Wild-type Ca Research Articles

Acetylation halts missense mutant p53 aggregation and rescues tumor suppression in non-small cell lung cancers

TP53 mutations are ubiquitous with tumorigenesis in non-small cell lung cancers (NSCLC). By analyzing the TCGA database, we reported that TP53 missense mutations are correlated with chromosomal instability and tumor mutation burden in NSCLC. The inability of wild-type nor mutant p53 expression can't predict survival in lung cancer cohorts, however, an examination of primary NSCLC tissues found that acetylated p53 did yield an association with improved survival outcomes. Molecularly, we demonstrated that acetylation drove the ubiquitination and degradation of mutant p53 but enhanced stability of wild-type p53. Moreover, acetylation of a missense p53 mutation prevented the gain of oncogenic function observed in typical TP53 mutant-expressing cells and enhanced tumor suppressor functions. Consequently, acetylation inducer targeting of missense mutant p53 may be a viable therapeutic goal for NSCLC treatment and may improve the accuracy of current efforts to utilize p53 mutations in a prognostic manner.

Localization and functions of native and eGFP-tagged capsid proteins in HIV-1 particles.

In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components.

Enhancement of C6–C10 fatty acid ethyl esters production in Saccharomyces cerevisiae CA by metabolic engineering

C6–C10 fatty acid ethyl esters (FAEEs) are important aromatic substances in alcoholic beverages and include ethyl hexanoate (EH), ethyl octanoate (EO), and ethyl decanoate (ED). However, the biosynthesis capacity of these esters are limited because of poor precursor C6–C10 fatty acyl-CoA accumulation and low alcohol acyltransferase (AATase) activity levels in Saccharomyces cerevisiae. In this study, S. cerevisiae CA stain was selected as the host stain because of its relatively high C6–C10 FAEE productivity. Ester production was improved by metabolic engineering strategies. Acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (ACS1) were overexpressed to enhance the acetyl-CoA precursor accumulation. Intracellular malonyl-CoA availability was also improved by acetyl-CoA decarboxylase (ACC1Ser659Ala, Ser1157Ala) overexpression. FAS1 and FAS2 (encoding fatty acid synthase) overexpression facilitated the condensation reactions to form C6–C10 fatty acyl-CoA from malonyl-CoA and acetyl-CoA precursors. Furthermore, the strawberry alcohol acyltransferase (SAAT) was introduced into S. cerevisiae CA to catalyze the formation of C6–C10 FAEEs from C6–C10 fatty acyl-CoA and ethanol substrates. As a consequence, the newly engineered strain achieved 25.89-, 7.27-, and 9.05-fold increases in extracellular EH (7.53 mg/L), EO (13.65 mg/L) and ED (13.87 mg/L) levels, respectively, compared to the wild-type strain CA.

L'amylose cardiaque à transthyrétine

Transthyretin (TTR) cardiac amyloidosis results from the dissociation of the tetrameric, liver-synthetized transport protein, either because of a mutation (hereditary CA), or spontaneously due to ageing (wild type CA). Monomers self-associate into amyloid fibrils within the myocardium, causing heart failure, arrhythmias and conduction defects. This overlooked disease must be recognized in case of unexplained increased thickness of the myocardium, particularly in subjects of African descent, in patients with heart failure and preserved ejection fraction, and in those with aortic stenosis. Some extra-cardiac symptoms must also be considered as red flags: carpal tunnel syndrome, lumbar canal stenosis, recent deafness, peripheral neuropathy, or dysautonomia. Medical assessment includes an electrocardiogram, biological assessment including troponin, natriuretic peptide and monoclonal protein assay, echocardiography with 2-D strain study, MRI and bone scintigraphy. Once the diagnosis established, cardiologic management must avoid beta-blockers and other rate-slowing drugs, which are deleterious in restrictive cardiomyopathy, and restrain the use of renin-angiotensin system inhibitors, of little use and often poorly tolerated. Congestion must be treated with diuretics. Anticoagulants are often necessary due to the risk of arrhythmias and stroke. Pacemaker or defibrillator implantation should be determined in patients with high risk of sudden death. Until now, etiologic treatments were liver and/or heart transplantation in some rare cases. Tafamidis, a TTR stabilizer has recently been approved, and new therapeutic approaches targeting TTR at the transcriptional level are under investigation.

