The Desensitization Gating of the MthK K+ Channel Is Governed by Its Cytoplasmic Amino Terminus

The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensitization and acid-induced inactivation. The acid inactivation is mediated by the C-terminal RCK domain assembly. Here, we report that the desensitization gating is governed by a desensitization domain (DD) of the cytoplasmic N-terminal 17 residues. Deletion of DD completely removes the desensitization, and the process can be fully restored by a synthetic DD peptide added in trans. Mutagenesis analyses reveal a sequence-specific determinant for desensitization within the initial hydrophobic segment of DD. Proton nuclear magnetic resonance (¹H NMR) spectroscopy analyses with synthetic peptides and isolated RCK show interactions between the two terminal domains. Additionally, we show that deletion of DD does not affect the acid-induced inactivation, indicating that the two inactivation processes are mutually independent. Our results demonstrate that the short N-terminal DD of MthK functions as a complete moveable module responsible for the desensitization. Its interaction with the C-terminal RCK domain may play a role in the gating process.     

Heat-induced Irreversible Denaturation of the Camelid Single Domain VHH Antibody Is Governed by Chemical Modifications

The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.     

Biased Signaling of the G-Protein-Coupled Receptor β2AR Is Governed by Conformational Exchange Kinetics

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Localization of Protein Aggregation in Escherichia coli Is Governed by Diffusion and Nucleoid Macromolecular Crowding Effect

Aggregates of misfolded proteins are a hallmark of many age-related diseases. Recently, they have been linked to aging of Escherichia coli (E. coli) where protein aggregates accumulate at the old pole region of the aging bacterium. Because of the potential of E. coli as a model organism, elucidating aging and protein aggregation in this bacterium may pave the way to significant advances in our global understanding of aging. A first obstacle along this path is to decipher the mechanisms by which protein aggregates are targeted to specific intercellular locations. Here, using an integrated approach based on individual-based modeling, time-lapse fluorescence microscopy and automated image analysis, we show that the movement of aging-related protein aggregates in E. coli is purely diffusive (Brownian). Using single-particle tracking of protein aggregates in live E. coli cells, we estimated the average size and diffusion constant of the aggregates. Our results provide evidence that the aggregates passively diffuse within the cell, with diffusion constants that depend on their size in agreement with the Stokes-Einstein law. However, the aggregate displacements along the cell long axis are confined to a region that roughly corresponds to the nucleoid-free space in the cell pole, thus confirming the importance of increased macromolecular crowding in the nucleoids. We thus used 3D individual-based modeling to show that these three ingredients (diffusion, aggregation and diffusion hindrance in the nucleoids) are sufficient and necessary to reproduce the available experimental data on aggregate localization in the cells. Taken together, our results strongly support the hypothesis that the localization of aging-related protein aggregates in the poles of E. coli results from the coupling of passive diffusion-aggregation with spatially non-homogeneous macromolecular crowding. They further support the importance of “soft” intracellular structuring (based on macromolecular crowding) in diffusion-based protein localization in E. coli.     

The Biogenesis of SRP RNA Is Modulated by an RNA Folding Intermediate Attained during Transcription

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TLR9 Activation Is Triggered by the Excess of Stimulatory versus Inhibitory Motifs Present in Trypanosomatidae DNA

DNA sequences purified from distinct organisms, e.g. non vertebrate versus vertebrate ones, were shown to differ in their TLR9 signalling properties especially when either mouse bone marrow-derived- or human dendritic cells (DCs) are probed as target cells. Here we found that the DC-targeting immunostimulatory property of Leishmania major DNA is shared by other Trypanosomatidae DNA, suggesting that this is a general trait of these eukaryotic single-celled parasites. We first documented, in vitro, that the low level of immunostimulatory activity by vertebrate DNA is not due to its limited access to DCs'' TLR9. In addition, vertebrate DNA inhibits the activation induced by the parasite DNA. This inhibition could result from the presence of competing elements for TLR9 activation and suggests that DNA from different species can be discriminated by mouse and human DCs. Second, using computational analysis of genomic DNA sequences, it was possible to detect the presence of over-represented inhibitory and under-represented stimulatory sequences in the vertebrate genomes, whereas L. major genome displays the opposite trend. Interestingly, this contrasting features between L. major and vertebrate genomes in the frequency of these motifs are shared by other Trypanosomatidae genomes (Trypanosoma cruzi, brucei and vivax). We also addressed the possibility that proteins expressed in DCs could interact with DNA and promote TLR9 activation. We found that TLR9 is specifically activated with L. major HMGB1-bound DNA and that HMGB1 preferentially binds to L. major compared to mouse DNA. Our results highlight that both DNA sequence and vertebrate DNA-binding proteins, such as the mouse HMGB1, allow the TLR9-signaling to be initiated and achieved by Trypanosomatidae DNA.     

