BLOC-2 targets recycling endosomal tubules to melanosomes for cargo delivery.

Hermansky-Pudlak syndrome (HPS) is a group of disorders characterized by the malformation of lysosome-related organelles, such as pigment cell melanosomes. Three of nine characterized HPS subtypes result from mutations in subunits of BLOC-2, a protein complex with no known molecular function. In this paper, we exploit melanocytes from mouse HPS models to place BLOC-2 within a cargo transport pathway from recycling endosomal domains to maturing melanosomes. In BLOC-2-deficient melanocytes, the melanosomal protein TYRP1 was largely depleted from pigment granules and underwent accelerated recycling from endosomes to the plasma membrane and to the Golgi. By live-cell imaging, recycling endosomal tubules of wild-type melanocytes made frequent and prolonged contacts with maturing melanosomes; in contrast, tubules from BLOC-2-deficient cells were shorter in length and made fewer, more transient contacts with melanosomes. These results support a model in which BLOC-2 functions to direct recycling endosomal tubular transport intermediates to maturing melanosomes and thereby promote cargo delivery and optimal pigmentation.

Rational design and characterization of platelet factor 4 antagonists for the study of heparin-induced thrombocytopenia.

Patients with heparin-induced thrombocytopenia (HIT) remain at risk for recurrent thromboembolic complications despite improvements in management. HIT is caused by antibodies that preferentially recognize ultralarge complexes (ULCs) of heparin and platelet factor 4 (PF4) tetramers. We demonstrated previously that a variant PF4(K50E) forms dimers but does not tetramerize or form ULCs. Here, we identified small molecules predicted to bind PF4 near the dimer-dimer interface and that interfere with PF4 tetramerization. Screening a library of small molecules in silico for binding at this site, we identified 4 compounds that inhibited tetramerization at micromolar concentrations, designated PF4 antagonists (PF4As). PF4As also inhibited formation of pathogenic ULCs, and 3 of these PF4As promoted the breakdown of preformed ULCs. To characterize the ability of PF4As to inhibit cellular activation, we developed a robust and reproducible assay that measures cellular activation by HIT antibodies via FcγRIIA using DT40 cells. PF4As inhibit FcγRIIA-dependent activation of DT40 cells by HIT antibodies as well as platelet activation, as measured by serotonin release. PF4As provide new tools to probe the pathophysiology of HIT. They also may provide insight into the development of novel, disease-specific therapeutics for the treatment of thromboembolic complications in HIT.

Genome wide ChIP-chip analyses reveal important roles for CTCF in Drosophila genome organization.

Insulators or chromatin boundary elements are defined by their ability to block transcriptional activation by an enhancer and to prevent the spread of active or silenced chromatin. Recent studies have increasingly suggested that insulator proteins play a role in large-scale genome organization. To better understand insulator function on the global scale, we conducted a genome-wide analysis of the binding sites for the insulator protein CTCF in Drosophila by Chromatin Immunoprecipitation (ChIP) followed by a tiling-array analysis. The analysis revealed CTCF binding to many known domain boundaries within the Abd-B gene of the BX-C including previously characterized Fab-8 and MCP insulators, and the Fab-6 region. Based on this finding, we characterized the Fab-6 insulator element. In genome-wide analysis, we found that dCTCF-binding sites are often situated between closely positioned gene promoters, consistent with the role of CTCF as an insulator protein. Importantly, CTCF tends to bind gene promoters just upstream of transcription start sites, in contrast to the predicted binding sites of the insulator protein Su(Hw). These findings suggest that CTCF plays more active roles in regulating gene activity and it functions differently from other insulator proteins in organizing the Drosophila genome.

Targeting of antigen to the herpesvirus entry mediator augments primary adaptive immune responses.