Truncated CPSF6 Forms Higher-Order Complexes That Bind and Disrupt HIV-1 Capsid.

Cleavage and polyadenylation specificity factor 6 (CPSF6) is a human protein that binds HIV-1 capsid and mediates nuclear transport and integration targeting of HIV-1 preintegration complexes. Truncation of the protein at its C-terminal nuclear-targeting arginine/serine-rich (RS) domain produces a protein, CPSF6-358, that potently inhibits HIV-1 infection by targeting the capsid and inhibiting nuclear entry. To understand the molecular mechanism behind this restriction, the interaction between CPSF6-358 and HIV-1 capsid was characterized using in vitro and in vivo assays. Purified CPSF6-358 protein formed oligomers and bound in vitro-assembled wild-type (WT) capsid protein (CA) tubes, but not CA tubes containing a mutation in the putative binding site of CPSF6. Intriguingly, binding of CPSF6-358 oligomers to WT CA tubes physically disrupted the tubular assemblies into small fragments. Furthermore, fixed- and live-cell imaging showed that stably expressed CPSF6-358 forms cytoplasmic puncta upon WT HIV-1 infection and leads to capsid permeabilization. These events did not occur when the HIV-1 capsid contained a mutation known to prevent CPSF6 binding, nor did they occur in the presence of a small-molecule inhibitor of capsid binding to CPSF6-358. Together, our in vitro biochemical and transmission electron microscopy data and in vivo intracellular imaging results provide the first direct evidence for an oligomeric nature of CPSF6-358 and suggest a plausible mechanism for restriction of HIV-1 infection by CPSF6-358.IMPORTANCE After entry into cells, the HIV-1 capsid, which contains the viral genome, interacts with numerous host cell factors to facilitate crucial events required for replication, including uncoating. One such host cell factor, called CPSF6, is predominantly located in the cell nucleus and interacts with HIV-1 capsid. The interaction between CA and CPSF6 is critical during HIV-1 replication in vivo Truncation of CPSF6 leads to its localization to the cell cytoplasm and inhibition of HIV-1 infection. Here, we determined that truncated CPSF6 protein forms large higher-order complexes that bind directly to HIV-1 capsid, leading to its disruption. Truncated CPSF6 expression in cells leads to premature capsid uncoating that is detrimental to HIV-1 infection. Our study provides the first direct evidence for an oligomeric nature of truncated CPSF6 and insights into the highly regulated process of HIV-1 capsid uncoating.

Ras-Raf-MAPK signaling promotes nuclear localization of FOXA transcription factor SGF1 via Ser91 phosphorylation

Ras-Raf-MAPK signaling promotes cell proliferation and cell survival. We previously reported that Ras1CA overexpression, specifically in the posterior silk glands (PSGs) of the silkworm Bombyx mori, increased fibroin synthesis and cell size, resulting in improved silk yields. In this study, we compared the iTRAQ-based phosphoproteomic profiles of PSGs from wild-type and Ras1CA-overexpressing silkworms. Silk gland factor 1 (SGF1), a FOXA transcription factor that plays a critical role in activating fibroin gene expression, was identified as a phosphoprotein harboring Ser91 as a potential MAPK phosphorylation site. Ser91 phosphorylation of SGF1 was enhanced by Ras1CA overexpression, and this finding was verified by selected reaction monitoring. Consistently, MAPK activity is well correlated with Ser91 phosphorylation of SGF1 and its nuclear localization in PSG cells during silkworm development. Ras1CA overexpression and treatment with inhibitors of Ras signaling promoted or inhibited SGF1 nuclear localization, respectively; mutation of Ser91 to Ala91 eliminated SGF1 nuclear localization. Moreover, MAPK binds to SGF1 and directly phosphorylates Ser91, demonstrating Ser91 as a MAPK phosphorylation site in SGF1. In conclusion, Ras-Raf-MAPK signaling promotes SGF1 nuclear localization for transactivation via Ser91 phosphorylation in silkworms, showing that FOXA transcription factors are regulated via MAPK phosphorylation in animals.

