dysfunction in insulin-secreting INS-1E cells.

Insight into polyproline II helical bundle stability in an antifreeze protein denatured state

The use of polyproline II (PPII) helices in protein design is currently hindered by limitations in our understanding of their conformational stability and folding. Recent studies of the snow flea antifreeze protein (sfAFP), a useful model system composed of six PPII helices, suggested that a low denatured state entropy contributes to folding thermodynamics. Here, circular dichroism spectroscopy revealed minor populations of PPII like conformers at low temperature. To get atomic level information on the conformational ensemble and entropy of the reduced, denatured state of sfAFP, we have analyzed its chemical shifts and {1H}-15N relaxation parameters by NMR spectroscopy at four experimental conditions. No significant populations of stable secondary structure were detected. The stiffening of certain N-terminal residues at neutral versus acidic pH and shifted pKa values leads us to suggest that favorable charge-charge interactions could bias the conformational ensemble to favor the formation the C1-C28 disulfide bond during nascent folding, although no evidence for preferred contacts between these positions was detected by paramagnetic relaxation enhancement under denaturing conditions. Despite a high content of flexible glycine residues, the mobility of the sfAFP denatured ensemble is similar for denatured α/β proteins both on fast ps/ns as well as slower μs/ms timescales. These results are in line with a conformational entropy in the denatured ensemble resembling that of typical proteins and suggest that new structures based on PPII helical bundles should be amenable to protein design.

The antimicrobial peptide Magainin-2 interacts with BamA impairing folding of E. coli membrane proteins.

Antimicrobial peptides (AMPs) are a unique and diverse group of molecules endowed with a broad spectrum of antibiotics properties that are being considered as new alternative therapeutic agents. Most of these peptides are membrane-active molecules, killing bacteria by membrane disruption. However, recently an increasing number of AMPs was shown to enter bacterial cells and target intracellular processes fundamental for bacterial life. In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent β-barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling, in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. Functional implications of this interactions were investigated by two alternative and complementary approaches. The amount of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation were evaluated by both western blot analysis and quantitative tandem mass spectrometry in Multiple Reaction Monitoring scan mode. In both experiments a gradual decrease in outer membrane proteins production with time was observed as a consequence of Mag-2 treatment. These results suggested BamA as a possible good target for the rational design of new antibiotics since this protein is responsible for a crucial biological event of bacterial life and is absent in humans.

Extended disordered regions of ribosome-associated NAC proteins paralogs belong only to the germline in Drosophila melanogaster

The nascent polypeptide-associated complex (NAC) consisting of α- and β-subunits is an essential ribosome-associated protein conserved in eukaryotes. NAC is a ubiquitously expressed co-translational regulator of nascent protein folding and sorting providing for homeostasis of cellular proteins. Here we report on discovering the germline-specific NACαβ paralogs (gNACs), whose β-subunits, non-distinguishable by ordinary immunodetection, are encoded by five highly homologous gene copies, while the α-subunit is encoded by a single αNAC gene. The gNAC expression is detected in the primordial embryonic and adult gonads via immunostaining. The germline-specific α and β subunits differ from the ubiquitously expressed paralogs by the extended intrinsically disordered regions (IDRs) acquired at the N- and C-termini of the coding regions, predicted to be phosphorylated. The presence of distinct phosphorylated isoforms of gNAC-β subunits is confirmed by comparing of their profiles by 2D-isoeletrofocusing resolution before and after phosphatase treatment of testis ribosomes. We revealed that the predicted S/T sites of phosphorylation in the individual orthologous IDRs of gNAC-β sequences of Drosophila species are positionally conserved despite these disordered regions are drastically different. We propose the IDR-dependent molecular crowding and specific coordination of NAC and other proteostasis regulatory factors at the ribosomes of germinal cells. Our findings imply that there may be a functional crosstalk between the germinal and ubiquitous α- and β-subunits based on assessing their depletion effects on the fly viability and gonad development.

METTL18-mediated histidine methylation of RPL3 modulates translation elongation for proteostasis maintenance.

