Homodimeric Protein Research Articles

Aberrant function of pathogenic STAT3 mutant proteins is linked to altered stability of monomers and homodimers

AbstractSTAT3 mutations, predominantly in the DNA-binding domain (DBD) and Src-homology 2 domain (SH2D), cause rare cases of immunodeficiency, malignancy, and autoimmunity. The exact mechanisms by which these mutations abrogate or enhance STAT3 function are not completely understood. Here, we examined how loss-of-function (LOF) and gain-of-function (GOF) STAT3 mutations within the DBD and SH2D affect monomer and homodimer protein stability as well as their effect on key STAT3 activation events, including recruitment to phosphotyrosine (pY) sites within peptide hormone receptors, tyrosine phosphorylation at Y705, dimerization, nuclear translocation, and DNA binding. The DBD LOF mutants showed reduced DNA binding when homodimerized, whereas the DBD GOF mutants showed increased DNA binding. DBD LOF and GOF mutants showed minimal changes in other STAT3 functions or in monomer or homodimer protein stability. However, SH2D LOF mutants demonstrated reduced conformational stability as either monomers or homodimers, leading to decreased pY-peptide recruitment, tyrosine phosphorylation, dimerization, nuclear localization, and DNA binding. In contrast, cancer-causing SH2D GOF mutants showed increased STAT3 homodimer stability, which increased their DNA binding. Of note, a small-molecule inhibitor of STAT3 that targets the tyrosine phosphopeptide–binding pocket within the STAT3 SH2D potently inhibited cell proliferation driven by STAT3 SH2D GOF mutants. These findings indicate that the stability of STAT3 protein monomer and homodimer is critical for the pathogenesis of diseases caused by SH2D LOF and GOF mutations and suggest that agents that modulate STAT3 monomer and/or homodimer protein stability may have therapeutic value in diseases caused by these mutations.

Isolation of a Natural Killer Group 2D Small-Molecule Ligand from DNA-Encoded Chemical Libraries.

Natural Killer Group 2D (NKG2D) is a homo-dimeric transmembrane protein which is typically expressed on the surface of natural killer (NK) cells, natural killer T (NKT) cells, gamma delta T (γδT) cells, activated CD8 positive T-cells and activated macrophages. Bispecific molecules, capable of bridging NKG2D with a target protein expressed on the surface of tumor cells, may be used to redirect the cytotoxic activity of NK-cells towards antigen-positive malignant T-cells. In this work, we report the discovery of a novel NKG2D small molecule binder [KD =(410±60) nM], isolated from a DNA-Encoded Chemical Library (DEL). The discovery of small organic NKG2D ligands may facilitate the generation of fully synthetic bispecific adaptors, which may serve as an alternative to bispecific antibody products and which may benefit from better tumor targeting properties.

Tetramerization of the S100B Chaperone Spawns a Ca2+ Independent Regulatory Surface that Enhances Anti-aggregation Activity and Client Specificity

Alzheimer’s disease (AD) hallmarks include the aggregation of amyloid-β (Aβ), tau and neuroinflammation promoted by several alarmins. Among these is S100B, a small astrocytic homodimeric protein, upregulated in AD, whose multiple biological activities depend on localization, concentration, and assembly state. S100B was reported to inhibit the aggregation and toxicity of Aβ42 and tau similarly to a holdase-type chaperone. This activity is dependent of Ca2+-binding, which triggers the exposure of a regulatory binding cleft at the S100B dimer interface with which amyloidogenic clients dynamically interact. Although the dimer prevails, a significant portion of secreted S100B in the human brain occurs as higher order multimers, whose protective functions remain uncharacterized and which we here investigate. Resorting to ThT-monitored aggregation kinetics, we determined that unlike the dimer, tetrameric S100B inhibits Aβ42 aggregation at sub/equimolar ratios, an effect that persists in the absence of Ca2+ binding. Structural analysis revealed that S100B tetramerization spawns a novel extended cleft accommodating an aggregation-prone surface that mediates interactions with monomeric Aβ client via hydrophobic interactions, as corroborated by Bis-ANS fluorescence and docking analysis. Correspondingly, at high ionic strength that reduces solvation and favours hydrophobic contacts, the inhibition of Aβ42 aggregation by tetrameric S100B is 3-fold increased. Interestingly, this extended Ca2+-independent surface favours Aβ42 as substrate, as tau K18 aggregation is not inhibited by the apo tetramer. Overall, results illustrate a mechanism through which oligomerization of the S100B chaperone fine-tunes anti-aggregation activity and client specificity, highlighting the potential functional relevance of S100B multimers in the regulation of AD proteotoxicity.

