Selective arginines are important for the antibacterial activity and host cell interaction of human α-defensin 5

Defensins constitute a major family of natural antimicrobial peptides that protect the host against microbial invasion. Here, we report on the antibacterial properties and cellular interaction of Human Defensin 5 as a function of its positive charge and hydrophobicity. We find that selective replacement of arginine residues in HD-5 by alanine or charge-neutral lysine residues reduces antibacterial killing as well as host cell interaction. We identify arginines at positions 9 and 28 in the HD-5 sequence as particularly important for its function. Replacement of arginine at position 13 to Histidine, as observed in a Crohn’s disease patient, reduced bacterial killing strain-selectively. Finally, we find that HD-5 interacts with host cells via receptor-mediated mechanisms.     

Inhibitory effect of human TRIM5α on HIV-1 production

Tripartite motif-containing 5 isoform-α (TRIM5α), a host restriction factor, blocks infection of some retroviruses at a post-entry, pre-integration stage in a species-specific manner. A recent report by Sakuma et al. describes a second antiretroviral activity of rhesus macaque TRIM5α, which blocks HIV-1 production through rapid degradation of HIV-1 Gag polyproteins. Here, we find that human TRIM5α limits HIV-1 production. Transient expression of TRIM5α decreased HIV-1 production, whereas knockdown of TRIM5α in human cells increased virion release. A single amino acid substitution (R437C) in the SPRY domain diminished the restriction effect. Moderate levels of human wild-type TRIM5α and a little amount of R437C mutant were incorporated into HIV-1 virions. The R437C mutant also lost restriction activity against N-tropic murine leukemia virus infection. However, the corresponding R to C mutation in rhesus macaque TRIM5α had no effect on the restriction ability. Our findings suggest human TRIM5α is an intrinsic immunity factor against HIV-1 infection. The importance of arginine at 437 aa in SPRY domain for the late restriction is species-specific.     

Homology-based identification of capsid determinants that protect HIV1 from human TRIM5α restriction

The tropism of retroviruses relies on their ability to exploit cellular factors for their replication as well as to avoid host-encoded inhibitory activities such as TRIM5α. N-tropic murine leukemia virus is sensitive to human TRIM5α (huTRIM5α) restriction, whereas human immunodeficiency virus type 1 (HIV1) escapes this antiviral factor. We previously revealed that mutation of four critical amino acid residues within the capsid can render murine leukemia virus resistant to huTRIM5α. Here, we exploit the high degree of conservation in the tertiary structure of retroviral capsids to map the corresponding positions on the HIV1 capsid. We then demonstrated that, when changes were introduced at some of these positions, HIV1 becomes sensitive to huTRIM5α restriction, a phenomenon reinforced by additionally mutating the nearby cyclophilin A binding loop of the viral protein. These results indicate that retroviruses have evolved similar mechanisms to escape TRIM5α restriction via the interference of structurally homologous determinants in the viral capsid.     

Generation of mutants of CK2α which are dependent on the β-subunit for catalytic activity

To shed light on the structural features underlying high constitutive activity of protein kinase CK2 a number of mutants of the human CK2 subunit altered in the interactions between the N-terminal segment and the activation loop have been generated and shown to be defective in catalytic activity. In particular the truncated mutant 2-12 displays under standard conditions an almost complete loss of catalytic activity accounted for by a dramatic rise in its Km for ATP (from 10 to 206 M) and a reduced Kcat. Such a drop in efficiency is paralleled by conformational disorganization, as judged from Superdex 75 gel filtration profile. Both catalytic properties and gel filtration behaviour similar to those of wild type CK2 were restored upon association with the regulatory -subunit, suggesting that constitutive activity is conferred to CK2 and to CK2 holoenzyme through different molecular mechanisms. In the holoenzyme an assumable release of tension at the backbone of Ala-193 (as seems to be indicated by a comparison of the crystal structures of maize CK2 alone vs. a CK2– peptide complex) may result in the ability of the activation loop to adopt its proper conformation independently of interactions with the N-terminal segment.     

Is changing hypothalamic activity important for control of ovulation?

