Enhanced Virus Clearance by Early Inducible Lymphocytic Choriomeningitis Virus-Neutralizing Antibodies in Immunoglobulin-Transgenic Mice

Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin μ heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host’s capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin μ heavy and the κ light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.Against many cytopathic viruses such as poliovirus, influenza virus, rabies virus, and vesicular stomatitis virus, protective virus-neutralizing antibodies are generated early, within 1 week after infection (3, 31, 36, 44, 49). In contrast, several noncytopathic viruses (e.g., human immunodeficiency virus and hepatitis viruses B and C in humans or lymphocytic choriomeningitis virus [LCMV] in mice) elicit poor and delayed virus-neutralizing antibody responses (1, 7, 20, 24, 27, 35, 45, 48).In the mouse, the natural host of LCMV, the acute LCMV infection is predominantly controlled by cytotoxic T lymphocytes (CTLs) in an obligatory perforin-dependent manner (13, 18, 28, 50). In addition to the CTL response, LCMV-specific antibodies are generated. Early after infection (by day 8), a strong antibody response specific for the internal viral nucleoprotein (NP) is mounted (7, 19, 23, 28). These early LCMV NP-specific antibodies exhibit no virus-neutralizing capacity (7, 10). Results from studies of B-cell-depleted mice and B-cell-deficient mice implied that the early LCMV NP-specific antibodies are not involved in the clearance of LCMV (8, 11, 12, 40). Late after infection (between days 30 and day 60), LCMV-neutralizing antibodies develop (7, 19, 22, 28, 33); these antibodies are directed against the surface glycoprotein (GP) of LCMV (9, 10). LCMV-neutralizing antibodies have an important function in protection against reinfection (4, 6, 38, 41, 47).In some viral infections, subprotective virus-neutralizing antibody titers can enhance disease rather than promote host recovery (i.e., exhibit antibody-dependent enhancement of disease [ADE] [14, 15, 21, 46]). For example, neutralizing antibodies are involved in the resolution of a primary dengue virus infection and in the protection against reinfection. However, if subprotective neutralizing antibody titers are present at the time of reinfection, a severe form of the disease (dengue hemorrhagic fever/dengue shock syndrome [15, 21]), which might be caused by Fc receptor-mediated uptake of virus-antibody complexes leading to an enhanced infection of monocytes (15, 16, 25, 39), can develop. Similarly, an enhancement of disease after intracerebral (i.c.) LCMV infection was observed in mice which had been treated with virus-neutralizing antibodies before the virus challenge (6). ADE in LCMV-infected mice was either due to an enhanced infection of monocytes by Fc receptor-mediated uptake of antibody-virus complexes or due to CTL-mediated immunopathology caused by an imbalanced virus spread and CTL response.To analyze whether LCMV-neutralizing antibodies generated early after infection improve the host’s capacity to clear the virus or enhance immunopathological disease, immunoglobulin (Ig)-transgenic mice expressing LCMV-neutralizing IgM antibodies were generated. After LCMV infection of transgenic mice expressing the Ig heavy chain (H25 transgenic mice), LCMV-neutralizing serum antibodies were mounted within 8 days, which significantly improved the host’s capacity to eliminate LCMV. H25 transgenic mice did not show any signs of ADE after i.c. LCMV infection.Transgenic mice expressing the Ig heavy and light chains (HL25 transgenic mice) exhibited spontaneous LCMV-neutralizing serum antibodies and confirmed the protective role of preexisting LCMV-neutralizing antibodies, even though the neutralizing serum antibodies were of the IgM isotype. Similar to mice which had been treated with LCMV-neutralizing antibodies, HL25 transgenic mice developed an enhanced disease after i.c. LCMV infection, which indicated that ADE was due to an imbalance between virus spread and CTL response. Thus, the early-inducible LCMV-neutralizing antibody response significantly enhanced clearance of the acute infection without any risk of causing ADE.     

