Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic, anti-oxidative, and anti-inflammatory properties. It is also one of the most potent endogenous inhibitors of angiogenesis, playing an important role in restricting tumor growth, invasion, and metastasis. Studies show that PEDF binds to cell surface proteins, but little is known about how it exerts its effects. Recently, research identified phospholipase A 2 /nutrin/patatin-like phospholipase domaincontaining 2 as one PEDF receptor. To identify other receptors, we performed yeast two-hybrid screening using PEDF as bait and discovered that the non-integrin 37/67-kDa laminin receptor (LR) is another PEDF receptor. Co-immunoprecipitation, His tag pulldown, and surface plasmon resonance assays confirmed the interaction between PEDF and LR. Using the yeast two-hybrid method, we further restricted the LR-interacting domain on PEDF to a 34-amino acid (aa) peptide (aa 44 -77) and the PEDF-interacting domain on LR to a 91-aa fragment (aa 120 -210). A 25-mer peptide named P46 (aa 46 -70), derived from 34-mer, interacts with LR in surface plasmon resonance assays and binds to endothelial cell (EC) membranes. This peptide induces EC apoptosis and inhibits EC migration, tube-like network formation in vitro, and retinal angiogenesis ex vivo, like PEDF. Our results suggest that LR is a real PEDF receptor that mediates PEDF angiogenesis inhibition.Pigmented epithelium-derived factor (PEDF), 2 also known as SERPIN F1 and EPC1, is a 50-kDa serpin-like peptide. Although first identified in cultured pigment epithelial cells from fetal human retinas (1), we now know that liver, kidney, heart, testis, and lung tissues also express PEDF (2). PEDF influences many biological processes. It is anti-angiogenic, anti-tumorigenic, anti-inflammatory, anti-oxidative, neurotrophic, and neuroprotective, and it exhibits anti-vasopermeability properties (3-9). These diverse actions affect many cell types, including retinal cells (10), neuronal cells (11), endothelial cells (12), and hematopoietic stem cells (13). X-ray diffraction studies show that PEDF has an asymmetrical charge distribution (14). A high density of basic residues on one side of the molecule (positive) interact with heparin and glycosaminoglycans, whereas acidic residues on the opposite side (negative) interact with type-1 collagen (15)(16)(17)(18)(19)). Yet the mechanisms explaining the diverse biological activities of PEDF remain unclear.A ligand/receptor interaction at the cell membrane seemed likely, in addition to interactions within extracellular matrices, because of the diverse effects and ubiquitous expression of PEDF and the fact that most PEDF deposits remain within extracellular matrices (20). We suspected that distinct PEDF receptors elicit divergent signals to cause different biological effects. Indeed, evidence shows that PEDF binds at least two receptors: a 60-kDa receptor in ECs and an 80-kDa receptor in neuronal cells (21)(22)(23)(24). Research has identified two functional epi...

Soluble oligomers of the amyloid beta-protein (Abeta) are linked to Alzheimer's disease. Irrespective of the nature of the nucleus before fibril growth, dimers are essential species in Abeta assembly, but their transient character has precluded, thus far, high-resolution structure determination. We have investigated the effects of the point mutation A21G on Abeta dimers by performing high temperature all-atom molecular dynamics simulations of Abeta(40), Abeta(42), and their Flemish variants (A21G) starting from their fibrillar conformations. Abeta dimers are found in equilibrium between various topologies, and the absence of common structural features shared by the four species makes problematic the design of a unique inhibitor for blocking dimers. We also show that the impact of the point mutation A21G on Abeta structure and dynamics varies from Abeta(40) to Abeta(42). Finally, we provide a possible structural explanation for the reduced aggregation rate of Abeta fibrils containing the Flemish disease-causing mutation.

The herpesvirus capsid is a complex protein assembly that includes hundreds of copies of four major subunits and lesser numbers of several minor proteins, all essential for infectivity. Cryo-electron microscopy is uniquely suited for studying interactions that govern the assembly and function of such large and functional complexes. Here we report two high quality capsid structures, from human herpes simplex virus type 1 (HSV-1) and the animal pseudorabies virus (PRV), imaged inside intact virions at ~7 Å resolution. From these we developed a complete model of subunit and domainal organization and identified extensive networks of subunit contacts that underpin capsid stability and form a pathway that may signal the completion of DNA packaging from the capsid interior to outer surface for initiating nuclear egress. Differences in folding and orientation of subunit domains between herpesvirus capsids suggest that common elements have been modified for specific functions.