Reduction of a Heme Cofactor Initiates N-Nitroglycine Degradation by NnlA.

Linear nitramines are potentially carcinogenic environmental contaminants. The NnlA enzyme from Variovorax sp. strain JS1663 degrades the nitramine N-nitroglycine (NNG)-a natural product produced by some bacteria-to glyoxylate and nitrite (NO2-). Ammonium (NH4+) was predicted as the third product of this reaction. A source of nonheme FeII was shown to be required for initiation of NnlA activity. However, the role of this FeII for NnlA activity was unclear. This study reveals that NnlA contains a b-type heme cofactor. Reduction of this heme-either by a nonheme iron source or dithionite-is required to initiate NnlA activity. Therefore, FeII is not an essential substrate for holoenzyme activity. Our data show that reduced NnlA (FeII-NnlA) catalyzes at least 100 turnovers and does not require O2. Finally, NH4+ was verified as the third product, accounting for the complete nitrogen mass balance. Size exclusion chromatography showed that NnlA is a dimer in solution. Additionally, FeII-NnlA is oxidized by O2 and NO2- and stably binds carbon monoxide (CO) and nitric oxide (NO). These are characteristics shared with heme-binding PAS domains. Furthermore, a structural homology model of NnlA was generated using the PAS domain from Pseudomonas aeruginosa Aer2 as a template. The structural homology model suggested His73 is the axial ligand of the NnlA heme. Site-directed mutagenesis of His73 to alanine decreased the heme occupancy of NnlA and eliminated NNG activity, validating the homology model. We conclude that NnlA forms a homodimeric heme-binding PAS domain protein that requires reduction for initiation of the activity. IMPORTANCE Linear nitramines are potential carcinogens. These compounds result from environmental degradation of high-energy cyclic nitramines and as by-products of carbon capture technologies. Mechanistic understanding of the biodegradation of these compounds is critical to inform strategies for their remediation. Biodegradation of NNG by NnlA from Variovorax sp. strain JS 1663 requires nonheme iron, but its role is unclear. This study shows that nonheme iron is unnecessary. Instead, our study reveals that NnlA contains a heme cofactor, the reduction of which is critical for activating NNG degradation activity. These studies constrain the proposals for NnlA reaction mechanisms, thereby informing mechanistic studies of degradation of anthropogenic nitramine contaminants. In addition, these results will inform future work to design biocatalysts to degrade these nitramine contaminants.

De novo design of protein homodimers containing tunable symmetric protein pockets

Function follows form in biology, and the binding of small molecules requires proteins with pockets that match the shape of the ligand. For design of binding to symmetric ligands, protein homo-oligomers with matching symmetry are advantageous as each protein subunit can make identical interactions with the ligand. Here, we describe a general approach to designing hyperstable C2 symmetric proteins with pockets of diverse size and shape. We first designed repeat proteins that sample a continuum of curvatures but have low helical rise, then docked these into C2 symmetric homodimers to generate an extensive range of C2 symmetric cavities. We used this approach to design thousands of C2 symmetric homodimers, and characterized 101 of them experimentally. Of these, the geometry of 31 were confirmed by small angle X-ray scattering and 2 were shown by crystallographic analyses to be in close agreement with the computational design models. These scaffolds provide a rich set of starting points for binding a wide range of C2 symmetric compounds.