Neutralization Sensitivity of Human Immunodeficiency Virus Type 1 Primary Isolates to Antibodies and CD4-Based Reagents Is Independent of Coreceptor Usage

We have investigated whether the identity of the coreceptor (CCR5, CXCR4, or both) used by primary human immunodeficiency virus type 1 (HIV-1) isolates to enter CD4⁺ cells influences the sensitivity of these isolates to neutralization by monoclonal antibodies and CD4-based agents. Coreceptor usage was not an important determinant of neutralization titer for primary isolates in peripheral blood mononuclear cells. We also studied whether dualtropic primary isolates (able to use both CCR5 and CXCR4) were differentially sensitive to neutralization by the same antibodies when entering U87MG-CD4 cells stably expressing either CCR5 or CXCR4. Again, we found that the coreceptor used by a virus did not greatly affect its neutralization sensitivity. Similar results were obtained for CCR5- or CXCR4-expressing HOS cell lines engineered to express green fluorescent protein as a reporter of HIV-1 entry. Neutralizing antibodies are therefore unlikely to be the major selection pressure which drives the phenotypic evolution (change in coreceptor usage) of HIV-1 that can occur in vivo. In addition, the increase in neutralization sensitivity found when primary isolates adapt to growth in transformed cell lines in vitro has little to do with alterations in coreceptor usage.Human immunodeficiency virus type 1 (HIV-1) enters CD4⁺ T cells via an interaction with CD4 and coreceptor molecules, the most important of which yet identified are the chemokine receptors CXCR4 and CCR5 (4, 12, 23, 26, 28, 32). CXCR4 is used by T-cell line-tropic (T-tropic) primary isolates or T-cell line-adapted (TCLA) lab strains, whereas CCR5 is used by primary isolates of the macrophage-tropic (M-tropic) phenotype (4, 12, 23, 26, 28, 32). Most T-tropic isolates and some TCLA strains are actually dualtropic in that they can use both CXCR4 and CCR5 (and often other coreceptors such as CCR3, Bonzo/STRL33, and BOB/gpr15), at least in coreceptor-transfected cells (18, 24, 30, 54, 89). The M-tropic and T-tropic/dualtropic nomenclature has often been used interchangeably with the terms “non-syncytium-inducing” (NSI) and “syncytium-inducing” (SI), although it is semantically imprecise to do so.M-tropic viruses are those most commonly transmitted sexually (3, 33, 87, 106) and from mother to infant (2, 72, 81). If T-tropic strains are transmitted, or when they emerge, this is associated with a more rapid course of disease in both adults (17, 37, 46, 51, 52, 76, 78, 82, 92, 101) and children (6, 45, 84, 90). However, T-tropic viruses emerge in only about 40% of infected people, usually only several years after infection (76, 78). A well-documented, albeit anecdotal, study found that when a T-tropic strain was transmitted by direct transfer of blood, its replication was rapidly suppressed: the T-tropic virus was eliminated from the body, and M-tropic strains predominated (20). These results suggest that there is a counterselection pressure against the emergence of T-tropic strains during the early stages of HIV-1 infection in most people. But what is this pressure?Since the M-tropic and T-tropic phenotypes are properties mediated by the envelope glycoproteins whose function is to associate with CD4 and the coreceptors, a selection pressure differentially exerted on M- and T-tropic viruses could, in principle, act at the level of virus entry. In other words, neutralizing antibodies to the envelope glycoproteins, or the chemokine ligands of the coreceptors, could theoretically interfere more potently with the interactions of T-tropic strains with CXCR4 than with M-tropic viruses and CCR5. A differential effect of this nature could suppress the emergence of T-tropic viruses. Consistent with this possibility, neutralizing antibodies are capable of preventing the CD4-dependent association of gp120 with CCR5 (42, 94, 103), and chemokines can also prevent the coreceptor interactions of HIV-1 (8, 13, 23, 28, 70).Here, we explore whether the