Rac-GAP-dependent inhibition of breast cancer cell proliferation by {beta}2-chimerin

beta2-Chimerin is a member of the "non-protein kinase C" intracellular receptors for the second messenger diacylglycerol and the phorbol esters that is yet poorly characterized, particularly in the context of signaling pathways involved in proliferation and cancer progression. beta2-Chimerin possesses a C-terminal Rac-GAP (GTPase-activating protein) domain that accelerates the hydrolysis of GTP from the Rac GTPase, leading to its inactivation. We found that beta2-chimerin messenger levels are significantly down-regulated in human breast cancer cell lines as well as in breast tumors. Adenoviral delivery of beta2-chimerin into MCF-7 breast cancer cells leads to inhibition of proliferation and G(1) cell cycle arrest. Mechanistic studies show that the effect involves the reduction in Rac-GTP levels, cyclin D1 expression, and retinoblastoma dephosphorylation. Studies using the mutated forms of beta2-chimerin revealed that these effects were entirely dependent on its C-terminal GAP domain and Rac-GAP activity. Moreover, MCF-7 cells stably expressing active Rac (V12Rac1) but not RhoA (V14RhoA) were insensitive to beta2-chimerin-induced inhibition of proliferation and cell cycle progression. The modulation of G(1)/S progression by beta2-chimerin not only implies an essential role for Rac in breast cancer cell proliferation but also raises the intriguing possibility that diacylglycerol-regulated non-protein kinase C pathways can negatively impact proliferation mechanisms controlled by Rho GTPases.     

The "promiscuous drug concept" with applications to Alzheimer's disease

Arguably, Alzheimer's disease (AD) is a multifactorial syndrome, rather than single disease, arising from a complex array of neurochemical factors. Numerous studies on the molecular pathogenesis of AD implicate a diversity of factors ranging from neurotoxic peptides (beta-amyloid) to inflammatory processes (interleukins), but all culminating in a common neuropathology. This diversity of molecular causation is an impediment to the design of effective therapies for AD. To address this design problem, we sought to identify a single, common motif (a "common receptor") shared by multiple structurally and functionally diverse proteins implicated in AD. This search revealed the presence of a common BBXB peptide motif and upon refinement, an AXBBXB motif; these regions can be exploited for the design of a "promiscuous drug" that exploits a "one-drug-multiple-receptors" therapeutic strategy for AD.     

Processing of pulmonary surfactant protein B by napsin and cathepsin H

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2-terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2-terminal propeptide between Leu178 and Pro179, 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.     

N-terminally extended surfactant protein (SP) C isolated from SP-B-deficient children has reduced surface activity and inhibited lipopolysaccharide binding

In both humans and mice, a deficiency of surfactant protein B (SP-B) is associated with a decreased concentration of mature SP-C and accumulation of a larger SP-C peptide, denoted SP-C(i), which is not observed under normal conditions. Isolation of hydrophobic polypeptides from the lungs of children who died with two different SP-B mutations yielded pure SP-C(i) and showed only trace amounts of mature SP-C. Determination of the SP-C(i) covalent structure revealed a 12-residue N-terminal peptide segment, followed by a 35-residue segment that is identical to mature SP-C. The SP-C(i) structure determined herein is similar to that of a proposed late intermediate in the processing of proSP-C, suggesting that SP-C(i) is the immediate precursor of SP-C. In bronchoalveolar lavage fluid from transgenic mice with a focal deficiency of SP-B, SP-C(i) was detected in the biophysically active, large aggregate fraction and was associated with membrane structures that are typical for a large aggregate surfactant. However, unlike SP-C, SP-C(i) exhibited a very poor ability to promote phospholipid adsorption, gave high surface tension during cyclic film compression, and did not bind lipopolysaccharide in vitro. SP-C(i) is thus capable of associating with surfactant lipids, but its N-terminal dodecapeptide segment must be proteolytically removed to generate a biologically functional peptide. The results of this study indicate that the early postnatal fatal respiratory distress seen in SP-B-deficient children is combined with the near absence of active variants of SP-C.     

Phylogenetic analyses of Texas isolates indicate an evolving subtype of the clade B feline immunodeficiency viruses

Rigorous phylogenetic analyses were used to compare the nucleotide sequences of feline immunodeficiency virus strains isolated from Texas and throughout the world. The envelope V3-V4 sequences and capsid gene of the Texas isolates formed a cluster between subtypes B and E. Statistical comparisons with other published sequences confirmed that the Texas group is a unique cluster, possibly a new subtype, arising from subtype B.     