Disrupting ACE2 Dimerization Mitigates the Infection by SARS-CoV-2 Pseudovirus

The coronavirus disease 2019 pandemic has caused over million death and 500 million reported cases globally. More effective antiviral medications are needed to curb the continued spread of this disease. The infection by SARS-CoV-2 virus is initiated via the interaction between the receptor binding domain (RBD) of the viral glycoprotein Spike (S protein) and the N-term peptidase domain of the angiotensin-converting enzyme 2 (ACE2) expressed on the host cell membrane. ACE2 forms a protein homodimer primarily through its ferredoxin-like fold domain (a.k.a., Neck-domain). We investigated whether the dimerization of ACE2 receptor plays a role in SARS-CoV-2 virus infection. We report here that the ACE2 receptor dimerization enhances the recognition of SARS-CoV-2 S protein. A 43-amino-acid peptide based on the N-term of Neck-domain could block the ACE2 dimerization and hence the interaction between RBD and ACE2 and mitigate the SARS-CoV-2 S protein pseudotyped virus/host cell interaction. Our study illustrated a new route to develop potential therapeutics for the prevention and treatment of SARS-CoV-2 viral infection.

Preeclampsia is Associated With Reduced ISG15 Levels Impairing Extravillous Trophoblast Invasion.

Among several interleukin (IL)-6 family members, only IL-6 and IL-11 require a gp130 protein homodimer for intracellular signaling due to lack of intracellular signaling domain in the IL-6 receptor (IL-6R) and IL-11R. We previously reported enhanced decidual IL-6 and IL-11 levels at the maternal-fetal interface with significantly higher peri-membranous IL-6 immunostaining in adjacent interstitial trophoblasts in preeclampsia (PE) vs. gestational age (GA)-matched controls. This led us to hypothesize that competitive binding of these cytokines to the gp130 impairs extravillous trophoblast (EVT) differentiation, proliferation and/or invasion. Using global microarray analysis, the current study identified inhibition of interferon-stimulated gene 15 (ISG15) as the only gene affected by both IL-6 plus IL-11 vs. control or IL-6 or IL-11 treatment of primary human cytotrophoblast cultures. ISG15 immunostaining was specific to EVTs among other trophoblast types in the first and third trimester placental specimens, and significantly lower ISG15 levels were observed in EVT from PE vs. GA-matched control placentae (p = 0.006). Induction of primary trophoblastic stem cell cultures toward EVT linage increased ISG15 mRNA levels by 7.8-fold (p = 0.004). ISG15 silencing in HTR8/SVneo cultures, a first trimester EVT cell line, inhibited invasion, proliferation, expression of ITGB1 (a cell migration receptor) and filamentous actin while increasing expression of ITGB4 (a receptor for hemi-desmosomal adhesion). Moreover, ISG15 silencing further enhanced levels of IL-1β-induced pro-inflammatory cytokines (CXCL8, IL-6 and CCL2) in HTR8/SVneo cells. Collectively, these results indicate that ISG15 acts as a critical regulator of EVT morphology and function and that diminished ISG15 expression is associated with PE, potentially mediating reduced interstitial trophoblast invasion and enhancing local inflammation at the maternal-fetal interface. Thus, agents inducing ISG15 expression may provide a novel therapeutic approach in PE.

NADP+-dependent l-arginine dehydrogenase from Pseudomonas velonii: Purification, characterization and application to an l-arginine assay

l-Arginine dehydrogenase (L-ArgDH) is an amino acid dehydrogenase which catalyzes the reversible oxidative deamination of l-arginine to the oxo analog in the presence of NAD(P)+. We here found the gene homolog of L-ArgDH in genome data of Pseudomonas veronii and succeeded in expression of P. veronii JCM11942 gene in E. coli. The gene product exhibited strong NADP+-dependent L-ArgDH activity. The enzyme was unstable, but markedly stabilized by the addition of 10% glycerol. The enzyme first purified to homogeneity consisted of a homodimeric protein with a molecular mass of about 65 kDa. The enzyme selectively catalyzed NADP+-dependent l-arginine oxidation with maximal activity at pH 9.5. The apparent Km values for l-arginine and NADP+ were 2.5 and 0.21 mM, respectively. The nucleotide sequence coding the enzyme gene was determined and the amino acid sequence was deduced from the nucleotide sequence. The simple colorimetric microassay for l-arginine using the enzyme was achieved.