The Transition from Quiescent to Activated States in Human Hematopoietic Stem Cells Is Governed by Dynamic 3D Genome Reorganization

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Stability of the Plasmodium falciparum AMA1-RON2 Complex Is Governed by the Domain II (DII) Loop

Plasmodium falciparum is an obligate intracellular protozoan parasite that employs a highly sophisticated mechanism to access the protective environment of the host cells. Key to this mechanism is the formation of an electron dense ring at the parasite-host cell interface called the Moving Junction (MJ) through which the parasite invades. The MJ incorporates two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, the latter one being targeted to the host cell membrane during invasion. Crystal structures of AMA1 have shown that a partially mobile loop, termed the DII loop, forms part of a deep groove in domain I and overlaps with the RON2 binding site. To investigate the mechanism by which the DII loop influences RON2 binding, we measured the kinetics of association and dissociation and binding equilibria of a PfRON2sp1 peptide with both PfAMA1 and an engineered form of PfAMA1 where the flexible region of the DII loop was replaced by a short Gly-Ser linker (ΔDII-PfAMA1). The reactions were tracked by fluorescence anisotropy as a function of temperature and concentration and globally fitted to acquire the rate constants and corresponding thermodynamic profiles. Our results indicate that both PfAMA1 constructs bound to the PfRON2sp1 peptide with the formation of one intermediate in a sequential reversible reaction: A↔B↔C. Consistent with Isothermal Titration Calorimetry measurements, final complex formation was enthalpically driven and slightly entropically unfavorable. Importantly, our experimental data shows that the DII loop lengthened the complex half-life time by 18-fold (900 s and 48 s at 25°C for Pf and ΔDII-Pf complex, respectively). The longer half-life of the Pf complex appeared to be driven by a slower dissociation process. These data highlight a new influential role for the DII loop in kinetically locking the functional binary complex to enable host cell invasion.     

The Intermediate Disturbance Hypothesis of Species Coexistence Is Based on Patch Dynamics

The 'Intermediate Disturbance Hypothesis' (IDH) is one mechanism suggested to explain indefinite species coexistence. Hutchinson's original concept of the IDH was of a mechanism based on patch dynamics, and logical consideration shows that IDH works only if interpreted this way. Dependence on patch dynamics distinguishes IDH from Gradual Climate Change (GCC), though they are distinct also in terms of premature death of individuals, species selectivity, and the suddenness and transience of the perturbation. The application of the concepts of 'disturbance' and of IDH to phytoplankton communities is questioned.     

The Native 67-Kilodalton Minor Fimbria of Porphyromonas gingivalis Is a Novel Glycoprotein with DC-SIGN-Targeting Motifs

