Repeat domains of melanosome matrix protein Pmel17 orthologs form amyloid fibrils at the acidic melanosomal pH

Most amyloids are pathological, but fragments of Pmel17 form a functional amyloid in vertebrate melanosomes essential for melanin synthesis and deposition. We previously reported that only at the mildly acidic pH (4-5.5) typical of melanosomes, the repeat domain (RPT) of human Pmel17 can form amyloid in vitro. Combined with the known presence of RPT in the melanosome filaments and the requirement of this domain for filament formation, we proposed that RPT may be the core of the amyloid formed in vivo. Although most of Pmel17 is highly conserved across a broad range of vertebrates, the RPT domains vary dramatically, with no apparent homology in some cases. Here, we report that the RPT domains of mouse and zebrafish, as well as a small splice variant of human Pmel17, all form amyloid specifically at mildly acid pH (pH ～5.0). Protease digestion, mass per unit length measurements, and solid-state NMR experiments suggest that amyloid of the mouse RPT has an in-register parallel β-sheet architecture with two RPT molecules per layer, similar to amyloid of the Aβ peptide. Although there is no sequence conservation between human and zebrafish RPT, amyloid formation at acid pH is conserved.     

Binding of full-length HIV-1 gp120 to CD4 induces structural reorientation around the gp120 core

Small-angle x-ray scattering data on the unliganded full-length fully glycosylated HIV-1 gp120, the soluble CD4 (domains 1-2) receptor, and their complex in solution are presented. Ab initio structure restorations using these data provides the first look at the envelope shape for the unliganded and the complexed gp120 molecule. Fitting known crystal structures of the unliganded SIV and the complexed HIV gp120 core regions within our resultant shape constraints reveals movement of the V3 loop upon binding.     

Multiple interactions across the surface of the gp120 core structure determine the global neutralization resistance phenotype of human immunodeficiency virus type 1

Resistance to neutralization is an important characteristic of primary isolates of human immunodeficiency virus type 1 (HIV-1) that relates to the potential for successful vaccination to prevent infection and use of immunotherapeutics for treatment of established infection. In order to further elucidate mechanisms responsible for neutralization resistance, we studied the molecular mechanisms that determine the resistance of the primary virus isolate of the strain HIV-1 MN to neutralization by soluble CD4 (sCD4). As is the case for the global neutralization resistance phenotype, sCD4 resistance depended upon sequences in the amino-terminal heptad repeat region of gp41 (HR1), as well as on multiple functional interactions within the envelope complex. The functional interactions that determined the resistance included interactions between the variable loop 1 and 2 (V1/V2) region and sequences in or near the CD4 binding site (CD4bs) and with the V3 loop. Additionally, the V3 loop region was found to interact functionally with sequences in the outer domain of gp120, distant from the CD4bs and coreceptor-binding site, as well as with a residue thought to be located centrally in the coreceptor-binding site. These and previous results provide the basis for a model by which functional signals that determine the neutralization resistance, high-infectivity phenotype depend upon interactions occurring across the surface of the gp120 core structure and involving variable loop structures and gp41. This model should be useful in efforts to define epitopes that may be important for primary virus neutralization.     

HIV-1 Tat raises an adjuvant-free humoral immune response controlled by its core region and its ability to form cysteine-mediated oligomers

Proteins are poor immunogens that require an adjuvant to raise an immune response. Here we show that the human immunodeficiency virus, type 1 Tat protein possesses an autoadjuvant property, and we have identified the determinants and the molecular events that are associated with this unusual property. Using a series of chemically synthesized Tat101 derivatives, we show that the core region controls the autoadjuvant phenomenon independently of the B-cell recognition and T-cell stimulation that are associated with epitopes respectively located on the N-terminal region and the cysteine-rich region. We also show that cysteine-mediated oligomerization is a key molecular event of the adjuvant-free antibody response. In particular, a Tat dimer formed by the oxidation of two cysteine residues, at position 34 only, raises an adjuvant-free antibody response that is comparable with that observed with the wild-type protein. Unlike the parent protein, the Tat dimer has no transactivating activity and remains homogeneous for several weeks in solution. This construct might be of value for the design of an adjuvant-free Tat-based vaccine. Furthermore, we suggest that the specific autoadjuvanticity determinant of Tat could be used to provide other proteins with adjuvant-free immunogenicity.     