Effects of reduced carbonic anhydrase activity on CO2 assimilation rates in Setaria viridis: a transgenic analysis.

In C4 species, the major β-carbonic anhydrase (β-CA) localized in the mesophyll cytosol catalyses the hydration of CO2 to HCO3-, which phosphoenolpyruvate carboxylase uses in the first step of C4 photosynthesis. To address the role of CA in C4 photosynthesis, we generated transgenic Setaria viridis depleted in β-CA. Independent lines were identified with as little as 13% of wild-type CA. No photosynthetic defect was observed in the transformed lines at ambient CO2 partial pressure (pCO2). At low pCO2, a strong correlation between CO2 assimilation rates and CA hydration rates was observed. C18O16O isotope discrimination was used to estimate the mesophyll conductance to CO2 diffusion from the intercellular air space to the mesophyll cytosol (gm) in control plants, which allowed us to calculate CA activities in the mesophyll cytosol (Cm). This revealed a strong relationship between the initial slope of the response of the CO2 assimilation rate to cytosolic pCO2 (ACm) and cytosolic CA activity. However, the relationship between the initial slope of the response of CO2 assimilation to intercellular pCO2 (ACi) and cytosolic CA activity was curvilinear. This indicated that in S. viridis, mesophyll conductance may be a contributing limiting factor alongside CA activity to CO2 assimilation rates at low pCO2.

Major Variations in HIV-1 Capsid Assembly Morphologies Involve Minor Variations in Molecular Structures of Structurally Ordered Protein Segments

We present the results of solid state nuclear magnetic resonance (NMR) experiments on HIV-1 capsid protein (CA) assemblies with three different morphologies, namely wild-type CA (WT-CA) tubes with 35-60 nm diameters, planar sheets formed by the Arg(18)-Leu mutant (R18L-CA), and R18L-CA spheres with 20-100 nm diameters. The experiments are intended to elucidate molecular structural variations that underlie these variations in CA assembly morphology. We find that multidimensional solid state NMR spectra of (15)N,(13)C-labeled CA assemblies are remarkably similar for the three morphologies, with only small differences in (15)N and (13)C chemical shifts, no significant differences in NMR line widths, and few differences in the number of detectable NMR cross-peaks. Thus, the pronounced differences in morphology do not involve major differences in the conformations and identities of structurally ordered protein segments. Instead, morphological variations are attributable to variations in conformational distributions within disordered segments, which do not contribute to the solid state NMR spectra. Variations in solid state NMR signals from certain amino acid side chains are also observed, suggesting differences in the intermolecular dimerization interface between curved and planar CA lattices, as well as possible differences in intramolecular helix-helix packing.

Essential Opposite Roles of ERK and Akt Signaling in Cardiac Steroid-Induced Increase in Heart Contractility.

Interaction of cardiac steroids (CS) with the Na(+), K(+)-ATPase elicits, in addition to inhibition of the enzyme's activity, the activation of intracellular signaling such as extracellular signal-regulated (ERK) and protein kinase B (Akt). We hypothesized that the activities of these pathways are involved in CS-induced increase in heart contractility. This hypothesis was tested using in vivo and ex vivo wild type (WT) and sarcoplasmic reticulum Ca(2+) atpase1a-deficient zebrafish (accordion, acc mutant) experimental model. Heart contractility was measured in vivo and in primary cardiomyocytes in WT zebrafish larvae and acc mutant. Ca(2+) transients were determined ex vivo in adult zebrafish hearts. CS dose dependently augmented the force of contraction of larvae heart muscle and cardiomyocytes and increased Ca(2+) transients in WT but not in acc mutant. CS in vivo increased the phosphorylation rate of ERK and Akt in the adult zebrafish heart of the two strains. Pretreatment of WT zebrafish larvae or cardiomyocytes with specific MAPK inhibitors completely abolished the CS-induced increase in contractility. On the contrary, pretreatment with Akt inhibitor significantly enhanced the CS-induced increase in heart contractility both in vivo and ex vivo without affecting CS-induced Ca(2+) transients. Furthermore, pretreatment of the acc mutant larvae or cardiomyocytes with Akt inhibitor restored the CS-induced increase in heart contractility also without affecting Ca(2+) transients. These results support the notion that the activity of MAPK pathway is obligatory for CS-induced increases in heart muscle contractility. Akt activity, on the other hand, plays a negative role, via Ca(2+) independent mechanisms, in CS action. These findings point to novel potential pharmacological intervention to increase CS efficacy.