efficiency of HIV-1 neutralization is affected by coreceptor usage. Although earlier studies have not found T-tropic strains to be inherently more neutralization sensitive than M-tropic ones (20, 40, 44), previously available reagents and techniques may not have been adequate to fully address this question. One major problem is that even single residue changes can drastically affect both antibody binding to neutralization epitopes and the HIV-1 phenotype (25, 55, 62, 67, 83, 91), and so studies using relatively unrelated viruses and a fixed antibody (polyclonal or monoclonal) preparation have two variables to contend with: the viral phenotype (coreceptor use) and the antigenic structure of the virus and hence the efficiency of the antibody-virion interaction.We have used a new experimental strategy to explore whether coreceptor usage affects neutralization sensitivity in the absence of other confounding variables: the use of dualtropic viruses able to enter CD4⁺ cells via either CCR5 or CXCR4. By using a constant HIV-1 isolate or clone and the same monoclonal antibodies (MAbs) or CD4-based reagents as neutralizing agents, we can ensure that the only variable under study in the neutralization reaction is the nature of the coreceptor used for entry. Our major conclusion is that there is no strong association between coreceptor usage and neutralization sensitivity for primary HIV-1 isolates. Independent studies have reached the same conclusion (53a, 59). The emergence of T-tropic (SI) viruses in vivo may be unlikely to be due to escape from antibody-mediated selection pressure.     

Monoclonal Antibodies Recognize Distinct Conformational Epitopes Formed by Polyglutamine in a Mutant Huntingtin Fragment

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) domain in the N-terminal region of huntingtin (htt). PolyQ expansion above 35–40 results in disease associated with htt aggregation into inclusion bodies. It has been hypothesized that expanded polyQ domains adopt multiple potentially toxic conformations that belong to different aggregation pathways. Here, we used atomic force microscopy to analyze the effect of a panel of anti-htt antibodies (MW1–MW5, MW7, MW8, and 3B5H10) on aggregate formation and the stability of a mutant htt-exon1 fragment. Two antibodies, MW7 (polyproline-specific) and 3B5H10 (polyQ-specific), completely inhibited fibril formation and disaggregated preformed fibrils, whereas other polyQ-specific antibodies had widely varying effects on aggregation. These results suggest that expanded polyQ domains adopt multiple conformations in solution that can be readily distinguished by monoclonal antibodies, which has important implications for understanding the structural basis for polyQ toxicity and the development of intrabody-based therapeutics for HD.Huntington disease (HD)⁵ is a fatal neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the protein huntingtin (htt), which leads to its aggregation into fibrils (1). HD is part of a growing group of diseases that are classified as “conformational diseases,” which include Alzheimer disease (AD), Parkinson disease (PD), the prion encephalopathies, and many more (2–4). The length of polyQ expansion in HD is tightly correlated with disease onset, and a critical threshold of 35–40 glutamine residues is required for disease manifestation (5). Biochemical and electron microscopic studies with htt fragments demonstrated that expanded polyQ repeats (>39) form detergent-insoluble aggregates that share characteristics with amyloid fibrils (6–8), and the formation of amyloid-like fibrils by polyQ was confirmed by studies with synthetic polyQ peptides (9). Collectively, these studies demonstrated a correlation between polyQ length and the kinetics of aggregation. This phenomenon has been recapitulated in cell-culture models that express htt fragments (10–12). Although it is clear that proteins with expanded polyQ repeats assemble into fibrils in vitro, recent studies have reported that htt fragments can also assemble into spherical and annular oligomeric structures (13–16) similar