Protein methylation occurs predominantly on lysine and arginine residues, but histidine also serves as a methylation substrate. However, a limited number of enzymes responsible for this modification have been reported. Moreover, the biological role of histidine methylation has remained poorly understood to date. Here, we report that human METTL18 is a histidine methyltransferase for the ribosomal protein RPL3 and that the modification specifically slows ribosome traversal on Tyr codons, allowing the proper folding of synthesized proteins. By performing an in vitro methylation assay with a methyl donor analog and quantitative mass spectrometry, we found that His245 of RPL3 is methylated at the τ-N position by METTL18. Structural comparison of the modified and unmodified ribosomes showed stoichiometric modification and suggested a role in translation reactions. Indeed, genome-wide ribosome profiling and an in vitro translation assay revealed that translation elongation at Tyr codons was suppressed by RPL3 methylation. Because the slower elongation provides enough time for nascent protein folding, RPL3 methylation protects cells from the cellular aggregation of Tyr-rich proteins. Our results reveal histidine methylation as an example of a ribosome modification that ensures proteome integrity in cells.

Transfection of Heat Shock Protein 70 kDa (HSP70)

Heat shock protein 70 kDa (HSP70) is a major protein family in the cell protections against stress-induced denaturation and aggregation and in the folding of nascent proteins. It is a highly conserved protein that can be found in most organisms and is strongly connected to several intracellular pathways such as protein folding and refolding, protein degradation and regulation, and protection against intense stress. Cellular delivery of HSP70 would be of high impact for clarification of its role in these cellular processes.PepFect14 is a cell-penetrating peptide known to be able to mediate the transfection of various oligonucleotides to multiple cell lines with a higher efficacy than most commercially available transfection agents and without inducing significant toxic effects.In this study we demonstrated that PepFect14 was able to form a complex with HSP70 and to deliver it inside cells in the same fashion with oligonucleotide delivery. The delivered HSP70 showed an effect in the cell regulation indicating that the protein was biologically available in the cytoplasm and the interactions with PepFect14 did not impeach its active sites once the plasma barrier crossed.This study reports the first successful delivery of HSP70 to our knowledge and the first protein transfection mediated by PepFect14. It opens new fields of research for both PepFect14 as a delivery agent and HSP70 as a therapeutic agent; with potential in peptide aggregation caused diseases such as Parkinson’s and Alzheimer’s diseases.

Allosteric Probes for Modulating Essential Bacterial Chaperone‐Cofactor Interactions

DnaK is the bacterial homolog of Hsp70, an ATP‐dependent chaperone that assists cofactor proteins to catalyze nascent protein folding and salvage misfolded proteins. In the pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), DnaK and its cofactor proteins DnaJ1, DnaJ2 and GrpE are proposed drug targets. Despite the importance of chaperone function in human cancers and infectious disease, there are limited chemical probes or inhibitors that enable in vivo studies on the function of individual chaperones/cofactors. Here, we describe the discovery of small molecule inhibitors of mycobacterial DnaK from a re‐purposed drug library, most notably a peptidomimetic called telaprevir, which is able to inhibit chaperone function through interactions with the peptide‐binding cleft of DnaK. Binding of telaprevir leads to allosteric conformational changes that prevent ATP hydrolysis in a distal domain. We find that telaprevir also inhibits E. coli DnaK and human Hsp70 chaperones due to high conservation of Hsp70 sequences. Using in vitro and in vivo chaperone assays, we demonstrate that telaprevir modulates the function of mycobacterial DnaK and its cofactor protein DnaJ2 in cells, which disrupts cellular proteostasis. Co‐treatment of mycobacteria with telaprevir and aminoglycosides, which further stress the proteome, enhances the potency of these antibiotics. In addition, telaprevir combats mycobacterial resistance to the frontline TB drug rifampin, as DnaK‐DnaJ2 function is required for stabilization of protein mutants that confer drug tolerance. This work sets the stage for the design of peptide‐based inhibitors with higher selectivity for bacterial chaperones to probe chaperone‐protein interactions and explore resulting synergy with different classes of antibiotics.