Beta(2)-microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro.

Amyloid fibrils formed by incubation of recombinant wild-type human beta(2)-microglobulin (beta(2)M) ab initio in vitro at low pH and high ionic strength are short and highly curved. By contrast, fibrils extracted from patients suffering from haemodialysis-related amyloidosis and those formed by seeding growth of the wild-type protein in vitro with fibrils ex vivo are longer and straighter than those previously produced ab initio in vitro. Here we explore the effect of growth conditions on morphology of beta(2)M fibrils formed ab initio in vitro from the wild-type protein, as well as a variant form of beta(2)M in which Asn17 is deamidated to Asp (N17D). We show that deamidation results in significant destabilisation of beta(2)M at neutral pH. Despite this, acidification is still necessary to form amyloid from the mutant protein in vitro. Interestingly, at low pH and low ionic strength long, straight fibrils of recombinant beta(2)M are formed in vitro. The fibrils comprise three distinct morphological types when examined using electron microscopy (EM) and atomic force microscopy (AFM) that vary in periodicity and the number of constituent protofibrils. Using kinetic experiments we suggest that the immature fibrils observed previously do not represent intermediates in the assembly of fully mature amyloid, at least under the conditions studied here.     

Characterization of the conformational state and flexibility of HIV-1 glycoprotein gp120 core domain

gp120 is key to the human immunodeficiency virus type 1 viral cell entry. Knowledge of the detailed conformational states of gp120 is crucial to intervention, yet the unbound form is still resistant to structural characterization probably because of its flexibility. Toward this goal, we performed molecular dynamics simulations on the wild type gp120 core domain extracted from its ternary crystal structure and on a modeled mutant, S375W, that experimentally has a significantly different phenotype from the wild type. Although the mutant retained a bound-like conformation, the wild type drifted to a different conformational state. The wild type beta strands 2 and 3 of the bridging sheet were very mobile and partially unfolded, and the organization among the inner and outer domains and beta strands 20 and 21 of the bridging sheet, near the mutation site, was more open than in the bound form, although the overall structure was maintained. These differences were apparently a result of the strengthening of the hydrophobic core in the mutant. This stabilization further explains the experimentally significantly different thermodynamic properties between the wild type and the mutant. Taken together, our results suggest that the free form, although different from the bound state, shares many of the bound structural features. The observed loss of freedom near the binding site, rather than the previously hypothesized more dramatic conformational transition from the unbound to the bound state, appears to be the major contributor to the large entropy cost for the CD4 binding to the wild type.     

A salt bridge between an N-terminal coiled coil of gp41 and an antiviral agent targeted to the gp41 core is important for anti-HIV-1 activity

HIV-1 envelope glycoprotein transmembrane subunit gp41 play a critical role in the fusion of viral and target cell membranes. The gp41 C-terminal heptad repeat region interacts with the N-terminal coiled-coil region to form a six-stranded core structure. Peptides derived from gp41 C-terminal heptad repeat region (C-peptides) are potent HIV-1 entry inhibitors by binding to gp41 N-terminal coiled-coil region. Most recently, we have identified two small organic compounds that inhibit HIV-1-mediated membrane fusion by blocking the formation of gp41 core. These two active compounds contain both hydrophobic and acidic groups while the inactive compounds only have hydrophobic groups. Analysis by computer modeling indicate that the acidic groups in the active compounds can form salt bridge with Lys 574 in the N-terminal coiled-coil region of gp41. Asp 632 in a C-peptide can also form a salt bridge with Lys 574. Replacement of Asp 632 with positively charged residues or hydrophobic residues resulted in significant decrease of HIV-1 inhibitory activity. These results suggest that a salt bridge between an N-terminal coiled coil of the gp41 and an antiviral agent targeted to the gp41 core is important for anti-HIV-1 activity.     