The activity of the hypothalamic gonadotrophin releasing hormone pulse generator in women with regular ovulatory and anovulatory menstrual cycles was assessed to see whether changes therein are important determinants of normal and impaired ovarian function. Endogenous gonadotrophin releasing hormone secretion was inferred by measurement of the pituitary luteinising hormone response by characterisation of pulsatile luteinising hormone release over eight hours on three occasions during the course of follicular development and once during the luteal stage of the same cycles. In 13 ovulatory cycles (serum progesterone concentration greater than 25 nmol/l) confirmed by ovarian ultrasonography a pronounced variability in luteinising hormone pulse patterns among subjects was compatible with ovulation. In the luteal stage of ovulatory cycles the luteinising hormone interpeak interval (85 min, range 42-125) was significantly longer than that during the early follicular (64 min, 40-103), mid-follicular (62 min, 37-107), and late follicular (59 min, 39-80) stages of the same cycles. Thus in ovulatory cycles no increase in frequency of the gonadotrophin releasing hormone pulse generator was detected during follicular development, though this activity decreased in the luteal stage. In five late follicular stage studies in which part of the preovulatory luteinising hormone surge was captured no change in pulse frequency of luteinising hormone was detected compared with the mid-follicular stage of the same cycles or when compared with the late follicular stage of other cycles when no luteinising hormone surge was captured. Though mean luteinising hormone concentrations in luteinising hormone surge series (36 IU/l) were high, the amplitude of luteinising hormone pulses (165%) was only slightly greater than during non-surge late follicular stage studies (145%). Hence no change in hypothalamic gonadotrophin releasing hormone activity is required to generate the preovulatory discharge of luteinising hormone in man, which occurs as a result of the sensitising action of rising oestradiol concentrations on pituitary responsiveness to the same hypothalamic input signal. Luteinising hormone pulse frequency, peak amplitude, and mean serum luteinising hormone concentrations in seven anovulatory cycles (progesterone concentration less than 10 nmol/l) were not different from those at comparable stages of ovulatory cycles. These data suggest that the primary abnormality in this group of regularly menstruating anovulatory women lies in the ovary rather than in the hypothalamic control of the anterior pituitary.     

Evidence for Restriction of Ancient Primate Gammaretroviruses by APOBEC3 but Not TRIM5α Proteins

Because of evolutionary pressures imposed through episodic colonization by retroviruses, many mammals express factors, such as TRIM5α and APOBEC3 proteins, that directly restrict retroviral replication. TRIM5 and APOBEC restriction factors are most often studied in the context of modern primate lentiviruses, but it is likely that ancient retroviruses imposed the selective pressure that is evident in primate TRIM5 and APOBEC3 genes. Moreover, these antiretroviral factors have been shown to act against a variety of retroviruses, including gammaretroviruses. Endogenous retroviruses can provide a ‘fossil record’ of extinct retroviruses and perhaps evidence of ancient TRIM5 and APOBEC3 antiviral activity. Here, we investigate whether TRIM5 and APOBEC3 proteins restricted the replication of two groups of gammaretroviruses that were endogenized in the past few million years. These endogenous retroviruses appear quite widespread in the genomes of old world primates but failed to colonize the human germline. Our analyses suggest that TRIM5α proteins did not pose a major barrier to the cross-species transmission of these two families of gammaretroviruses, and did not contribute to their extinction. However, we uncovered extensive evidence for inactivation of ancient gammaretroviruses through the action of APOBEC3 cytidine deaminases. Interestingly, the identities of the cytidine deaminases responsible for inactivation appear to have varied in both a virus and host species–dependent manner. Overall, sequence analyses and reconstitution of ancient retroviruses from remnants that have been preserved in the genomes of modern organisms offer the opportunity to probe and potentially explain the evolutionary history of host defenses against retroviruses.     

Cytoplasmic Body Component TRIM5α Requires Lipid-enriched Microdomains for Efficient HIV-1 Restriction

TRIM5α is a member of the tripartite motif (TRIM) family of proteins and affects both early and late phases of the retroviral life cycle. Although TRIM5α multimerizes to form cytoplasmic bodies, which are thought to play an important role in viral restriction, the identity of TRIM5α-containing cytoplasmic bodies remains elusive. To better understand TRIM5α cytoplasmic body constituents and the cellular proteins that could be involved in the TRIM5α-mediated antiviral activities, we sought TRIM5α-binding factors. We identified a lipid microdomain protein flotillin-1/Reggie-2 as an interacting partner of TRIM5α via co-immunoprecipitation. Immunohistochemistry studies confirmed the co-localization of rhesus monkey TRIM5α (TRIM5αrh) cytoplasmic bodies with flotillin-1/Reggie-2. Caveolin-1, another lipid microdomain-associated protein, also co-localized with TRIM5α cytoplasmic bodies. Intriguingly, disruption of cellular cholesterol by cyclodextrin perturbed TRIM5α cytoplasmic body formation. Furthermore, lipid starvation partially relieved the endogenous post-entry restriction of HIV-1 infection, which could be subsequently restored by lipid repletion. These observations indicate the involvement of cellular lipids in TRIM5α-mediated antiviral activities. Given that many viruses utilize cellular lipid microdomains for viral entry and assembly, it is plausible that lipid-enriched domains provide microenvironments where TRIM5α recognizes retroviral components.     