Interactions between the herpesvirus entry mediator (HVEM) and the B- and T-lymphocyte attenuator (BTLA) inhibit B and T cell activation. HVEM-BTLA interactions are blocked by herpes simplex virus (HSV) glycoprotein D (gD) through binding of its N-terminal domain to the BTLA binding site of HVEM. In this study, we inserted viral antigens into the C-terminal domain of gD and expressed these antigens with plasmid or E1-deleted (replication-defective) adenovirus vectors. Viral antigens fused to gD induced T and B cell responses to the antigen that were far more potent than those elicited by the same antigen expressed without gD. The immunopotentiating effect required binding of the gD chimeric protein to HVEM. Overall, the studies demonstrate that targeting of antigen to the BTLA binding site of HVEM augments the immunogenicity of vaccines.

Large-scale purification and crystallization of adenovirus hexon.

This chapter provides a protocol for the large-scale purification of adenovirus type 2 and 5 virions and the soluble major coat protein hexon. The purified virus particles remain intact and are suitable for vector, vaccine, or structural studies and can also be used as seed stock for further rounds of infection. The hexon may be used to produce crystals suitable for high-resolution X-ray crystallographic studies. Briefly, virus is propagated in HeLa cell suspension cultures. The infected cells are lysed, virions and hexon are separated by centrifugation, and the protein is then further purified by anion exchange chromatography. The entire purification procedure takes approx 1 wk and typically yields 10(13) virus particles and 10-20 mg of highly purified hexon.

The proteome of the mouse photoreceptor sensory cilium complex.

Primary cilia play critical roles in many aspects of biology. Specialized versions of primary cilia are involved in many aspects of sensation. The single photoreceptor sensory cilium (PSC) or outer segment elaborated by each rod and cone photoreceptor cell of the retina is a classic example. Mutations in genes that encode cilia components are common causes of disease, including retinal degenerations. The protein components of mammalian primary and sensory cilia have not been defined previously. Here we report a detailed proteomics analysis of the mouse PSC complex. The PSC complex comprises the outer segment and its cytoskeleton, including the axoneme, basal body, and ciliary rootlet, which extends into the inner segment of photoreceptor cells. The PSC complex proteome contains 1968 proteins represented by three or more unique peptides, including approximately 1500 proteins not detected in cilia from lower organisms. This includes 105 hypothetical proteins and 60 proteins encoded by genes that map within the critical intervals for 23 inherited cilia-related disorders, increasing their priority as candidate genes. The PSC complex proteome also contains many cilia proteins not identified previously in photoreceptors, including 13 proteins produced by genes that harbor mutations that cause cilia disease and seven intraflagellar transport proteins. Analyses of PSC complexes from rootletin knock-out mice, which lack ciliary rootlets, confirmed that 1185 of the identified PSC complex proteins are derived from the outer segment. The mass spectrometry data, benchmarked by 15 well characterized outer segment proteins, were used to quantify the copy number of each protein in a mouse rod outer segment. These results reveal mammalian cilia to be several times more complex than the cilia of unicellular organisms and open novel avenues for studies of how cilia are built and maintained and how these processes are disrupted in human disease.

CD44 regulates vascular gene expression in a proatherogenic environment.

OBJECTIVE: To identify early changes in vascular gene expression mediated by CD44 that promote atherosclerotic disease in apolipoprotein E (apoE)-deficient (apoE-/-) mice. METHODS AND RESULTS: We demonstrate that CD44 is upregulated and functionally activated in aortic arch in the atherogenic environment of apoE-/- mice relative to wild-type (C57BL/6) controls. Moreover, CD44 activation even in apoE-/- mice is selective to lesion-prone regions because neither the thoracic aorta from apoE-/- mice nor the aortic arch of C57BL/6 mice exhibited upregulation of CD44 compared with thoracic aorta of CD57BL/6 mice. Consistent with these observations, gene expression profiling using cDNA microarrays and quantitative polymerase chain reaction revealed that approximately 155 of 19,200 genes analyzed were differentially regulated in the aortic arch, but not in the thoracic aorta, in apoE-/- CD44-/- mice compared with apoE-/- CD44+/+ mice. However, these genes were not regulated by CD44 in the context of a C57BL/6 background, illustrating the selective impact of CD44 on gene expression in a proatherogenic environment. The patterns of differential gene expression implicate CD44 in focal adhesion formation, extracellular matrix deposition, and angiogenesis, processes critical to atherosclerosis. CONCLUSIONS: CD44 is an early mediator of atherogenesis by virtue of its ability to regulate vascular gene expression in response to a proatherogenic environment.