Modulation of CaV2.1 channels by neuronal calcium sensor-1 induces short-term synaptic facilitation.

Facilitation and inactivation of P/Q-type Ca2+ currents mediated by Ca2+/calmodulin binding to Ca(V)2.1 channels contribute to facilitation and rapid depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin from its binding site and differentially modulate P/Q-type Ca2 + currents, resulting in diverse patterns of short-term synaptic plasticity. Neuronal calcium sensor-1 (NCS-1, frequenin) has been shown to enhance synaptic facilitation, but the underlying mechanism is unclear. We report here that NCS-1 directly interacts with IQ-like motif and calmodulin-binding domain in the C-terminal domain of Ca(V)2.1 channel. NCS-1 reduces Ca2 +-dependent inactivation of P/Q-type Ca2+ current through interaction with the IQ-like motif and calmodulin-binding domain without affecting peak current or activation kinetics. Expression of NCS-1 in presynaptic superior cervical ganglion neurons has no effect on synaptic transmission, eliminating effects of this calcium sensor protein on endogenous N-type Ca2+ currents and the endogenous neurotransmitter release machinery. However, in superior cervical ganglion neurons expressing wild-type Ca(V)2.1 channels, co-expression of NCS-1 induces facilitation of synaptic transmission in response to paired pulses and trains of depolarizing stimuli, and this effect is lost in Ca(V)2.1 channels with mutations in the IQ-like motif and calmodulin-binding domain. These results reveal that NCS-1 directly modulates Ca(V)2.1 channels to induce short-term synaptic facilitation and further demonstrate that CaS proteins are crucial in fine-tuning short-term synaptic plasticity.

Critical Roles of Interdomain Interactions for Modulatory ATP Binding to Sarcoplasmic Reticulum Ca2+-ATPase

ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca(2+)-ATPase. Upon binding to the Ca2E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca(2+) transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser(186) and Asp(203) interact with Glu(439) (N-domain) and Arg(678) (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp(203) and Arg(678) rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser(186) and Glu(439). By taking advantage of the ability of wild type and mutant Ca(2+)-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca(2+)-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp(203)-Arg(678) and Ser(186)-Glu(439) interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser(186)-Glu(439) bond are mutually exclusive in the E2P ground state.

Human carbonic anhydrase II-cyanate inhibitor complex: putting the debate to rest.

The binding of anions to carbonic anhydrase II (CA II) has been attributed to high affinity for the active-site zinc. An anion of interest is cyanate, for which contrasting binding modes have been reported in the literature. Previous spectroscopic data have shown cyanate behaving as an inhibitor, directly binding to the zinc, in contrast to previous crystallographic data that implied that cyanate acts as a substrate mimic that is not directly bound to the zinc but overlaps with the binding site of the substrate CO2. Wild-type and the V207I variant of CA II have been expressed and X-ray crystal structures of their cyanate complexes have been determined to 1.7 and 1.5 Å resolution, respectively. The rationale for the V207I CA II variant was its close proximity to the CO2-binding site. Both structures clearly show that the cyanate binds directly to the zinc. In addition, inhibition constants (∼40 µM) were measured using (18)O-exchange mass spectrometry for wild-type and V207I CA II and were similar to those determined previously (Supuran et al., 1997). Hence, it is concluded that under the conditions of these experiments the binding of cyanate to CA II is directly to the zinc, displacing the zinc-bound solvent molecule, and not in a site that overlaps with the CO2 substrate-binding site.