The Astonishing Large Family of HSP40/DnaJ Proteins Existing in Leishmania.

Abrupt environmental changes are faced by Leishmania parasites during transmission from a poikilothermic insect vector to a warm-blooded host. Adaptation to harsh environmental conditions, such as nutrient deprivation, hypoxia, oxidative stress and heat shock needs to be accomplished by rapid reconfiguration of gene expression and remodeling of protein interaction networks. Chaperones play a central role in the maintenance of cellular homeostasis, and they are responsible for crucial tasks such as correct folding of nascent proteins, protein translocation across different subcellular compartments, avoiding protein aggregates and elimination of damaged proteins. Nearly one percent of the gene content in the Leishmania genome corresponds to members of the HSP40 family, a group of proteins that assist HSP70s in a variety of cellular functions. Despite their expected relevance in the parasite biology and infectivity, little is known about their functions or partnership with the different Leishmania HSP70s. Here, we summarize the structural features of the 72 HSP40 proteins encoded in the Leishmania infantum genome and their classification into four categories. A review of proteomic data, together with orthology analyses, allow us to postulate cellular locations and possible functional roles for some of them. A detailed study of the members of this family would provide valuable information and opportunities for drug discovery and improvement of current treatments against leishmaniasis.

Comparative Toxicity of Interferon Beta-1a Impurities of Heavy Metal Ions.

Background and Objectives: Providing a proper quality control of drugs is essential for efficient treatment of various diseases minimizing the possible side effects of pharmaceutical active substances and potential impurities. Recent in vitro and in vivo studies have shown that certain heavy metalloids and metals interfere with protein folding of nascent proteins in cells and their biological function can be altered. It is unknown whether the drug impurities including heavy metals may affect the tertiary protein structure. Materials and Methods: ReciGen and Rebif are pharmaceutical interferon beta-1a (IFNβ-1a) contained in preparations that are used for parenteral administration. Heavy metal impurities of these samples have been studied by gel electrophoresis, Fourier-transform infrared spectroscopy (FTIR) and inductively coupled plasma mass spectrometry analysis (ICP MS). The concentration of heavy metals including mercury, arsenic, nickel, chromium, iron, and aluminum did not exceed permitted levels established by International Council for Harmonisation guideline for elemental impurities. Results: The ICP MS analysis revealed the presence of heavy metals, moreover zeta potential was significantly different for IFNβ-1a, which can be an indirect indication of the difference in composition of ReciGen and Rebif samples, respectively. FTIR analysis revealed very similar amide I and II bonds at 1654 and 1560 cm−1 attributed to the peptide absorption peaks of IFNβ-1a in Rebif and ReciGen. Conclusions: It was hypothesized that the IFNβ-1a complex binds heavy metals affecting the tertiary protein structure and may lead to some side effects of drug administration. Further testing of IFNβ-1a bioequivalence for parenteral application is necessary.

Life in the freezer: protein metabolism in Antarctic fish.

Whole-animal, in vivo protein metabolism rates have been reported in temperate and tropical, but not Antarctic fish. Growth in Antarctic species is generally slower than lower latitude species. Protein metabolism data for Antarctic invertebrates show low rates of protein synthesis and unusually high rates of protein degradation. Additionally, in Antarctic fish, increasing evidence suggests a lower frequency of successful folding of nascent proteins and reduced protein stability. This study reports the first whole-animal protein metabolism data for an Antarctic fish. Groups of Antarctic, Harpagifer antarcticus, and temperate, Lipophrys pholis, fish were acclimatized to a range of overlapping water temperatures and food consumption, whole-animal growth and protein metabolism measured. The rates of protein synthesis and growth in Antarctic, but not temperate fish, were relatively insensitive to temperature and were significantly lower in H. antarcticus at 3°C than in L. pholis. Protein degradation was independent of temperature in H. antarcticus and not significantly different to L. pholis at 3°C, while protein synthesis retention efficiency was significantly higher in L. pholis than H. antarcticus at 3°C. These results suggest Antarctic fish degrade a significantly larger proportion of synthesized protein than temperate fish, with fundamental energetic implications for growth at low temperatures.