The orphan ligand, activin C, signals through activin receptor-like kinase 7.

Activin ligands are formed from two disulfide-linked inhibin β (Inhβ) subunit chains. They exist as homodimeric proteins, as in the case of activin A (ActA; InhβA/InhβA) or activin C (ActC; InhβC/InhβC), or as heterodimers, as with activin AC (ActAC; InhβA:InhβC). While the biological functions of ActA and activin B (ActB) have been well characterized, little is known about the biological functions of ActC or ActAC. One thought is that the InhβC chain functions to interfere with ActA production by forming less active ActAC heterodimers. Here, we assessed and characterized the signaling capacity of ligands containing the InhβC chain. ActC and ActAC activated SMAD2/3-dependent signaling via the type I receptor, activin receptor-like kinase 7 (ALK7). Relative to ActA and ActB, ActC exhibited lower affinity for the cognate activin type II receptors and was resistant to neutralization by the extracellular antagonist, follistatin. In mature murine adipocytes, which exhibit high ALK7 expression, ActC elicited a SMAD2/3 response similar to ActB, which can also signal via ALK7. Collectively, these results establish that ActC and ActAC are active ligands that exhibit a distinct signaling receptor and antagonist profile compared to other activins.

AlphaFold2 models indicate that protein sequence determines both structure and dynamics

AlphaFold 2 (AF2) has placed Molecular Biology in a new era where we can visualize, analyze and interpret the structures and functions of all proteins solely from their primary sequences. We performed AF2 structure predictions for various protein systems, including globular proteins, a multi-domain protein, an intrinsically disordered protein (IDP), a randomized protein, two larger proteins (> 1000 AA), a heterodimer and a homodimer protein complex. Our results show that along with the three dimensional (3D) structures, AF2 also decodes protein sequences into residue flexibilities via both the predicted local distance difference test (pLDDT) scores of the models, and the predicted aligned error (PAE) maps. We show that PAE maps from AF2 are correlated with the distance variation (DV) matrices from molecular dynamics (MD) simulations, which reveals that the PAE maps can predict the dynamical nature of protein residues. Here, we introduce the AF2-scores, which are simply derived from pLDDT scores and are in the range of [0, 1]. We found that for most protein models, including large proteins and protein complexes, the AF2-scores are highly correlated with the root mean square fluctuations (RMSF) calculated from MD simulations. However, for an IDP and a randomized protein, the AF2-scores do not correlate with the RMSF from MD, especially for the IDP. Our results indicate that the protein structures predicted by AF2 also convey information of the residue flexibility, i.e., protein dynamics.

Precise O-Glycosylation Site Localization of CD24Fc by LC-MS Workflows.

CD24Fc is a homodimeric recombinant Fc-fusion protein comprised of human CD24 connected to immunoglobulin G1 (IgG1) Fc fragment. CD24 is heavily glycosylated, and its biological function is considered mainly mediated by its glycosylation. Identification of the O-glycosylation sites would facilitate an in-depth understanding of the functional role of O-glycans in CD24. However, the presence of clustered mucin-type O-glycans together with N-glycans makes the determination of O-glycosylation sites and abundance very challenging. In this study, two sets of liquid chromatography-mass spectrometry (LC-MS) workflows were developed for the comprehensive characterization of O-glycosylation in CD24: (1) Fractionation and collision-induced dissociation (CID) workflow involving multienzyme digestion, fractionation, OpeRATOR/SialEXO digestion, and CID analysis; (2) Direct OpeRATOR/SialEXO digestion followed by electron-transfer/higher-energy collision dissociation (EThcD) analysis. The precise O-glycosylation sites were identified in CD24 for the first time, and the site occupancy was assessed. A total of 12 O-glycosylation sites were identified. Seven glycosylation sites were identified by both workflows, and five additional sites were identified only by the EThcD workflow. The predominant O-glycosylation site in CD24 was Thr25 followed by Thr15. The CID workflow provided an overall relative quantitation of O-glycoforms at the CD24 level and site localization for singly O-glycosylated peptides. The EThcD workflow directly identified glycosylation sites by tandem mass spectrometry (MS/MS) for singly, doubly, and triply O-glycosylated peptides. Together, both workflows validated each other's results and can be applied to a complex mucin-type O-glycosylation site analysis of other glycoproteins and Fc-fusion therapeutics.