A Limited Role for Carbonic Anhydrase in C4 Photosynthesis as Revealed by a ca1ca2 Double Mutant in Maize

Carbonic anhydrase (CA) catalyzes the first biochemical step of the carbon-concentrating mechanism of C4 plants, and in C4 monocots it has been suggested that CA activity is near limiting for photosynthesis. Here, we test this hypothesis through the characterization of transposon-induced mutant alleles of Ca1 and Ca2 in maize (Zea mays). These two isoforms account for more than 85% of the CA transcript pool. A significant change in isotopic discrimination is observed in mutant plants, which have as little as 3% of wild-type CA activity, but surprisingly, photosynthesis is not reduced under current or elevated CO2 partial pressure (pCO2). However, growth and rates of photosynthesis under subambient pCO2 are significantly impaired in the mutants. These findings suggest that, while CA is not limiting for C4 photosynthesis in maize at current pCO2, it likely maintains high rates of photosynthesis when CO2 availability is reduced. Current atmospheric CO2 levels now exceed 400 ppm (approximately 40.53 Pa) and contrast with the low-pCO2 conditions under which C4 plants expanded their range approximately 10 million years ago, when the global atmospheric CO2 was below 300 ppm (approximately 30.4 Pa). Thus, as CO2 levels continue to rise, selective pressures for high levels of CA may be limited to arid climates where stomatal closure reduces CO2 availability to the leaf.

DESIGN OF <i>CANDIDA ANTARCTICA</i> LIPASE B THERMOSTABILITY IMPROVEMENT BY INTRODUCING EXTRA DISULFIDE BOND INTO THE ENZYME

Candida Antarctica Lipase B (CALB) is extensively studied in enzymatic production of biodiesel, pharmaceutical products, detergents and other chemi cals. One drawback of using CALB is its relatively low optimum temperature at 313 K (40°C). The objective of this research is to design CALB mutant with improved thermostability by introducing extra disul fide bond. Molecular dynamic simulation was conducted to get better insight into the process of thermal denaturation or unfolding in CALB. Thermal denaturation of CALB was accelerated by conducting simulation at high temperature. Molecular dynamic simulation of CALB was performed with GROMACS software package at 300-700 K. Prediction of possible mutation was done using “Disulfide by Desi gn TM ” software. Selection of mutated residues was based on flexibility analysis of CALB. From those a nalyses, three mutants were designed, which are Mutant-1 (73LeuCys/151AlaCys), Mutant-2 (155TrpCys/294GluCys) and Mutant-3 (43ThrCys/67SerCys). Parameters that were used to compare the thermostability of mutant with wild type enzyme were Root Mean Square Deviations (RMSD), Solvent Accessible Surface Area (SASA), Radius of gyration (Rg) and secondary structure. Molecular dynamic simulation conducted on those three mutants showed that Mutant-1 has better thermostability compared to wild type CA LB. We proposed the order of mutant thermostability improvement as follows: Mutant-1, Mutant-2 and Mutant-3, with Mutant-1 having better potential thermostability improvement and Mutant-3, the least stable.

Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli

BackgroundHIV genome is packaged and organized in a conical capsid, which is made up of ~1,500 copies of the viral capsid protein p24 (CA). Being a primary structural component and due to its critical roles in both late and early stages of the HIV replication cycle, CA has attracted increased interest as a drug discovery target in recent years. Drug discovery studies require large amounts of highly pure and biologically active protein. It is therefore desirable to establish a simple and reproducible process for efficient production of HIV-1 CA.ResultIn this work, 6-His-tagged wild type CA from HIV-1 (NL4.3) was expressed in rare tRNA-supplemented NiCo21(DE3) Escherichia coli, and its production was studied in shake flask culture condition of expression. Influences of various key cultivation parameters were examined to identify optimal conditions for HIV-1 CA production. It was found that a culture temperature of 22°C and induction with 0.05 mM IPTG at the early stage of growth were ideal, leading to a maximum biomass yield when grown in Super broth supplemented with 1% glucose. With optimized culture conditions, a final biomass concentration of ~27.7 g L-1 (based on optical density) was obtained in 12 hours post-induction, leading to a yield of about ~170 mg L-1 HIV-1 CA. A two-step purification strategy (chitin beads + IMAC) was employed, which efficiently removed metal affinity resin-binding bacterial proteins that contaminate recombinant His-tagged protein preparation, and resulted in highly pure HIV-1 CA. The purified protein was capable of polymerization when tested in an in vitro polymerization assay.ConclusionsBy using this optimized expression and purification procedure, milligram amounts of highly pure and polymerization-competent recombinant HIV-1 CA can be produced at the lab-scale and thus used for further biochemical studies.

Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits

The host restriction factors TRIM5α and TRIMCyp potently inhibit retrovirus infection by binding to the incoming retrovirus capsid. TRIM5 proteins are dimeric, and their association with the viral capsid appears to be enhanced by avidity effects owing to formation of higher-order oligomeric complexes. We examined the stoichiometric requirement for TRIM5 functional recognition by quantifying the efficiencies of restriction of HIV-1 and murine leukemia virus (MLV) particles containing various proportions of restriction-sensitive and -insensitive CA subunits. Both TRIMCyp and TRIM5α inhibited infection of retrovirus particles containing as little as 25% of the restriction-sensitive CA protein. Accordingly, we also observed efficient binding of TRIMCyp in vitro to capsid assemblies containing as little as one-fourth wild-type CA protein. Paradoxically, the ability of HIV-1 particles to abrogate TRIMCyp restriction in trans was more strongly dependent on the fraction of wild-type CA than was restriction of infection. Collectively, our results indicate that TRIM5 restriction factors bind to retroviral capsids in a highly cooperative manner and suggest that TRIM5 can engage a capsid lattice containing a minimum of three or fewer recognizable subunits per hexamer. Our study supports a model in which localized binding of TRIM5 to the viral capsid nucleates rapid polymerization of a TRIM5 lattice on the capsid surface.

TNFα Levels and Macrophages Expression Reflect an Inflammatory Potential of Trigeminal Ganglia in a Mouse Model of Familial Hemiplegic Migraine

Latent changes in trigeminal ganglion structure and function resembling inflammatory conditions may predispose to acute attacks of migraine pain. Here, we investigated whether, in trigeminal sensory ganglia, cytokines such as TNFα might contribute to a local inflammatory phenotype of a transgenic knock-in (KI) mouse model of familial hemiplegic migraine type-1 (FHM-1). To this end, macrophage occurrence and cytokine expression in trigeminal ganglia were compared between wild type (WT) and R192Q mutant CaV2.1 Ca2+ channel (R192Q KI) mice, a genetic model of FHM-1. Cellular and molecular characterization was performed using a combination of confocal immunohistochemistry and cytokine assays. With respect to WT, R192Q KI trigeminal ganglia were enriched in activated macrophages as suggested by their morphology and immunoreactivity to the markers Iba1, CD11b, and ED1. R192Q KI trigeminal ganglia constitutively expressed higher mRNA levels of IL1β, IL6, IL10 and TNFα cytokines and the MCP-1 chemokine. Consistent with the report that TNFα is a major factor to sensitize trigeminal ganglia, we observed that, following an inflammatory reaction evoked by LPS injection, TNFα expression and macrophage occurrence were significantly higher in R192Q KI ganglia with respect to WT ganglia. Our data suggest that, in KI trigeminal ganglia, the complex cellular and molecular environment could support a new tissue phenotype compatible with a neuroinflammatory profile. We propose that, in FHM patients, this condition might contribute to trigeminal pain pathophysiology through release of soluble mediators, including TNFα, that may modulate the crosstalk between sensory neurons and resident glia, underlying the process of neuronal sensitisation.