Structure-based identification of a major neutralizing site in an adenovirus hexon.

Virus-specific neutralizing antibodies present an obstacle to the effective use of adenovirus vectors for gene therapy and vaccination. The specific sites recognized by neutralizing antibodies have not been identified for any adenovirus, but they have been proposed to reside within the hexon, in small regions of the molecule that are exposed on the capsid surface and possess sequences that vary among serotypes. We have mapped the epitopes recognized by a panel of seven hexon-specific monoclonal antibodies that neutralize the chimpanzee adenovirus 68 (AdC68). Surface plasmon resonance experiments revealed that the antibodies compete for a single hexon binding site, and experiments with synthetic peptides indicated that this site resides within just one small surface loop. Mutations within this loop (but not in other surface loops) permitted virus to escape neutralization by all seven monoclonal antibodies and to resist neutralization by polyclonal antisera obtained from animals immunized against AdC68. These results indicate that a single small surface loop defines a major neutralization site for AdC68 hexon.

Sense and antisense transcripts of the apolipoprotein E gene in normal and ApoE knockout mice, their expression after spinal cord injury and corresponding human transcripts.

The apolipoprotein E (ApoE) gene has been linked to maladies such as hypercholesterolemia, CNS injury and disease. In this study, we present evidence that, in addition to the known transcript (ApoE S1) that translates into ApoE, there are three additional transcripts in mice. Two of these transcripts, ApoE S2 and ApoE S3, which are predicted to be transmembrane proteins, are transcribed from the sense strand. ApoE AS1 is transcribed from the antisense strand and is complementary to exon 4 of ApoE S1. The open reading frame of ApoE AS1 is conserved between human and mouse. The antisense transcript falls within the region of the human epsilon 4 allele that has been linked to the familial onset form of Alzheimer's disease. We also demonstrate the expression of ApoE S3 and ApoE AS1 in ApoE knockout mice, and ApoE S1 and ApoE S2 do not get transcribed. We had previously identified ApoE S1 as being upregulated in mice after spinal cord injury. In this study, we show that in spinal cord-injured C57BL/6 mice, both ApoE S1 and ApoE S3 transcripts are 10-fold upregulated and the antisense ApoE AS1 is 100-fold upregulated compared with normal levels. Such data suggest that these alternate transcripts are involved in the molecular pathogenesis of CNS disease and perhaps in ApoE expression in general, as we show that ApoE S2 and AS1 are also transcribed in human.

A novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes.

A novel strategy, termed protein array pixelation, is described for comprehensive profiling of human plasma and serum proteomes. This strategy consists of three sequential high-resolution protein prefractionation methods (major protein depletion, solution isoelectrofocusing, and 1-DE) followed by nanocapillary RP tryptic peptide separation prior to MS/MS analysis. The analysis generates a 2-D protein array where each pixel in the array contains a group of proteins with known pI and molecular weight range. Analysis of the HUPO samples using this strategy resulted in 575 and 2890 protein identifications from plasma and serum, respectively, based on HUPO-approved criteria for high-confidence protein assignments. Most importantly, a substantial number of low-abundance proteins (low ng/mL - pg/mL range) were identified. Although larger volumes were used in initial prefractionation steps, the protein identifications were derived from fractions equivalent to approximately 0.6 microL (45 microg) of plasma and 2.4 microL (204 microg) of serum. The time required for analyzing the entire protein array for each sample is comparable to some published shotgun analyses of plasma and serum proteomes. Therefore, protein array pixelation is a highly sensitive method capable of detecting proteins differing in abundance by up to nine orders of magnitude. With further refinement, this method has the potential for even higher capacity and higher throughput.

Gene specific siRNA selector.

Small interfering RNA (siRNA) is used in functional genomics applications to decrease the expression of a target gene, which may yield a biological effect that suggests a function for the target gene. The siRNA design tool scans a target gene for candidate siRNA sequences that satisfy user-adjustable rules. Selected candidates are then screened to identify those siRNA sequences that are specific to the gene of interest.