Bacteriophage T5 represents a large family of lytic Siphoviridae infecting Gram-negative bacteria. The low-resolution structure of T5 showed the T‫31؍‬ geometry of the capsid and the unusual trimeric organization of the tail tube, and the assembly pathway of the capsid was established. Although major structural proteins of T5 have been identified in these studies, most of the genes encoding the morphogenesis proteins remained to be identified. Here, we combine a proteomic analysis of T5 particles with a bioinformatic study and electron microscopic immunolocalization to assign function to the genes encoding the structural proteins, the packaging proteins, and other nonstructural components required for T5 assembly. A head maturation protease that likely accounts for the cleavage of the different capsid proteins is identified. Two other proteins involved in capsid maturation add originality to the T5 capsid assembly mechanism: the single head-to-tail joining protein, which closes the T5 capsid after DNA packaging, and the nicking endonuclease responsible for the single-strand interruptions in the T5 genome. We localize most of the tail proteins that were hitherto uncharacterized and provide a detailed description of the tail tip composition. Our findings highlight novel variations of viral assembly strategies and of virion particle architecture. They further recommend T5 for exploring phage structure and assembly and for deciphering conformational rearrangements that accompany DNA transfer from the capsid to the host cytoplasm. Bacteriophage T5 is a member of the Siphoviridae family infecting Escherichia coli. It consists of an icosahedral capsid containing a large molecule of double-stranded DNA (dsDNA) (121.75 kbp) attached to a long flexible noncontractile tail. The complete genomes of two wild-type T5 strains (GenBank accession numbers AY587007 [1] and AY543070) and of the heat-stable deletion mutant T5st0 (GenBank accession number AY692264 [this study]) have been sequenced. Moreover, the genomes of T5-related phages H8 (2), EPS7 (3), SPC35 (4), AKVF3 (5), pVp-1 (6), and My1 (7) exhibit high sequence similarity to T5. Of the 159 to 174 genes predicted in each genome, about 70 were assigned functions on the basis of similarity searches and/or previous genetic studies. Most of the identified genes are related to nucleotide metabolism, DNA replication, recombination, and various enzymatic functions. Despite the fact that the major structural proteins of T5 have been identified (8), the functions of 13 of the 23 late genes encoding the structural and morphogenesis proteins remain to be ascertained.The overall structure of T5 was solved by cryo-electron microscopy (cryo-EM) and image reconstruction (9). The large icosahedral capsid consists of the coat protein pb8, arranged as 11 pentamers at the vertices and 120 hexamers on the faces. The 12th vertex is occupied by a dodecamer of the portal protein pb7. The early events of T5 capsid assembly have been partly deciphered (10). The initial shell (prohead I) is assemb...

Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBDSARS-CoV. Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics.

Large icosahedral viruses that infect bacteria represent an extreme of the coevolution of capsids and the genomes they accommodate. One subset of these large viruses is the jumbophages, tailed phages with double-stranded DNA genomes of at least 200,000 bp. We explored the mechanism leading to increased capsid and genome sizes by characterizing structures of several jumbophage capsids and the DNA packaged within them. Capsid structures determined for six jumbophages were consistent with the canonical phage HK97 fold, and three had capsid geometries with novel triangulation numbers (T=25, T=28, and T=52). Packaged DNA (chromosome) sizes were larger than the genome sizes, indicating that all jumbophages use a head-full DNA packaging mechanism. For two phages (PAU and G), the sizes appeared very much larger than their genome length. We used two-dimensional DNA gel electrophoresis to show that these two DNAs migrated abnormally due to base modifications and to allow us to calculate their actual chromosome sizes. Our results support a ratchet model of capsid and genome coevolution whereby mutations lead to increased capsid volume and allow the acquisition of additional genes. Once the added genes and larger capsid are established, mutations that restore the smaller size are disfavored.

A recently identified class of myopathies is produced by abnormal desmin, and is characterized by a disorganization of the desmin filament network, the accumulation of insoluble desmin-containing aggregates, and destructive changes in the sarcomeric organization of striated muscles.

The packaging of DNA into preformed capsids is a critical step during herpesvirus infection. For herpes simplex virus, this process requires the products of seven viral genes: the terminase proteins pUL15, pUL28 and pUL33; the capsid vertex specific component (CVSC) proteins pUL17 and pUL25; the portal protein pUL6 and pUL32. The pUL6 portal dodecamer is anchored at one vertex of the capsid by interactions with the adjacent triplexes as well as helical density attributed to the pUL17 and pUL25 subunits of the CVSC. To define the roles and structures of the CVSC proteins in virus assembly and DNA packaging we isolated a number of recombinant viruses expressing pUL25, pUL17 and pUL36 fused with green or red fluorescent proteins as well as viruses with specific deletions in the CVSC genes. Biochemical and structural studies of these mutants demonstrated that: (i) four of the helices in the CVSC helix bundle can be attributed to two copies each of pUL36 and pUL25; (ii) pUL17 and pUL6 are required for capsid binding of the terminase complex in the nucleus; (iii) pUL17 is important for determining the site of the first cleavage reaction generating replicated genomes with termini derived from the long-arm component of the HSV-1 genome; (iv) pUL36 serves no direct role in cleavage/packaging; (v) cleavage and stable packaging of the viral genome involves an ordered interaction of the terminase complex and pUL25 with pUL17 at the portal vertex; and (vi) packaging of the viral genome results in a dramatic displacement of the portal. Importance Herpes simplex virus type I (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. A critical step during productive HSV-1 infection is the cleavage and packaging of replicated, concatemeric viral DNA into preformed capsids. A key knowledge gap is how the capsid engages the replicated viral genome and the subsequent packaging of a unit length HSV genome. Here biochemical and structural studies focused on the unique portal vertex of wild type HSV and packaging mutants provide insights into the mechanism of HSV genome packaging. The significance of our research is in identifying the portal protein pUL6 and pUL17 as key viral factors for engaging the terminase complex with the capsid and the subsequent cleavage, packaging, and stable incorporation of the viral genome in the HSV-1 capsid.