We recently reported that the oral mucosal pathogen Porphyromonas gingivalis, through its 67-kDa Mfa1 (minor) fimbria, targets the C-type lectin receptor DC-SIGN for invasion and persistence within human monocyte-derived dendritic cells (DCs). The DCs respond by inducing an immunosuppressive and Th2-biased CD4⁺ T-cell response. We have now purified the native minor fimbria by ion-exchange chromatography and sequenced the fimbria by tandem mass spectrometry (MS/MS), confirming its identity and revealing two putative N-glycosylation motifs as well as numerous putative O-glycosylation sites. We further show that the minor fimbria is glycosylated by ProQ staining and that glycosylation is partially removed by treatment with β(1-4)-galactosidase, but not by classic N- and O-linked deglycosidases. Further monosaccharide analysis by gas chromatography-mass spectrometry (GC-MS) confirmed that the minor fimbria contains the DC-SIGN-targeting carbohydrates fucose (1.35 nmol/mg), mannose (2.68 nmol/mg), N-acetylglucosamine (2.27 nmol/mg), and N-acetylgalactosamine (0.652 nmol/mg). Analysis by transmission electron microscopy revealed that the minor fimbria forms fibers approximately 200 nm in length that could be involved in targeting or cross-linking DC-SIGN. These findings shed further light on molecular mechanisms of invasion and immunosuppression by this unique mucosal pathogen.Porphyromonas gingivalis is one of several mucosal pathogens that have been implicated in chronic periodontitis (CP), a common oral disease that may affect 40 to 60% of the U.S. population (7). P. gingivalis utilizes a myriad of virulence factors that contribute to chronic periodontitis. Among these are a polysaccharide capsule, fimbriae, proteases for opsonins C3 and IgG, gingipains (21, 30, 43, 52), bacterial lipopolysaccharides (LPS) (22, 44), and toxins and hemagglutinins (10, 25).The fimbriae of P. gingivalis play a crucial role in adhesion to and invasion of host cells. We have shown that optimum entry of P. gingivalis into human dendritic cells (DCs) requires the presence of two fimbriae, termed the major and minor fimbriae. The major fimbria is composed of a 41-kDa protein termed fimbrillin, encoded by the fimA gene (65). Much less is known about the minor fimbria, the focus of this paper. The minor fimbria is comprised of a 67-kDa protein (19) that is encoded by the mfa1 gene. The major and minor fimbriae are antigenically distinct, and they also differ based on amino acid composition and size (5, 19). Very little is understood about the formation and secretion of the minor fimbriae and about possible posttranslational modifications of these fimbriae. Formation and secretion of the major fimbriae is a complex reaction consisting of numerous steps required for transfer of prefimbrillin proteins from the cytoplasm to the periplasm, cleavage of the N-terminal signal peptide (24, 50), transport of prefimbrillin to the outer face of the outer membrane, and assembly into fimbria structures (23, 24, 34).Deciphering the cellular receptors for the fimbriae is an active area of research. Evidence suggests that the cellular targets of the major fimbriae are the β-1 integrins (CD29) (32, 66). Others have proposed a role for β-2 integrins (CD18) (17, 18, 55) in the cellular response to major fimbriae. In contrast, little is known of the cellular receptors for the minor fimbriae. Lamont et al. in 2002 showed that the minor fimbria of P. gingivalis intimately interacts with the SspB protein of Streptococcus gordonii (26). This interaction might aid in P. gingivalis colonization of plaque biofilm before it invades gingival tissue (26, 41). We recently showed that the minor fimbria targets DC-SIGN on DCs for entry into DCs and that this targeting has the immunological consequence of dampening the immune response (68).DC-SIGN is a type II membrane protein on DCs in which the extracellular domain consists of a stalk that promotes tetramerization (13). DC-SIGN contains a C-terminal carbohydrate-recognizing domain (CRD) that belongs to the C-type lectin superfamily (13). Early studies by Feinberg et al. in 2001 showed that the DC-SIGN CRD preferentially binds to the high-mannose N-linked oligosaccharides GlcNAc (N-acetylglucosamine) and Manα1-3[Manα1-6] Man (mannose) (13). Furthermore, Appelmelk et al. showed that DC-SIGN also binds to fucose-containing Lewis blood antigens (4). Guo et al. utilized an extensive glycan array and showed that DC-SIGN will bind high-mannose-containing glycans or glycans that contain terminal fucose residues (16). Previous studies showed that DC-SIGN on DCs is used by microorganisms such as Neisseria gonorrhoeae, Mycobacterium tuberculosis, Mycobacterium leprae, HIV, and Helicobacter pylori for entry into DCs and induction of immunosuppression (4, 27, 42, 51, 69). Like P. gingivalis, many of these pathogens can induce chronic life-long infections.Our previously published work established that the minor fimbria is necessary for targeting DC-SIGN, resulting in entry of P. gingivalis into DCs (68). We were able to abrogate minor fimbria-mediated DC-SIGN ligation by using DC-SIGN-blocking agents or agonists, including fucose, mannose, and mannan (68). Additionally, we described that the minor fimbria is able to induce immunosuppression of DCs via its interaction with DC-SIGN, which was blocked by sugars (68). Further, we demonstrated that minor fimbriated strains of P. gingivalis inhibited DC maturation and suppressed proinflammatory cytokine secretion (68). Moreover, DCs that were pulsed with minor fimbriated strains of P. gingivalis and then cocultured with autologous T cells shifted the T-cell effector phenotype to a Th2 effector phenotype, as evidenced by high interleukin-4 (IL-4) production (68).Our previous results, described above, suggested that the minor fimbria-DC-SIGN interaction was mediated by glycosylated proteins. We therefore set out to identify the carbohydrate moieties on the minor fimbria that could account for its DC-SIGN-targeting function. The intact native minor fimbria was purified and analyzed for glycosylation and for the presence of relevant monosaccharides. We show here by a combination of ProQ gel staining and gas chromatography-mass spectrometry (GC-MS) analysis that the minor fimbria is glycosylated and expresses the DC-SIGN ligands fucose, mannose, GlcNAc, and GalNAc. Use of classic N- and O-linked deglycosidases on the native minor fimbria revealed a novel glycoprotein structure. Overall, these results indicate that the minor fimbria is glycosylated with DC-SIGN-binding motifs that likely account for the reported ability of P. gingivalis to bind to and invade DCs, resulting in an immunosuppressive DC response.     