Protein kinase Cα activity is important for contraction-induced FXYD1 phosphorylation in skeletal muscle

Exercise-induced phosphorylation of FXYD1 is a potential important regulator of Na(+)-K(+)-pump activity. It was investigated whether skeletal muscle contractions induce phosphorylation of FXYD1 and whether protein kinase Cα (PKCα) activity is a prerequisite for this possible mechanism. In part 1, human muscle biopsies were obtained at rest, after 30 s of high-intensity exercise (166 ± 31% of Vo(2max)) and after a subsequent 20 min of moderate-intensity exercise (79 ± 8% of Vo(2max)). In general, FXYD1 phosphorylation was increased compared with rest both after 30 s (P < 0.05) and 20 min (P < 0.001), and more so after 20 min compared with 30 s (P < 0.05). Specifically, FXYD1 ser63, ser68, and combined ser68 and thr69 phosphorylation were 26-45% higher (P < 0.05) after 20 min of exercise than at rest. In part 2, FXYD1 phosphorylation was investigated in electrically stimulated soleus and EDL muscles from PKCα knockout (KO) and wild-type (WT) mice. Contractile activity caused FXYD1 ser68 phosphorylation to be increased (P < 0.001) in WT soleus muscles but to be reduced (P < 0.001) in WT extensor digitorum longus. In contrast, contractile activity did not affect FXYD1 ser68 phosphorylation in the KO mice. In conclusion, exercise induces FXYD1 phosphorylation at multiple sites in human skeletal muscle. In mouse muscles, contraction-induced changes in FXYD1 ser68 phosphorylation are fiber-type specific and dependent on PKCα activity.     

Structure–activity relationships of the thujaplicins for inhibition of human tyrosinase

Tyrosinase inhibitors have become increasingly critical agents in cosmetic, agricultural, and medicinal products. Although a large number of tyrosinase inhibitors have been reported, almost all the inhibitors were unfortunately evaluated by using commercial available mushroom tyrosinase. Here, we examined the inhibitory effects of three isomers of thujaplicin (α, β, and γ) on human tyrosinase and analyzed their binding modes using homology model and docking studies. As the results, γ-thujaplicin was found to strongly inhibit human tyrosinase with the IC₅₀ of 1.15 μM, extremely superior to a well-known tyrosinase inhibitor kojic acid (IC₅₀ = 571.17 μM). MM-GB/SA binding free energy decomposition analyses suggested that the potent inhibitory activity of γ-thujaplicin may be due to the interactions with His367, Ile368, and Val377 (hot spot amino acid residues) in human tyrosinase. Furthermore, the binding mode of α-thujaplicin indicated that Val377 and Ser380 may cause van der Waals clashes with the isopropyl group of α-thujaplicin. These results provide a novel structural insight into the hot spot of human tyrosinase for the specific binding of γ-thujaplicin and a way to optimize not only thujaplicins but also other lead compounds as specific inhibitors for human tyrosinase in a rational manner.     

Expression of recombinant human interferon-γ with antiviral activity in the bi-cistronic baculovirus-insect/larval system

A bi-cistronic baculovirus-insect/larval system containing a polyhedron promoter, an internal ribosome entry site (IRES), and an egfp gene was developed as a cost-effective platform for the production of recombinant human interferon gamma (rhIFN-γ). There was no significant difference between the amounts of rhIFN-γ produced in the baculovirus-infected Spodoptera frugiferda 21 cells grown in serum-free medium and the serum-supplemented medium, while the Trichoplusia ni (T. ni) and Spodoptera exigua (S. exigua) larvae afforded rhIFN-γ amounting to 1.08±0.04 and 9.74±0.35 μg/mg protein respectively. The presence of non-glycosylated and glycosylated rhIFN-γ was confirmed by immunoblot and lectin blot. The immunological activity of purified rhIFN-γ, with 96% purity by Nickel (II)-nitrilotriacetic acid (Ni-NTA) affinity chromatography, was similar to that commercially available. Moreover, the rhIFN-γ protein from T. ni had more potent antiviral activity. These findings suggest that this IRES-based expression system is a simple and inexpensive alternative for large-scale protein production in anti-viral research.