to those formed by Aβ and α-synuclein, which are implicated in AD and PD, respectively.While the major hallmark of HD is the formation of intranuclear and cytoplasmic inclusion bodies of aggregated htt (17), the role of these structures in the etiology of HD remains controversial. For instance, the onset of symptoms in a transgenic mouse model of HD follows the appearance of inclusion bodies (18), while other studies indicate that inclusion body formation may protect against toxicity by sequestering diffuse, soluble forms of htt (10, 19, 20). Based on the direct correlation between polyQ length, htt aggregation propensity, and toxicity (6), it has been hypothesized that the aggregation of htt may mediate neurodegeneration in HD. However, there is no clear consensus on the aggregate form(s) that underlie toxicity, and there likely exist bioactive, oligomeric aggregates undetectable by traditional biochemical and electron microscopic approaches whose formation precedes disease symptoms. Although identification of the one or more toxic species of htt that trigger neurodegeneration in HD remains elusive, such species might exist in a diffuse, mobile fraction rather than in inclusion bodies (19). A thioredoxin-polyQ fusion protein was recently reported to exhibit toxicity in a meta-stable, β-sheet-rich, monomeric conformation (21), suggesting that polyQ can adopt multiple monomeric conformations, only some of which may be toxic. Consistent with such a scenario, molecular dynamic simulations and fluorescence correlation spectroscopy experiments with synthetic polyQ peptides indicate that polyQ domains can adopt a heterogeneous collection of collapsed conformations that are in equilibrium before aggregation (22–25).Although biochemical, biophysical, and computational approaches have yielded insight into the structures formed by polyQ in vitro, whether such structures form in vivo remains largely unknown. Indeed, determining the conformational state of any misfolded/aggregated protein in situ and/or in vivo remains a major technical challenge.Toward this goal, antibodies have been explored as a potentially powerful tool for detecting specific conformations or multimeric states of aggregated proteins in situ. Antibodies specific for amyloid fibrils often do not react with natively folded globular proteins from which they are derived, suggesting that such antibodies recognize a conformational epitope (26, 27). Several antibodies display conformation-dependent interactions with amyloids, aggregation intermediates, or natively folded precursor proteins. For example, there are antibodies specific for paired helical filaments of Tau (28–31), of aggregated forms of Aβ ranging from dimers to fibrils (32–34), and of native (35) or disease-related (36) forms of the prion protein. Antibodies have also been developed that are specific for common structural motifs associated with amyloid diseases, such as oligomers (37) and fibrils (38), independent of the peptide sequence of the amyloid forming protein from which they are derived, suggesting the potential for a common mechanism of aggregation and toxicity for these diseases.With regard to htt, several antibodies (MW1, MW2, MW3, MW4, MW5, IC2, and IF8), which are specific for polyQ repeats, stain Western blots of htt with expanded polyQ repeats much more strongly than htt with normal polyQ length (39, 40), suggesting that these antibodies may recognize abnormal polyQ conformations. Furthermore, these polyQ-specific antibodies have distinct staining patterns in immunohistochemical studies of brain tissue sections (39). In one study, the affinity and stoichiometry of MW1 binding to htt increased with polyQ length, suggesting a “linear lattice” model for polyQ (41). This model is supported by a crystal structure of polyQ bound to MW1, which showed that polyQ can adopt an extended, coil-like structure (42). However, an independent structural study showed that the anti-polyQ antibody 3B5H10 binds to a compact β-sheet-like structure of polyQ in a monomeric htt fragment.