Adenovirus structure.

Structural studies continue to play an essential role as the focus of adenovirus research shifts in emphasis from basic biology to adenovirus-based vector technologies. A crucial step in developing novel therapeutics for gene replacement, cancer, and vaccines is often to modify the virion. Such engineered changes are designed to retarget the virus, or to reduce the immunological responses to infection. These efforts are far more effective when they are based on detailed structural knowledge. This minireview provides a brief summary of the wealth of information that has been obtained from the combined application of X-ray crystallography and electron microscopy. This knowledge now includes a good working model for the architectural organization of the virion, and atomic resolution molecular structures for all the major capsid proteins, hexon, penton, and fiber. We highlight new developments, which include the structure of the penton base and the discovery that adenovirus has several relatives. We sketch how the structural information can be used to engineer novel virions and conclude with the prospects for future progress.

Lentiviral vectors pseudotyped with minimal filovirus envelopes increased gene transfer in murine lung.

A human immunodeficiency virus (HIV)-based vector pseudotyped with the Ebola Zaire (EboZ) viral envelope glycoprotein (GP) was recently shown to transduce murine airway epithelia cells in vivo. In this study, the vector was further redesigned to improve gene transfer and also to increase safety. We used mutant EboZ envelopes for pseudotyping, which resulted in higher titers and increased transduction of airway cells in vivo compared to vectors pseudotyped with wild-type EboZ GP. As these envelopes lack regions associated with toxicity of the wild-type EboZ GP, they should also be safer to use for pseudotyping of lentiviral vectors. In addition, lentiviral vectors were created based on feline immunodeficiency virus and shown to have similar efficiency of transduction compared to HIV-based vectors. The creation of lentiviral vectors with highly engineered EboZ envelopes improved the performance of the system and should also increase its safety since only minimal regions of the EboZ envelope, which lack the toxic domain, are used.

Structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic, molecular modeling, and sequence-based methods.

A major impediment to the use of adenovirus as a gene therapy vector and for vaccine applications is the host immune response to adenovirus hexon-the major protein component of the icosahedral capsid. A solution may lie in novel vectors with modified or chimeric hexons designed to evade the immune response. To facilitate this approach, we have distinguished the portion of hexon that all serotypes have in common from the hypervariable regions that are responsible for capsid diversity and type-specific immunogenicity. The common hexon core-conserved because it forms the viral capsid-sets boundaries to the regions where modifications can be made to produce nonnative hexons. The core has been defined from the large and diverse set of known hexon sequences by an accurate alignment based on the newly refined crystal structures of human adenovirus types 2 (Ad2) and Ad5 hexon. Comparison of the two hexon models, which are the most accurate so far, reveals that over 90% of the residues in each have three-dimensional positions that closely match. Structures for more distant hexons were predicted by building molecular models of human Ad4, chimpanzee adenovirus (AdC68), and fowl adenovirus 1 (FAV1 or CELO). The five structures were then used to guide the alignment of the 40 full-length (>900 residues) hexon sequences in public databases. Distance- and parsimony-based phylogenetic trees are consistent and reveal evolutionary relationships between adenovirus types that parallel those of their animal hosts. The combination of crystallography, molecular modeling, and phylogenetic analysis defines a conserved molecular core that can serve as the armature for the directed design of novel hexons.

Adeno-associated viruses undergo substantial evolution in primates during natural infections.

Adeno-associated viruses (AAVs) are single-stranded DNA viruses that are endemic in human populations without known clinical sequelae and are being evaluated as vectors for human gene therapy. To better understand the biology of this virus, we examined a number of nonhuman primate species for the presence of previously uncharacterized AAVs and characterized their structure and distribution. AAV genomes were widely disseminated throughout multiple tissues of a variety of nonhuman primate species. Surprising diversity of sequence, primarily localized to hypervariable regions of the capsid protein, was detected. This diversity of sequence is caused, in part, by homologous recombination of co-infecting parental viruses that modify the serologic reactivity and tropism of the virus. This is an example of rapid molecular evolution of a DNA virus in a way that was formerly thought to be restricted to RNA viruses.