MART-1 is required for the function of the melanosomal matrix protein PMEL17/GP100 and the maturation of melanosomes

More than 125 genes that regulate pigmentation have been identified to date. Of those, MART-1 has been widely studied as a melanoma-specific antigen and as a melanosome-specific marker. Whereas the functions of other melanosomal proteins, such as tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, and Pmel17, are known, the function of MART-1 in melanogenesis, is unclear. A role for MART-1 in pigmentation is expected because its expression pattern and subcellular distribution is quite similar to the other melanosomal proteins and usually correlates with melanin content. We investigated the function of MART-1 using a multidisciplinary approach, including the use of siRNA to inhibit MART-1 function and the use of transfection to re-express MART-1 in MART-1-negative cells. We show that MART-1 forms a complex with Pmel17 and affects its expression, stability, trafficking, and the processing which is required for melanosome structure and maturation. We conclude that MART-1 is indispensable for Pmel17 function and thus plays an important role in regulating mammalian pigmentation.     

Hexuronates influence the oligomeric form of the Dps structural protein of bacterial nucleoid and its ability to bind to linear DNA fragments

Proteins of the Dps family perform a dual function in bacterial cells. As ferritins, they protect cells from destructive effects of Fe²⁺ ions, while interacting with DNA they condense the genome in the absence of nutrients. The ability of Dps to self-aggregate is of a great importance. The way of genome remodelling from the condensed state to the active one is not yet known. Here, the effects of two sugar ligands on Dps interaction with DNA have been studied in vitro. For the first time it was demonstrated that D-glucuronate and D-galacturonate, but not D-glucose, can decompose the dodecameric structure of the protein and D-glucuronate stimulated the formation of binary complexes with the linear DNA fragments. As a result of flexible molecular docking, it was found that the molecules of all three sugars potentially can form clusters inside the protein cavity of Dps, but D-glucuronate and D-galacturonate were also bound in the region of intersubunit contacts of oligomer. The consequent destabilization of the intersubunit bonding network can, thus, be the main factor provoking the protein decay to the smaller oligomeric forms. Such a structural rearrangement, leading to a reduction in aggregation, may play a key role in genome decondensation during cell transition to the phase of rapid growth.     

Expression studies of the core+1 protein of the hepatitis C virus 1a in mammalian cells. The influence of the core protein and proteasomes on the intracellular levels of core+1

Recent studies have suggested the existence of a novel protein of hepatitis C virus (HCV) encoded by an ORF overlapping the core gene in the +1 frame (core+1 ORF). Two alternative translation mechanisms have been proposed for expression of the core+1 ORF of HCV-1a in cultured cells; a frameshift mechanism within codons 8-11, yielding a protein known as core+1/F, and/or translation initiation from internal codons in the core+1 ORF, yielding a shorter protein known as core+1/S. To date, the main evidence for the expression of this protein in vivo has been the specific humoral and cellular immune responses against the protein in HCV-infected patients, inasmuch as its detection in biopsies or the HCV infectious system remains elusive. In this study, we characterized the expression properties of the HCV-1a core+1 protein in mammalian cells in order to identify conditions that facilitate its detection. We showed that core+1/S is a very unstable protein, and that expression of the core protein in addition to proteosome activity can downregulate its intracellular levels. Also, we showed that in the Huh-7/T7 cytoplasmic expression system the core+1 ORF from the HCV-1 isolate supports the synthesis of both the core+1/S and core+1/F proteins. Finally, immunofluorescence and subcellular fractionation analyses indicated that core+1/S and core+1/F are cytoplasmic proteins with partial endoplasmic reticulum distribution in interphase cells, whereas in dividing cells they also localize to the microtubules of the mitotic spindle.     