⁶ These results clearly indicate that polyQ domains can fold into at least two unique, stable, monomeric conformations and suggest that the “linear lattice” model is not generally applicable to all polyQ structures.Not only are antibodies useful for understanding what polyQ structures exist in situ, especially in the diffuse htt fraction of neurons, but antibodies and/or intrabodies may also have potential as therapeutic agents. For example, several studies showed that intrabodies reduce htt toxicity in cellular models (44–49). Moreover, one intrabody (C4) slows htt aggregation and prolongs lifespan in a Drosophila model of HD (50, 51), while another (mEM48) ameliorates neurological symptoms in a mouse model of HD (48).Three of the antibodies examined in this study (MW1, MW2, and MW7) modulate htt-induced cell death when co-transfected as single-chain variable region fragment antibodies (scFvs) in 293 cells with htt exon 1 containing an expanded polyQ domain (46). In these studies MW1 and MW2, which bind to the polyQ repeat in htt, increased htt-induced toxicity and aggregation (46). Conversely, MW7, which binds to the polyproline (polyP) regions adjacent to the polyQ repeat in htt, decreased its aggregation and toxicity (46). Interestingly, MW7 has also been shown to increase the turnover of mutant htt in cultured cells and reduce its toxicity in corticostriatal brain slice explants (49).Given the difficulty in understanding which specie(s) of htt exist and mediate pathogenesis in the putative toxic diffuse fraction of neurons, we sought to rigorously characterize the conformational specificity of a panel of anti-htt antibodies, the best in situ probes currently available for distinguishing specie(s) of htt. We reasoned that if htt can adopt multiple conformations that mediate different aggregation pathways, then anti-htt antibodies should differentially alter htt aggregation pathways by stabilizing or sequestering the specific conformers or aggregates they recognize. We therefore examined the effects of various antibodies on mutant htt fragment fibril formation and stability by atomic force microscopy (AFM). Our results are consistent with the hypothesis that monoclonal antibodies recognize distinct conformational epitopes formed by polyQ in a mutant htt fragment.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Complex Lipid Requirements for SNARE- and SNARE Chaperone-dependent Membrane Fusion

Membrane fusion without lysis has been reconstituted with purified yeast vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the SNARE chaperones Sec17p/Sec18p and the multifunctional HOPS complex, which includes a subunit of the SNARE-interactive Sec1-Munc18 family, and vacuolar lipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), cardiolipin (CL), ergosterol (ERG), diacylglycerol (DAG), and phosphatidylinositol 3-phosphate (PI3P). We now report that many of these lipids are required for rapid and efficient fusion of the reconstituted SNARE proteoliposomes in the presence of SNARE chaperones. Omission of either PE, PA, or PI3P from the complete set of lipids strongly reduces fusion, and PC, PE, PA, and PI3P constitute a minimal set of lipids for fusion. PA could neither be replaced by other lipids with small headgroups such as DAG or ERG nor by the acidic lipids PS or PI. PA is needed for full association of HOPS and Sec18p with proteoliposomes having a minimal set of lipids. Strikingly, PA and PE are as essential for SNARE complex assembly as for fusion, suggesting that these lipids facilitate functional interactions among SNAREs and SNARE chaperones.Biological membrane fusion is the regulated rearrangement of the lipids in two apposed sealed membranes to form one bilayer while mixing lumenal contents without leakage or lysis. It is fundamental for intracellular vesicular traffic, cell growth and division, regulated secretion of hormones and other blood proteins, and neurotransmission and thus has attracted wide and sustained study (1, 2). Its fundamental mechanisms are conserved and employ a Rab-family GTPase, proteins which bind to the GTP-bound form of a