E Protein Transcription Factors as Suppressors of T Lymphocyte Acute Lymphoblastic Leukemia.

T Lymphocyte Acute Lymphoblastic Leukemia (ALL) is an aggressive disease arising from transformation of T lymphocytes during their development. The mutation spectrum of T-ALL has revealed critical regulators of the growth and differentiation of normal and leukemic T lymphocytes. Approximately, 60% of T-ALLs show aberrant expression of the hematopoietic stem cell-associated helix-loop-helix transcription factors TAL1 and LYL1. TAL1 and LYL1 function in multiprotein complexes that regulate gene expression in T-ALL but they also antagonize the function of the E protein homodimers that are critical regulators of T cell development. Mice lacking E2A, or ectopically expressing TAL1, LYL1, or other inhibitors of E protein function in T cell progenitors, also succumb to an aggressive T-ALL-like disease highlighting that E proteins promote T cell development and suppress leukemogenesis. In this review, we discuss the role of E2A in T cell development and how alterations in E protein function underlie leukemogenesis. We focus on the role of TAL1 and LYL1 and the genes that are dysregulated in E2a-/- T cell progenitors that contribute to human T-ALL. These studies reveal novel mechanisms of transformation and provide insights into potential therapeutic targets for intervention in this disease.

Dimerization-dependent membrane tethering by Atg23 is essential for yeast autophagy.

SUMMARYEukaryotes maintain cellular health through the engulfment and subsequent degradation of intracellular cargo using macroautophagy. The function of Atg23, despite being critical to the efficiency of this process, is unclear due to a lack of biochemical investigations and an absence of any structural information. In this study, we use a combination of in vitro and in vivo methods to show that Atg23 exists primarily as a homodimer, a conformation facilitated by a putative amphipathic helix. We utilize small-angle X-ray scattering to monitor the overall shape of Atg23, revealing that it contains an extended rod-like structure spanning approximately 320 Å. We also demonstrate that Atg23 interacts with membranes directly, primarily through electrostatic interactions, and that these interactions lead to vesicle tethering. Finally, mutation of the hydrophobic face of the putative amphipathic helix completely precludes dimer formation, leading to severely impaired subcellular localization, vesicle tethering, Atg9 binding, and autophagic efficiency.

SARS-CoV-2 Membrane Protein: From Genomic Data to Structural New Insights.

Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2) is composed of four structural proteins and several accessory non-structural proteins. SARS-CoV-2’s most abundant structural protein, Membrane (M) protein, has a pivotal role both during viral infection cycle and host interferon antagonism. This is a highly conserved viral protein, thus an interesting and suitable target for drug discovery. In this paper, we explain the structural nature of M protein homodimer. To do so, we developed and applied a detailed and robust in silico workflow to predict M protein dimeric structure, membrane orientation, and interface characterization. Single Nucleotide Polymorphisms (SNPs) in M protein were retrieved from over 1.2 M SARS-CoV-2 genomes and proteins from the Global Initiative on Sharing All Influenza Data (GISAID) database, 91 of which were located at the predicted dimer interface. Among those, we identified SNPs in Variants of Concern (VOC) and Variants of Interest (VOI). Binding free energy differences were evaluated for dimer interfacial SNPs to infer mutant protein stabilities. A few high-prevalent mutated residues were found to be especially relevant in VOC and VOI. This realization may be a game-changer to structure-driven formulation of new therapeutics for SARS-CoV-2.