A new form of crystalline rubisco and the conversion to its common dodecahedral form

In this paper, we present a new purification procedure that yields a new crystalline form of rubisco and has enabled us to completely remove this most abundant protein from tobacco leaf extract. The crystals formed within 48 h after refrigeration at 4 degrees C at pH 5.6. However, these crystals were not well-ordered crystals and lacked well-defined facets or edges. The remaining leaf extract (fraction 2 protein) was void of rubisco. Conversion of this new crystalline form of rubisco to its common dodecahedral form was achieved by dialysing the protein solution in Tris buffer at pH 8.0 or purified water. Since the molecular size of its large subunit of rubisco (55 kD) is similar to that of the papillomavirus capsid protein, L1 (57 kD), its complete removal from fraction 2-protein may facilitate the detection, purification, and recovery of the Li protein.     

Resistance to deglycosylation by ammonia of IgA1 O-glycopeptides: implications for the beta-elimination of O-glycans linked to serine and threonine

Pools of O-glycopeptides (and their deglycosylated analogues) derived from trypsin-digested normal human serum IgA1 have been treated with ammonia under conditions reported to result in complete liberation of O-glycans linked to serine and threonine residues in glycopeptides and glycoproteins. MALDI-TOF MS analysis has revealed that only one of the six glycosylated sites is susceptible to beta-elimination under these conditions. It is likely that resistance to beta-elimination is due to very close proximity of proline to the glycosylated serine or threonine residues. Preliminary results using 0.1M NaOH (instead of ammonia) to perform beta-elimination indicated that there was also selective de-O-glycosylation with this reagent, however, these results were complicated by the concomitant hydrolysis of the peptide bonds. These findings may have implications for similarly O-glycosylated peptides and proteins and possibly for other chemical methods that are used to carry out beta-eliminations of O-glycans.     

Analysis of the binding of high mobility group protein 17 to the nucleosome core particle by 1H NMR spectroscopy

The binding of high mobility group (HMG) protein 17 to the nucleosome core particle has been studied in D2O solution using 1H NMR at 500 MHz. Spectra were obtained for purified HMG 17, purified nucleosome core particles, and the reconstituted HMG 17-nucleosome core particle complex at 0.1, 0.2, 0.3, and 0.4 M NaCl. Subtraction of the core particle spectra from spectra of the core particle reconstituted with HMG 17 demonstrated those regions of HMG 17 which interact with the nucleosome at different ionic strengths; the resonance peaks of interacting groups are broadened due to their restricted mobility. At 0.1 M NaCl, the mobility of all the amino acid side chains of HMG 17 was restricted, indicating complete binding of HMG 17 to the much larger nucleosome core particle. At 0.2 M NaCl most of the amino acids were free with the exception of arginine and proline which are confined to or predominant in the basic central region of HMG 17. These amino acids were completely free only at 0.4 M NaCl. We conclude that the entire HMG 17 molecule interacts with the nucleosome core particle at physiological ionic strength. The acidic COOH-terminal region of HMG 17 is released from interaction with the core histones at an NaCl concentration between 0.1 and 0.2 M and so binds weakly at physiological ionic strength. The basic central region binds more strongly to the core particle DNA, being completely released only at much higher ionic strength, between 0.3 and 0.4 M NaCl.     

SEVI,the semen enhancer of HIV infection along with fragments from its central region,form amyloid fibrils that are toxic to neuronal cells

Semen-derived enhancer of viral infection (SEVI) is the term given to the amyloid fibrils formed by a 39-amino acid fragment (PAP248–286) of prostatic acidic phosphatase (PAP) found in human semen. SEVI enhances human immunodeficiency virus (HIV) infectivity by four to five orders of magnitude (Münch et al., 2007). Here, we show by various biophysical techniques including Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy that fragments encompassing the central region of SEVI, i.e. PAP248–271 and PAP257–267, form fibrils of similar morphology to SEVI. Our results show that the central region, residues PAP267–271, is crucially important in promoting SEVI fibril formation. Furthermore, SEVI and fibrillar forms of these peptide fragments are toxic to neuronal pheochromocytoma 12 cells but not to epithelial colon carcinoma cells. These findings imply that although SEVI assists in the attachment of HIV-1 to immune cells, it may not facilitate HIV entry by damaging the epithelial cell layer that presents a barrier to the HIV.     