A Trimer of Dimers Is the Basic Building Block for Human Immunodeficiency Virus-1 Capsid Assembly

Human immunodeficiency virus-1 (HIV-1) capsid protein (CA) has become a target of antiviral drug design in recent years. The recognition that binding of small molecules to the CA protein can result in the perturbation of capsid assembly or disassembly has led to mathematical modeling of the process. Although a number of capsid assembly models have been developed using biophysical parameters of the CA protein obtained experimentally, there is currently no model of CA polymerization that can be practically used to analyze in vitro CA polymerization data to facilitate drug discovery. Herein, we describe an equilibrium model of CA polymerization for the kinetic analysis of in vitro assembly of CA into polymer tubes. This new mathematical model has been used to assess whether a triangular trimer of dimers rather than a hexagonal hexamer can be the basic capsomere building block of CA polymer. The model allowed us to quantify for the first time the affinity for each of the four crucial interfaces involved in the polymerization process and indicated that the trimerization of CA dimers is a relatively slow step in CA polymerization in vitro. For wild-type CA, these four interfaces include the interface between two monomers of a CA dimer (K(D) = 6.6 μM), the interface between any two dimers within a CA trimer of dimers (K(D) = 32 nM), and two types of interfaces between neighboring trimers of dimers, either within the same ring around the perimeter of the polymer tube (K(D) = 438 nM) or from two adjacent rings (K(D) = 147 nM). A comparative analysis of the interface dissociation constants between wild-type and two mutant CA proteins, cross-linked hexamer (A14C/E45C/W184A/M185A) and A14C/E45C, yielded results that are consistent with the trimer of dimers with a triangular geometry being the capsomere building block involved in CA polymer growth. This work provides additional insights into the mechanism of HIV-1 CA assembly and may prove useful in elucidating how small molecule CA binding agents may disturb this essential step in the HIV-1 life cycle.

Smurf1 Protein Negatively Regulates Interferon-γ Signaling through Promoting STAT1 Protein Ubiquitination and Degradation

Interferons are important cytokines that mediate antiviral, antiproliferative, antitumor, and immunoregulatory activities. However, uncontrolled IFN signaling may lead to autoimmune diseases. Here we identified Smurf1 as a negative regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation. Smurf1 interacted with STAT1 through the WW domains of Smurf1 and the PY motif in STAT1 and catalyzed K48-linked polyubiquitination of STAT1. Interestingly, the Smurf1-mediated ubiquitination and degradation did not require STAT1 tyrosine and serine phosphorylation. Subsequently, overexpression of Smurf1 attenuated IFN-γ-mediated STAT1 activation and antiviral immune responses, whereas knockdown of Smurf1 enhanced IFN-γ-mediated STAT1 activation, expression of STAT1 target genes, and antiviral immune responses. Furthermore, IFN-γ stimulation led to enhanced expression of Smurf1. Therefore, our results demonstrate that Smurf1 is a negative feedback regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation.

Structure of a Monomeric Mutant of the HIV-1 Capsid Protein

The capsid protein (CA) of HIV-1 plays a significant role in the assembly of the immature virion and is the critical building block of its mature capsid. Thus, there has been significant interest in the CA protein as a target in the design of inhibitors of early and late stage events in the HIV-1 replication cycle. However, because of its inherent flexibility from the interdomain linker and the monomer-dimer equilibrium in solution, the HIV-1 wild-type CA monomer has defied structural determinations by X-ray crystallography and nuclear magnetic resonance spectroscopy. Here we report the detailed solution structure of full-length HIV-1 CA using a monomeric mutant that, though noninfective, preserves many of the critical properties of the wild-type protein. The structure shows independently folded N-terminal (NTD) and C-terminal domains (CTD) joined by a flexible linker. The CTD shows some differences from that of the dimeric wild-type CTD structures. This study provides insights into the molecular mechanism of the wild-type CA dimerization critical for capsid assembly. The monomeric mutant allows investigation of interactions of CA with human cellular proteins exploited by HIV-1, directly in solution without the complications associated with the monomer-dimer equilibrium of the wild-type protein. This structure also permits the design of inhibitors directed at a novel target, viz., interdomain flexibility, as well as inhibitors that target multiple interdomain interactions critical for assembly and interactions of CA with host cellular proteins that play significant roles within the replication cycle of HIV-1.