Mutations in Rab3a alter circadian period and homeostatic response to sleep loss in the mouse.

Rab3a is the most abundant Rab (ras-associated binding) protein in the brain and has a regulatory role in synaptic vesicle trafficking. Mice with a targeted loss-of-function mutation in Rab3a have defects in Ca(2+)-dependent synaptic transmission: the number of vesicles released in response to an action potential is greater than in wildtype mice, resulting in greater synaptic depression and the abolishment of CA3 mossy-fiber long term potentiation. The effect of these changes on behavior is unknown. In a screen for mouse mutants with abnormal rest-activity and sleep patterns, we identified a semidominant mutation, called earlybird, that shortens the circadian period of locomotor activity. Sequence analysis of Rab3a identified a point mutation in the conserved amino acid (Asp77Gly) within the GTP-binding domain of this protein in earlybird mutants, resulting in significantly reduced levels of Rab3a protein. Phenotypic assessment of earlybird mice and a null allele of Rab3a revealed anomalies in circadian period and sleep homeostasis, providing evidence that Rab3a-mediated synaptic transmission is involved in these behaviors.

A common DNA-binding site for SZF1 and the BRCA1-associated zinc finger protein, ZBRK1.

More than 220 Kruppel-associated box-zinc finger protein (KRAB-ZFP) genes are encoded in the human genome. KRAB-ZFPs function as transcriptionalrepressors by binding DNA through their tandem zinc finger motifs.Gene silencing is mediated by the highly conserved KRAB domain, which recruits histone deacetylase complexes, histone methylases, and heterochromatin proteins. However, little is known of the biological programs regulated by KRAB-ZFPs, in large part because of the difficulty in identifying DNA-binding sites recognized by long arrays of zinc fingers. In an attempt to identify the natural target genes for a KRAB-ZFP, we chose SZF1, a hematopoietic progenitor-restricted, KRAB-ZFP that contains only four C(2)H(2) zinc finger motifs. Using recombinant SZF1 protein and a PCR-based binding site selection strategy, we identified a 15-bp consensus DNA sequence recognized by SZF1. Remarkably, this sequence is similar to the core DNA-binding site described recently for ZBRK1, a KRAB-ZFP that binds to BRCA1 and is involved in coordinating the cellular DNA damage response. The SZF1 and ZBRK1 proteins bind to both the experimentally derived SZF1 site and the canonical ZBRK1 site. The KRAB domain from SZF1 bound directly to the KAP-1 corepressor and displayed intrinsic silencing activity. Moreover, full-length SZF1 repressed a promoter containing ZBRK1 recognition sequences. Thus, SZF1 and ZBRK1 may regulate a common set of target genes in vivo.

Domain-domain associations in cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR is a chloride channel whose activity requires protein kinase A-dependent phosphorylation of an intracellular regulatory domain (R-domain) and ATP hydrolysis at the nucleotide-binding domains (NBDs). To identify potential sites of domain-domain interaction within CFTR, we expressed, purified, and refolded histidine (His)- and glutathione-S-transferase (GST)-tagged cytoplasmic domains of CFTR. ATP-binding to his-NBD1 and his-NBD2 was demonstrated by measuring tryptophan fluorescence quenching. Tryptic digestion of in vitro phosphorylated his-NBD1-R and in situ phosphorylated CFTR generated the same phosphopeptides. An interaction between NBD1-R and NBD2 was assayed by tryptophan fluorescence quenching. Binding among all pairwise combinations of R-domain, NBD1, and NBD2 was demonstrated with an overlay assay. To identify specific sites of interaction between domains of CFTR, an overlay assay was used to probe an overlapping peptide library spanning all intracellular regions of CFTR with his-NBD1, his-NBD2, and GST-R-domain. By mapping peptides from NBD1 and NBD2 that bound to other intracellular domains onto crystal structures for HisP, MalK, and Rad50, probable sites of interaction between NBD1 and NBD2 were identified. Our data support a model where NBDs form dimers with the ATP-binding sites at the domain-domain interface.