A stochastic population model for Lepidium papilliferum (Brassicaceae), a rare desert ephemeral with a persistent seed bank

Population viability analysis (PVA) is a valuable tool for rare plant conservation, but PVA for plants with persistent seed banks is difficult without reliable information on seed bank processes. We modeled the population dynamics of the Snake River Plains ephemeral Lepidium papilliferum using data from an 11-yr artificial seed bank experiment to estimate age-specific vital rates for viability loss and germination. We related variation in postgermination demographic parameters to annual variation in precipitation patterns and used these relationships to construct a stochastic population model using precipitation driver variables. This enabled us to incorporate realistic levels of environmental variability into the model. A model incorporating best estimates for parameter values resulted in a mean trajectory for seed bank size that remained essentially stable through time, although there was a measurable risk of extinction over a 100-yr period for the study population under this scenario. Doubling the annual seed viability loss rate resulted in near-certain extinction, as did increasing first-year germination to 100%, showing the importance of the persistent seed bank. Interestingly, increasing environmental variance substantially decreased the risk of extinction, presumably because this plant relies on extremely good years to restock the persistent seed bank, while extremely bad years have little impact. If every year were average in this desert environment, the species could not persist. Simulated effects of livestock trampling resulted in greatly increased extinction risk, even over time frames as short as 15 years.     

Structure of the Recombinant Alphavirus Western Equine Encephalitis Virus Revealed by Cryoelectron Microscopy

Western equine encephalitis virus (WEEV; Togaviridae, Alphavirus) is an enveloped RNA virus that is typically transmitted to vertebrate hosts by infected mosquitoes. WEEV is an important cause of viral encephalitis in humans and horses in the Americas, and infection results in a range of disease, from mild flu-like illnesses to encephalitis, coma, and death. In addition to spreading via mosquito vectors, human WEEV infections can potentially occur directly via aerosol transmission. Due to its aerosol infectivity and virulence, WEEV is thus classified as a biological safety level 3 (BSL-3) agent. Because of its highly infectious nature and containment requirements, it has not been possible to investigate WEEV''s structure or assembly mechanism using standard structural biology techniques. Thus, to image WEEV and other BSL-3 agents, we have constructed a first-of-its-kind BSL-3 cryoelectron microscopy (cryoEM) containment facility. cryoEM images of WEEV were used to determine the first three-dimensional structure of this important human pathogen. The overall organization of WEEV is similar to those of other alphaviruses, consistent with the high sequence similarity among alphavirus structural proteins. Surprisingly, the nucleocapsid of WEEV, a New World virus, is more similar to the Old World alphavirus Sindbis virus than to other New World alphaviruses.The alphaviruses comprise a genus of single-stranded, plus-sense, enveloped RNA viruses that, together with rubella virus, comprise the family Togaviridae. The current classification of the genus Alphavirus includes 29 different species, with multiple subtypes and/or varieties represented within some species (30). These species can be grouped into 8 different complexes based on antigenic and/or genetic similarities (20). Most viruses from the New World are found in the Eastern, Venezuelan, and Western equine encephalitis (EEE, VEE, and WEE, respectively) complexes and cause encephalitis in humans and a variety of domesticated animals. Old World alphaviruses, on the other hand, typically cause only an arthralgia and rash syndrome that is rarely life threatening (5, 24). Among the New World alphaviruses, EEE, VEE, and WEE viruses (EEEV, VEEV, and WEEV, respectively) are potential biological weapons as well as naturally emerging pathogens and are therefore included on the category B Priority Pathogens list of the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (http://www.niaid.nih.gov/topics/biodefenserelated/biodefense/research/pages/cata.aspx).Alphaviruses replicate in the cytoplasm of infected cells after entry via receptor-mediated endocytosis (8). Following internalization, fusion of the viral envelope with the endocytic membrane is mediated by a low-pH-induced conformational change that exposes a fusion peptide found in the E1 envelope glycoprotein. The nucleocapsid then disassembles upon interactions with ribosomes, and an open reading frame (ORF) found in the 5′ two-thirds of the genome is translated. The resultant polyprotein is cleaved into 4 nonstructural proteins (nsP1 to -4) that mediate viral RNA replication, RNA capping, and polyprotein processing (Fig. ​(Fig.1).1). The structural proteins, including the two envelope glycoproteins E2 and E1 as well as the capsid protein, are encoded in a second ORF that is translated from a subgenomic message often referred to as 26S RNA. Following auto-cleavage of the capsid protein in the cytoplasm, the remaining polyprotein is inserted into the endoplasmic reticulum, where it is cleaved by host cell proteases and then processed through the secretory pathway, where the glycosylation of E2 and E1 occurs. Virion maturation occurs after E2/E1 heterodimers are inserted into the plasma membrane and 240 copies of the capsid protein interact with one copy of the genomic RNA to form nucleocapsids. These nucleocapsids then interact with a cytoplasmic domain of the E2 protein to initiate budding. The mature virion thus includes 240 copies of the capsid protein and 240 E2/E1 heterodimers arranged as trimeric spikes on the surface of the virus (8).Open in a separate windowFIG. 1.Diagram of the alphavirus genome, showing the 5′ cap, 5′ untranslated region, nonstructural polyprotein open reading frame, and major functions of the individual proteins, subgenomic promoter, structural polyprotein open reading frame, 3′ untranslated region, and poly(A) tail.The structures of several different alphaviruses, including Sindbis virus (SINV) (13), Ross River virus (RRV) (3, 35), Semliki Forest virus (SFV), (11), and VEEV (16), have been solved to subnanometer resolution using cryoelectron microscopy (cryoEM), and the X-ray crystallographic structure of the E1 protein from Semliki Forest virus has been determined to atomic resolution (9). The alphaviruses are ca. 700 Å in diameter, with 80 trimeric spikes on their surfaces. By fitting the E1 crystal structure into cryoEM reconstruction maps of whole viruses, the orientations of both envelope proteins within the spikes have been estimated (36). The E1 and E2 proteins are similar in shape, and the E2 proteins extend to the tips of the spikes, where most glycosylation and antibody-binding sites have been mapped (13). The underlying T=4 icosahedral capsid is constructed from regularly ordered capsomers arranged as hexons and pentons. These pentons and hexons consist of capsid protein monomers that apparently represent only the C-terminal half of the protein. Crystal structures of alphavirus capsid proteins also indicate that only the C terminus, including the protease domain, is ordered (25). cryoEM reconstructions of VEEV nucleocapsids isolated from virions have a less ordered structure, with density redistributed from the 3-fold to the 5-fold axis, suggesting that the envelope and/or the envelope glycoproteins constrain and stabilize the nucleocapsid in a compressed structure (15). Additionally, the VEEV nucleocapsids within viruses differ from those of Old World alphaviruses, with a counterclockwise rotation of the pentameric and hexameric capsomers in VEEV (16). Similar differences were observed in the capsid of Aura virus (AURAV), another New World alphavirus (34).In addition to being an important human and equine pathogen, WEEV is one of three alphaviruses that descended from a recombinant ancestor (6, 31). This ancestor derived its nonstructural and capsid protein genes from an ancestral EEEV strain, whereas its envelope glycoprotein genes were provided from an ancestral SINV. The recombination event was apparently followed by compensatory mutations in the cytoplasmic domain of the E2 protein that restored efficient interactions with the EEEV-like capsid protein (6). If this interpretation of the WEEV ancestral recombination event is correct, its nucleocapsids, constructed from capsid proteins derived from the New World EEEV ancestor, would be expected be more similar to those of the New World VEEV than to those of the Old World SINV, RRV, and SFV. To test this hypothesis and to investigate other structural features of interest related to its recombinant history and pathogenicity, we determined the structure of WEEV to a 13-Å resolution using cryoEM image reconstruction.     