Rab, termed its “effectors” (3), and SNARE³ (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins (4) with their attendant chaperones. SNAREs are integral or peripheral membrane proteins with characteristic heptad-repeat domains, which can associate in 4-helical coiled-coils (5), termed “cis-SNARE complexes,” if they are all anchored to the same membrane bilayer, or “trans-SNARE complexes” if they are anchored to apposed membranes.Stable membrane proximity (docking) does not suffice for fusion. Studies in model systems have shown that fusion can be promoted by any of several agents, which promote bilayer rearrangement, such as diacylglycerol (6), high levels of calcium (7), viral-encoded fusion proteins (8, 9), or SNAREs (10, 11). These studies frequently employed liposomes or proteoliposomes of simple lipid composition, suggesting that fusion may not have stringent requirements of lipid head group species. However, each of these model fusion reactions is accompanied by substantial lysis (12–15), whereas the preservation of subcellular compartments is a hallmark of physiological membrane fusion.We have studied membrane fusion with the vacuole (lysosome) of Saccharomyces cerevisiae (reviewed in Ref. 16). The fusion of isolated vacuoles requires the Rab Ypt7p, 4 SNAREs (Vam3p, Vti1p, Vam7p, and Nyv1p), the SNARE chaperones Sec17p (α-soluble N-ethylmaleimide-sensitive factor attachment protein)/Sec18p (N-ethylmaleimide-sensitive factor) and the hexameric HOPS complex (17), and key “regulatory” lipids including ERG, phosphoinositides, and DAG (18). HOPS interacts physically or functionally with each component of this fusion system. HOPS stably associates with Ypt7p in its GTP-bound state (19). One HOPS subunit, Vps33p, is a member of the Sec1-Munc18 family of SNARE-binding proteins, and HOPS exhibits direct affinity for SNAREs (17, 20–22) and proofreads correct vacuolar SNARE pairing (23). HOPS also has direct affinity for phosphoinositides (17). The SNAREs on isolated vacuoles are in cis-complexes, which are disassembled by Sec17p, Sec18p, and ATP (24). Docking requires Ypt7p (25) and HOPS (17). During docking, vacuoles are drawn against each other until each has a substantial membrane domain tightly apposed to the other. Each of the proteins (26) and lipids (18) required for fusion becomes enriched in a ring-shaped microdomain, the “vertex ring,” which surrounds the two tightly apposed membrane domains. Not only do the proteins depend on each other, in a cascade fashion, for vertex ring enrichment, and the lipids depend on each other for their vertex ring enrichment as well, but the lipids and proteins are mutually interdependent for their enrichment at this ring-shaped microdomain (18, 27). Fusion occurs around the ring, joining the two organelles. The fusion of vacuoles bearing physiological fusion constituents does not cause measurable organelle lysis, although fusion supported exclusively by higher levels of SNARE proteins is accompanied by massive lysis (28), in accord with model liposome studies (14). Thus fusion microdomain assembly and the coordinate action of SNAREs with other proteins and lipids to promote fusion without lysis are central topics in membrane fusion studies.Reconstitution of fusion with pure components allows chemical definition of essential elements of this biologically important reaction. Although SNAREs can drive a slow fusion of PC/PS proteoliposomes (29), this was not stimulated by HOPS and Sec17p/Sec18p (30). SNARE proteoliposomes bearing all the vacuolar lipids (18, 31–33), PC, PE, PI, PS, CL, PA, ERG, DAG, PI3P, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), showed rapid and efficient fusion that was fully dependent on Sec17p/Sec18p and HOPS (30). The omission of either DAG, ERG, or phosphoinositide from the liposomes caused a marked reduction in fusion (30). We now report that PE and PA are also necessary for rapid and efficient fusion, function in distinct manners, and are required for efficient assembly of newly formed SNARE complexes by the SNARE chaperones Sec17p/Sec18p and HOPS.     