High-pH structure of EmrE reveals the mechanism of proton-coupled substrate transport

The homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance. We recently determined the structure of substrate-bound EmrE in phospholipid bilayers by measuring hundreds of protein-ligand HN–F distances for a fluorinated substrate, 4-fluoro-tetraphenylphosphonium (F4-TPP+), using solid-state NMR. This structure was solved at low pH where one of the two proton-binding Glu14 residues is protonated. Here, to understand how substrate transport depends on pH, we determine the structure of the EmrE-TPP complex at high pH, where both Glu14 residues are deprotonated. The high-pH complex exhibits an elongated and hydrated binding pocket in which the substrate is similarly exposed to the two sides of the membrane. In contrast, the low-pH complex asymmetrically exposes the substrate to one side of the membrane. These pH-dependent EmrE conformations provide detailed insights into the alternating-access model, and suggest that the high-pH conformation may facilitate proton binding in the presence of the substrate, thus accelerating the conformational change of EmrE to export the substrate.

A deep dilated convolutional residual network for predicting interchain contacts of protein homodimers.

MotivationDeep learning has revolutionized protein tertiary structure prediction recently. The cutting-edge deep learning methods such as AlphaFold can predict high-accuracy tertiary structures for most individual protein chains. However, the accuracy of predicting quaternary structures of protein complexes consisting of multiple chains is still relatively low due to lack of advanced deep learning methods in the field. Because interchain residue–residue contacts can be used as distance restraints to guide quaternary structure modeling, here we develop a deep dilated convolutional residual network method (DRCon) to predict interchain residue–residue contacts in homodimers from residue–residue co-evolutionary signals derived from multiple sequence alignments of monomers, intrachain residue–residue contacts of monomers extracted from true/predicted tertiary structures or predicted by deep learning, and other sequence and structural features.ResultsTested on three homodimer test datasets (Homo_std dataset, DeepHomo dataset and CASP-CAPRI dataset), the precision of DRCon for top L/5 interchain contact predictions (L: length of monomer in a homodimer) is 43.46%, 47.10% and 33.50% respectively at 6 Å contact threshold, which is substantially better than DeepHomo and DNCON2_inter and similar to Glinter. Moreover, our experiments demonstrate that using predicted tertiary structure or intrachain contacts of monomers in the unbound state as input, DRCon still performs well, even though its accuracy is lower than using true tertiary structures in the bound state are used as input. Finally, our case study shows that good interchain contact predictions can be used to build high-accuracy quaternary structure models of homodimers.Availability and implementationThe source code of DRCon is available at https://github.com/jianlin-cheng/DRCon. The datasets are available at https://zenodo.org/record/5998532#.YgF70vXMKsB.Supplementary information Supplementary data are available at Bioinformatics online.

Comprehensive characterization and molecular insights into the salt tolerance of a Cu, Zn-superoxide dismutase from an Indian Mangrove, Avicennia marina

Superoxide dismutases are important group of antioxidant metallozyme and play important role in ROS homeostasis in salinity stress. The present study reports the biochemical properties of a salt-tolerant Cu, Zn-superoxide from Avicennia marina (Am_SOD). Am_SOD was purified from the leaf and identified by mass-spectrometry. Recombinant Am_SOD cDNA was bacterially expressed as a homodimeric protein. Enzyme kinetics revealed a high substrate affinity and specific activity of Am_SOD as compared to many earlier reported SODs. An electronic transition in 360–400 nm spectra of Am_SOD is indicative of Cu2+-binding. Am_SOD activity was potentially inhibited by diethyldithiocarbamate and H2O2, a characteristic of Cu, Zn-SOD. Am_SOD exhibited conformational and functional stability at high NaCl concentration as well in alkaline pH. Introgression of Am_SOD in E. coli conferred tolerance to oxidative stress under highly saline condition. Am_SOD was moderately thermostable and retained functional activity at ~ 60 °C. In-silico analyses revealed 5 solvent-accessible N-terminal residues of Am_SOD that were less hydrophobic than those at similar positions of non-halophilic SODs. Substituting these 5 residues with non-halophilic counterparts resulted in > 50% reduction in salt-tolerance of Am_SOD. This indicates a cumulative role of these residues in maintaining low surface hydrophobicity of Am_SOD and consequently high salt tolerance. The molecular information on antioxidant activity and salt-tolerance of Am_SOD may have potential application in biotechnology research. To our knowledge, this is the first report on salt-tolerant SOD from mangrove.