Laminin‐332–β1 integrin interactions negatively regulate invadopodia

Adhesion of epithelial cells to basement membranes (BM) occurs through two major structures: actin‐associated focal contacts and keratin‐associated hemidesmosomes, both of which form on laminin‐332 (Ln‐332). In epithelial‐derived cancer cells, additional actin‐linked structures with putative adhesive properties, invadopodia, are frequently present and mediate BM degradation. A recent study proposed that BM invasion requires a proper combination of focal contacts and invadopodia for invading cells to gain traction through degraded BM, and suggested that these structures may compete for common molecular components such as Src kinase. In this study, we tested the role of the Ln‐332 in regulating invadopodia in 804G rat bladder carcinoma cells, a cell line that secretes Ln‐332 and forms all three types of adhesions. Expression of shRNA to Ln‐332 γ2 chain (γ2‐kd) led to increased numbers of invadopodia and enhanced extracellular matrix degradation. Replating γ2‐kd cells on Ln‐332 or collagen‐I fully recovered cell spreading and inhibition of invadopodia. Inhibition of α3 or β1, but not α6 or β4, phenocopied the effect of γ2‐kd, suggesting that α3β1‐mediated focal contacts, rather than α6β4‐mediated hemidesmosome pathways, intersect with invadopodia regulation. γ2‐kd cells exhibited alterations in focal contact‐type structures and in activation of focal adhesion kinase (FAK) and Src kinase. Inhibition of FAK also increased invadopodia number, which was reversible with Src inhibition. These data are consistent with a model whereby actin‐based adhesions can limit the availability of active Src that is capable of invadopodia initiation and identifies Ln‐332‐β1 interactions as a potent upstream regulator that limits cell invasion. J. Cell. Physiol. 223: 134–142, 2010. © 2009 Wiley‐Liss, Inc.     

A single amino acid in human APOBEC3F alters susceptibility to HIV-1 Vif

Human APOBEC3F (huA3F) potently restricts the infectivity of HIV-1 in the absence of the viral accessory protein virion infectivity factor (Vif). Vif functions to preserve viral infectivity by triggering the degradation of huA3F but not rhesus macaque A3F (rhA3F). Here, we use a combination of deletions, chimeras, and systematic mutagenesis between huA3F and rhA3F to identify Glu(324) as a critical determinant of huA3F susceptibility to HIV-1 Vif-mediated degradation. A structural model of the C-terminal deaminase domain of huA3F indicates that Glu(324) is a surface residue within the α4 helix adjacent to residues corresponding to other known Vif susceptibility determinants in APOBEC3G and APOBEC3H. This structural clustering suggests that Vif may bind a conserved surface present in multiple APOBEC3 proteins.