Localization to Mature Melanosomes by Virtue of Cytoplasmic Dileucine Motifs Is Required for Human OCA2 Function

Oculocutaneous albinism type 2 is caused by defects in the gene OCA2, encoding a pigment cell-specific, 12-transmembrane domain protein with homology to ion permeases. The function of the OCA2 protein remains unknown, and its subcellular localization is under debate. Here, we show that endogenous OCA2 in melanocytic cells rapidly exits the endoplasmic reticulum (ER) and thus does not behave as a resident ER protein. Consistently, exogenously expressed OCA2 localizes within melanocytes to melanosomes, and, like other melanosomal proteins, localizes to lysosomes when expressed in nonpigment cells. Mutagenized OCA2 transgenes stimulate melanin synthesis in OCA2-deficient cells when localized to melanosomes but not when specifically retained in the ER, contradicting a proposed primary function for OCA2 in the ER. Steady-state melanosomal localization requires a conserved consensus acidic dileucine-based sorting motif within the cytoplasmic N-terminal region of OCA2. A second dileucine signal within this region confers steady-state lysosomal localization in melanocytes, suggesting that OCA2 might traverse multiple sequential or parallel trafficking routes. The two dileucine signals physically interact in a differential manner with cytoplasmic adaptors known to function in trafficking other proteins to melanosomes. We conclude that OCA2 is targeted to and functions within melanosomes but that residence within melanosomes may be regulated by secondary or alternative targeting to lysosomes.     

The Choice of Translation Initiation Site of the Rep Proteins from Goose Parvovirus P9-Generated mRNA Is Governed by Splicing and the Nature of the Excised Intron

The goose parvovirus (GPV) Rep 1 and Rep 2 proteins are encoded by P9-generated mRNAs that are either unspliced or spliced within the rep gene region, respectively. These mRNAs are present in an approximately equal ratio. The translation of Rep 1 was initiated from the first AUG in unspliced P9-generated mRNA; however, this AUG was bypassed in spliced P9-generated RNA and Rep 2 translation initiated predominately at the next initiating AUG downstream. We show that the choice of the site of initiation of translation of GPV Rep-encoding mRNAs is governed both by the splicing process itself and by the nature of the excised intron.Goose parvovirus (GPV) has identical hairpin termini, is most similar in both nucleotide sequence and protein homology to adeno-associated virus 2 (AAV2), and has been classified as a member of the Dependovirus genus (10-12); however, unlike the AAVs, GPV can replicate efficiently without the aid of a helper virus (12). The RNA expression profile of GPV is a surprising hybrid of features of the Parvovirus and Dependovirus genera of the Parvovirinae (7). Similar to the Dependovirus AAV5, RNAs transcribed from the GPV upstream P9 promoter, which encode the viral Rep protein(s), are polyadenylated at high efficiency at a polyadenylation [(pA)p] site located within the small intron in the center of the genome (7). No promoter analogous to the Dependovirus P19 promoter has been detected; however, similar to minute virus of mice (MVM) and other members of the Parvovirus genus, approximately half of the pre-mRNAs generated from the P9 promoter are additionally spliced within the putative GPV Rep coding region between a donor site located at nucleotide (nt) 814 and an acceptor site at nt 1198 (7). The GPV RNA profile has been shown to be the same in both human 293T and goose CGBQ cells (7). Thus, the mechanism that GPV uses for the expression of its nonstructural gene is more like that used by members of the autonomous Parvovirus group.In this report, we describe the coding strategy for the nonstructural proteins of GPV. We demonstrate that the large Rep 1 protein is encoded uninterruptedly in open reading frame 1 (ORF 1) from the unspliced P9-generated mRNA using an initiating AUG codon at nt 537. The smaller Rep 2 protein is encoded by the spliced P9-generated mRNA; it initiates in ORF 2 at an AUG at nt 650 and continues in ORF 1 after the splice. Strikingly, the first upstream AUG at nt 537 is not utilized in spliced P9-generated mRNA. We show that the choice of initiation site is governed by the splicing process itself and by the nature of the excised intron.