Structure and function of an atypical homodimeric actin capping protein from the malaria parasite

Apicomplexan parasites, such as Plasmodium spp., rely on an unusual actomyosin motor, termed glideosome, for motility and host cell invasion. The actin filaments are maintained by a small set of essential regulators, which provide control over actin dynamics in the different stages of the parasite life cycle. Actin filament capping proteins (CPs) are indispensable heterodimeric regulators of actin dynamics. CPs have been extensively characterized in higher eukaryotes, but their role and functional mechanism in Apicomplexa remain enigmatic. Here, we present the first crystal structure of a homodimeric CP from the malaria parasite and compare the homo- and heterodimeric CP structures in detail. Despite retaining several characteristics of a canonical CP, the homodimeric Plasmodium berghei (Pb)CP exhibits crucial differences to the canonical heterodimers. Both homo- and heterodimeric PbCPs regulate actin dynamics in an atypical manner, facilitating rapid turnover of parasite actin, without affecting its critical concentration. Homo- and heterodimeric PbCPs show partially redundant activities, possibly to rescue actin filament capping in life cycle stages where the β-subunit is downregulated. Our data suggest that the homodimeric PbCP also influences actin kinetics by recruiting lateral actin dimers. This unusual function could arise from the absence of a β-subunit, as the asymmetric PbCP homodimer lacks structural elements essential for canonical barbed end interactions suggesting a novel CP binding mode. These findings will facilitate further studies aimed at elucidating the precise actin filament capping mechanism in Plasmodium.

Monitoring the hepatitis B viral (HBV) capsid assembly by charge transfer spectroscopy

HBV virions are double-shelled particles, 40–42 nm in diameter containing a viral nucleocapsid, or the HBV core which is an ordered assembly of stable homo-dimeric HBV Core protein (Cp). Both the full-length Cp containing 183 residues as well as the first-149 residues fragment, Cp149 can assemble spontaneously in vitro into capsids triggered by high ionic strength (0.2-0.5M NaCl) into 120 Cp dimers in an icosahedral symmetry (T=4) or into 90 Cp dimers with (T=3) icosahedral symmetry. Both the Cp149/Cp183 capsids and their assembly-disassembly is generally studied by Size Exclusion Chromatography, Light Scattering, Electron microscopy, Atomic Force Microscopy and other high-end labour-intensive techniques.

Xanthine oxidoreductase activity in platelet-poor and rich plasma as a oxidative stress indicator in patients required renal replacement therapy

BackgroundXanthine oxidoreductase (XOR) is a hydroxylase enzyme involved in the metabolism of purines. XOR activity can vary: the homodimer protein can be converted into two different isoforms XD (antioxidant) and XO (prooxidant). Oxidative stress and inflammation that accompanying chronic kidney disease (CKD), dialysis, and kidney transplantation, resulted in platelet activation. Present study aimed to determine the influence of applied renal replacement therapy on xanthine oxidoreductase and its isoforms activity.Materials and MethodsThe study group consisted of 117 patients, divided into 4 groups: hemodialysis - 30 patients, peritoneal dialysis - 30 patients, kidney transplant patients - 27 and conservative treatment - 30 patients. The control group consisted of 30 healthy volunteers.ResultsSignificant differences were found in XOR activity in platelet-poor plasma (PPP) within the groups studied (p = 0.001). There was a relationship between the type of renal replacement therapy of all oxidoreductase isoforms in PPP (p < 0.001 all isoforms) and XD (p = 0.008), XO (p < 0.001) in platelet rich-plasma (PRP). A relationship was observed between the activity of all oxidoreductase isoforms in PPP and PRP, and the type of renal replacement therapy and the duration of dialysis and the age of patients. The cause of chronic kidney disease was also reflected differences in XD and XO activity in PPP.ConclusionsThe type of renal replacement therapy used in CKD patients, age of patients, duration of dialysis, CKD causes, and stage of progression significantly affect the activity of XOR and its isoforms.