Lipid Protein Interactions Couple Protonation to Conformation in a Conserved Cytosolic Domain of G Protein-coupled Receptors

The visual photoreceptor rhodopsin is a prototypical class I (rhodopsin-like) G protein-coupled receptor. Photoisomerization of the covalently bound ligand 11-cis-retinal leads to restructuring of the cytosolic face of rhodopsin. The ensuing protonation of Glu-134 in the class-conserved D(E)RY motif at the C-terminal end of transmembrane helix-3 promotes the formation of the G protein-activating state. Using transmembrane segments derived from helix-3 of bovine rhodopsin, we show that lipid protein interactions play a key role in this cytosolic “proton switch.” Infrared and fluorescence spectroscopic pK_a determinations reveal that the D(E)RY motif is an autonomous functional module coupling side chain neutralization to conformation and helix positioning as evidenced by side chain to lipid headgroup Foerster resonance energy transfer. The free enthalpies of helix stabilization and hydrophobic burial of the neutral carboxyl shift the side chain pK_a into the range typical of Glu-134 in photoactivated rhodopsin. The lipid-mediated coupling mechanism is independent of interhelical contacts allowing its conservation without interference with the diversity of ligand-specific interactions in class I G protein-coupled receptors.G protein-coupled receptors (GPCRs)² are hepta-helical membrane proteins that couple a large variety of extracellular signals to cell-specific responses via activation of G proteins. In the visual photoreceptor rhodopsin, a prototypical class I GPCR (1, 2), molecular activation processes can be monitored in real time by spectroscopic assays and analyzed in the context of several crystal structures (3–8). The primary signal for rhodopsin is the 11-cis to all-trans photoisomerization of retinal covalently bound to the apoprotein opsin through a protonated Schiff base to Lys²⁹⁶. Current models converge toward a picture in which “microdomains” act as conformational switches that are coupled to different degrees to the primary activation process. Two activating “proton switches” have been identified (9) as follows: breakage of an intramolecular salt bridge (10) by transfer of the Schiff base proton to its counter ion Glu-113 (11) is followed by movement of helix-6 (H6) (12, 13) in the metarhodopsin II_a (MII_a) to MII_b transition. The MII_b state takes up a proton at Glu-134 (14) in the class-conserved D(E)RY motif at the C-terminal end of helix-3 (H3) leading to the MII_bH⁺ intermediate (15, 16), which activates transducin (G_t), the G protein of the photoreceptor cell. Glu-134 regulates the pH sensitivity of receptor signaling (17) in membranes as reviewed previously (18), and in complex with G_t the protonated state of the carboxyl group becomes stabilized (19). This charge alteration is linked to the release of an “ionic lock,” originally described for the β₂-adrenergic receptor (20), which also in rhodopsin stabilizes the inactive state (16) through interactions between the cytosolic ends of H3 and H6 (21).In the absence of a lipidic bilayer, proton uptake and H6 movement become uncoupled (15). Lipidic composition affects MII formation, rhodopsin structure, and oligomerization (22–24) and differs at the rhodopsin membrane interface from the bulk lipidic phase (25). Likewise, MII formation specifically affects lipid structure (26). Although of fundamental importance for GPCR activation, the potential implication of lipid protein interactions in “proton switching” is not clear. A functional role of Glu-134 in lipid interactions has been originally derived from IR spectra where E134Q replacement abolished changes of lipid headgroup vibrations in the MIIG_t complex (19). Computational approaches emphasized the “strategic” location of the D(E)RY motif (27), and the Glu-134 carboxyl pK_a may critically depend on the lipid protein interface (28). However, the implications for proton switching are not evident, and the theoretical interest is contrasted by the lack of experimental data addressing the effect of the lipidic phase on side chain protonation, secondary structure, and membrane topology of the D(E)RY motif.We have studied the coupling between conformation and protonation in single transmembrane segments derived from H3 of bovine rhodopsin. We have assessed the “modular” function of the D(E)RY motif by determining parameters not evident from the crystal structures, i.e. the pK_a of the conserved carboxyl, its linkage to helical structure, and the effect of protonation on side chain to lipid headgroup distance. We show that the D(E)RY motif encodes an autonomous “proton switch” controlling side chain exposure and helix formation in the low dielectric of a lipidic phase. The data ascribe a functional role to lipid protein interactions that couple the chemical potential of protons to an activity-promoting GPCR conformation in a ligand-independent manner.     