Cooperative assembly of CYK-4/MgcRacGAP and ZEN-4/MKLP1 to form the centralspindlin complex

Cytokinesis in metazoan cells requires a set of antiparallel microtubules that become bundled upon anaphase onset to form a structure known as the central spindle. Bundling of these microtubules requires a protein complex, centralspindlin, that consists of the CYK-4/MgcRacGAP Rho-family GTPase-activating protein and the ZEN-4/MKLP1 kinesin-6 motor protein. Centralspindlin, but not its individual subunits, is sufficient to bundle microtubules in vitro. Here, we present a biochemical and genetic dissection of centralspindlin. We show that each of the two subunits of centralspindlin dimerize via a parallel coiled coil. The two homodimers assemble into a high-affinity heterotetrameric complex by virtue of two low-affinity interactions. Conditional mutations in the regions that mediate complex assembly can be readily suppressed by numerous second site mutations in the interacting regions. This unexpected plasticity explains the lack of primary sequence conservation of the regions critical for this essential protein-protein interaction.     

Membrane interactions of the synthetic N-terminal peptide of HIV-1 gp41 and its structural analogs

Structural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519-541), and also FP analogs (FP520V/E with Val-->Glu at residue 520; FP527L/R with Leu-->Arg at 527; FP529F/Y with Phe-->Tyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection.     

Expression of the full-length form of gp2 of equine herpesvirus 1 (EHV-1) completely restores respiratory virulence to the attenuated EHV-1 strain KyA in CBA mice

Wild-type equine herpesvirus 1 (EHV-1) strains express a large (250-kDa) glycoprotein, gp2, that is encoded by EUs4 (gene 71) located within the unique short region of the genome. DNA sequence analysis revealed that EUs4 of the pathogenic EHV-1 strain RacL11 is an open reading frame of 2,376 bp that encodes a protein of 791 amino acids. The attenuated EHV-1 vaccine strain KyA harbors an in-frame deletion of 1,242 bp from bp 222 to 1461 and expresses a truncated gp2 of 383 amino acids. To determine the relative contribution of gp2 to EHV-1 pathogenesis, we compared the course of respiratory infection of CBA mice infected with either wild-type RacL11, attenuated KyA, or a recombinant KyA that expresses the full-length gp2 protein (KyARgp2F). Mice infected with KyA lost a negligible amount of body weight (0.18% total weight loss) on day 1 postinfection and regained weight thereafter, whereas mice infected with KyARgp2F or RacL11 steadily lost weight beginning on day 1 and experienced a 20 and 18% loss in body weight, respectively, by day 3. Immunohistochemical and flow cytometric analyses revealed higher numbers of T and B lymphocytes and an extensive consolidation consisting of large numbers of Mac-1-positive cells in the lungs of animals infected with KyARgp2F compared to animals infected with KyA. RNase protection analyses revealed increased expression of numerous cytokines and chemokines, including interleukin-1beta (IL-1beta), IL-6, tumor necrosis factor alpha, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-2, interferon gamma-inducible protein, monocyte chemotactic protein 1, and T-cell activation gene 3 at 12 h postinfection with KyARgp2F. Three independent DNA array experiments confirmed these results and showed a 2- to 13-fold increase in the expression of 31 inflammatory genes at 8 and 12 h postinfection with KyARgp2F compared to infection with KyA. Taken together, the results indicate that expression of full-length gp2 is sufficient to restore full respiratory virulence to the attenuated KyA strain and raise caution concerning the inclusion of full-length gp2 in the development of EHV-1 vaccines.