F-Actin Dynamics in Neurospora crassa

This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.Actins are highly conserved proteins found in all eukaryotes and have an enormous variety of cellular roles. The monomeric form (globular actin, or G-actin) can self-assemble, with the aid of numerous actin-binding proteins (ABPs), into microfilaments (filamentous actin, or F-actin), which, together with microtubules, form the two major components of the fungal cytoskeleton. Numerous pharmacological and genetic studies of fungi have demonstrated crucial roles for F-actin in cell polarity, exocytosis, endocytosis, cytokinesis, and organelle movement (6, 7, 20, 34, 35, 51, 52, 59). Phalloidin staining, immunofluorescent labeling, and fluorescent-protein (FP)-based live-cell imaging have revealed three distinct subpopulations of F-actin-containing structures in fungi: patches, cables, and rings (1, 14, 28, 34, 60, 63, 64). Actin patches are associated with the plasma membrane and represent an accumulation of F-actin around endocytic vesicles (3, 26, 57). Actin cables are bundles of actin filaments stabilized with cross-linking proteins, such as tropomyosins and fimbrin, and are assembled by formins at sites of active growth, where they form tracks for myosin V-dependent polarized secretion and organelle transport (10, 16, 17, 27, 38, 47, 48). Cables, unlike patches, are absolutely required for polarized growth in the budding yeast Saccharomyces cerevisiae (34, 38). Contractile actomyosin rings are essential for cytokinesis in budding yeast, whereas in filamentous fungi, actin rings are less well studied but are known to be involved in septum formation (20, 28, 34, 39, 40).Actin cables and patches have been particularly well studied in budding yeast. However, there are likely to be important differences between F-actin architecture and dynamics in budding yeast and those in filamentous fungi, as budding yeasts display only a short period of polarized growth during bud formation, which is followed by isotropic growth over the bud surface (10). Sustained polarized growth during hyphal morphogenesis is a defining feature of filamentous fungi (21), making them attractive models for studying the roles of the actin cytoskeleton in cell polarization, tip growth, and organelle transport.In Neurospora crassa and other filamentous fungi, disruption of the actin cytoskeleton leads to rapid tip swelling, which indicates perturbation of polarized tip growth, demonstrating a critical role for F-actin in targeted secretion to particular sites on the plasma membrane (7, 22, 29, 56). Immunofluorescence studies of N. crassa have shown that F-actin localizes to hyphal tips as “clouds” and “plaques” (7, 54, 59). However, immunolabeling has failed to reveal actin cables in N. crassa and offers limited insights into F-actin dynamics. Live-cell imaging of F-actin architecture and dynamics has not been accomplished in N. crassa, yet it is expected to yield key insights into cell polarization, tip growth, and intracellular transport.We took advantage of a recently developed live-cell imaging probe for F-actin called Lifeact (43). Lifeact is a 17-amino-acid peptide derived from the N terminus of the budding yeast actin-binding protein Abp140 (5, 63) and has recently been demonstrated to be a universal live-cell imaging marker for F-actin in eukaryotes (43). Here, we report the successful application of fluorescent Lifeact fusion constructs for live-cell imaging of F-actin in N. crassa. We constructed two synthetic genes consisting of Lifeact fused to “synthetic” green fluorescent protein (sGFP) (S65T) (henceforth termed GFP) (12) or red fluorescent protein (TagRFP) (33) and expressed these constructs in various N. crassa strains. In all strain backgrounds, fluorescent Lifeact constructs clearly labeled actin patches, cables, and rings and revealed a direct association of F-actin structures with sites of cell polarization and active tip growth. Our results demonstrate the efficacy of Lifeact as a nontoxic live-cell imaging probe in N. crassa.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

The Wavelet-Based Cluster Analysis for Temporal Gene Expression Data

A variety of high-throughput methods have made it possible to generate detailed temporal expression data for a single gene or large numbers of genes. Common methods for analysis of these large data sets can be problematic. One challenge is the comparison of temporal expression data obtained from different growth conditions where the patterns of expression may be shifted in time. We propose the use of wavelet analysis to transform the data obtained under different growth conditions to permit comparison of expression patterns from experiments that have time shifts or delays. We demonstrate this approach using detailed temporal data for a single bacterial gene obtained under 72 different growth conditions. This general strategy can be applied in the analysis of data sets of thousands of genes under different conditions.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]