Mechanistic insights into RNase L through use of an MDMX-derived multi-functional protein domain.

RNase L is part of the innate immune response to viral infection. It is activated by a small oligonucleotide (2-5A) whose synthesis is initiated as part of the interferon response. Binding of 2-5A to the N-terminal regulatory region, the ANK domain, of RNase L activates its ribonuclease activity and results in cleavage of RNA in the cell, which ultimately leads to apoptosis of the infected cell. The mechanism by which 2-5A activates the ribonuclease activity of RNase L is currently unclear but 2-5A has been shown to induce dimerization of RNase L. To investigate the importance of dimerization of RNase L, we developed a 15kDa dimerization-inducing protein domain that was fused to the N-terminus of RNase L. From these studies we provide direct evidence that dimerization of RNase L occurs at physiologically relevant protein concentrations and correlates with activation of ribonuclease activity. We also show that the binding of 2-5A to RNase L promotes dimerization of the ANK domain and suggest how this could transmit a signal to the rest of the protein to activate ribonuclease activity. Finally, we show that the dimerization-inducing domain can be used as a general fusion partner to aid in protein expression and purification.

Spider glue proteins have distinct architectures compared with traditional spidroin family members.

Adhesive spider glues are required to perform a variety of tasks, including web construction, prey capture, and locomotion. To date, little is known regarding the molecular and structural features of spider glue proteins, in particular bioadhesives that interconnect dragline or scaffolding silks during three-dimensional web construction. Here we use biochemical and structural approaches to identify and characterize two aggregate gland specific gene products, AgSF1 and AgSF2, and demonstrate that these proteins co-localize to the connection joints of both webs and wrapping silks spun from the black widow spider, Latrodectus hesperus. Protein architectures are markedly divergent between AgSF1 and AgSF2, as well as traditional spider silk fibroin family members, suggesting connection joints consist of a complex proteinaceous network. AgSF2 represents a nonglycosylated 40-kDa protein that has novel internal amino acid block repeats with the consensus sequence NVNVN embedded in a glycine-rich matrix. Analysis of the amino acid sequence of AgSF1 reveals pentameric QPGSG iterations that are similar to conserved modular elements within mammalian elastin, a rubber-like elastomeric protein that interfaces with collagen. Wet-spinning methodology using purified recombinant proteins show AgSF1 has the potential to self-assemble into fibers. X-ray fiber diffraction studies performed on these synthetic fibers reveal the presence of noncrystalline domains that resemble classical rubber networks. Collectively, these data support that the aggregate gland serves to extrude a protein mixture that contains substances that allow for the self-assembly of fiber-like structures that interface with dragline silks to mediate prey capture.

Spider minor ampullate silk proteins are constituents of prey wrapping silk in the cob weaver Latrodectus hesperus.

Spiders spin high performance fibers with diverse biological functions and mechanical properties. Molecular and biochemical studies of spider prey wrapping silks have revealed the presence of the aciniform silk fibroin AcSp1-like. In our studies we demonstrate the presence of a second distinct polypeptide present within prey wrapping silk. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called MiSp1-like and demonstrate that its protein product is a constituent of prey wrap silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein database using the amino acid sequence of MiSp1-like revealed similarity to the conserved C-terminal domain of silk family members. In particular, MiSp1-like showed the highest degree of sequence similarity to the nonrepetitive C-termini of published orb-weaver minor ampullate fibroin molecules. Analysis of the internal amino acid sequence of the black widow MiSp1-like revealed polyalanine stretches interrupted by glycine residues and glycine-alanine couplets within MiSp1-like as well as repeats of the heptameric sequence AGGYGQG. Real-time quantitative PCR analysis demonstrates that the MiSp1-like gene displays a minor ampullate gland-restricted pattern of expression. Furthermore, amino acid composition analysis, coupled with scanning electron microscopy of raw wrapping silk, supports the assertion that minor ampullate silks are important constituents of black widow spider prey wrap silk. Collectively, our findings provide direct molecular evidence for the involvement of minor ampullate fibroins in swathing silks and suggest composite materials play an important role in the wrap attack process for cob-weavers.

Aciniform spidroin, a constituent of egg case sacs and wrapping silk fibers from the black widow spider Latrodectus hesperus.

Spiders produce high performance fibers with diverse mechanical properties and biological functions. Molecular and biochemical studies of spider egg case silk have revealed that the main constituent of the large diameter fiber contains the fibroin TuSp1. Here we demonstrate by SDS-PAGE and protein silver staining the presence of a distinct approximately 300-kDa polypeptide that is found in solubilized egg case sacs. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called AcSp1-like and demonstrate that its protein product is assembled into the small diameter fibers of egg case sacs and wrapping silks from the black widow spider, Latrodectus hesperus. BLAST searches of the NCBInr protein data base using the amino acid sequence of AcSp1-like revealed similarity to AcSp1, an inferred protein proposed to be a component of wrapping silk. However, the AcSp1-like protein was found to display more nonuniformity in its internal iterated repeat modules than the putative AcSp1 fibroin. Real time quantitative PCR analysis demonstrates that the AcSp1-like gene displays an aciniform gland-restricted pattern of expression. The amino acid composition of the fibroins extracted from the luminal contents of the aciniform glands was remarkably similar to the predicted amino acid composition of the AcSp1-like protein, which supports the assertion that AcSp1-like protein represents the major constituent stored within the aciniform gland. Collectively, our findings provide the first direct molecular evidence for the involvement of the aciniform gland in the production of a common fibroin that is assembled into the small diameter threads of egg case and wrapping silk of cob weavers.

Analysis of aqueous glue coating proteins on the silk fibers of the cob weaver, Latrodectus hesperus.

Elucidation of the molecular composition and physical properties of spider glue is necessary to understand its function in the mechanics of the web and prey capture. Previous reports have indicated that components of the adhesive coating contain inorganic molecules, phosphorylated glycoproteins, lipids, and organic low-molecular mass (LMM) compounds. Using a proteomic strategy, we have investigated the viscid, aqueous components that coat different silk fiber types from the black widow spider, Latrodectus hesperus. After in-solution tryptic digestion of the aqueous protein material extracted from egg case sacs, gumfooted lines, and the web scaffolding connection joints, followed by peptide analysis using MALDI tandem TOF mass spectrometry, we demonstrate that these fibers are coated with common peptides. Utilizing a reverse genetics approach, we have isolated the cDNAs encoding two distinct fiber coating products, which we have named spider coating peptide 1 and 2 (SCP-1 and SCP-2). Secreted forms of SCP-1 and SCP-2 contain 36 and 19 amino acids, respectively, and their primary sequences display no significant similarities to ensemble repeat units from traditional fibroins. Quantitative real-time reverse transcription PCR analyses show that these mRNAs are chiefly produced by the aggregate gland. Biochemical studies also demonstrate that the SCP-1 peptide has intrinsic metal binding properties, suggesting a role of peptide-metal ion interactions with the fiber constituents to enhance thread performance. Collectively, these investigations are the first to reveal a novel role for the aggregate gland in the production of peptides that coat spider silk threads.

Characterization of a novel class II bHLH transcription factor from the black widow spider, Latrodectus hesperus, with silk-gland restricted patterns of expression.

Members of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including lymphopoiesis, myogenesis, neurogenesis, and sex determination. Screening a cDNA library prepared from silk-producing glands of the black widow spider, we have identified a new bHLH transcription factor named SGSF. Within the bHLH region, SGSF showed considerable conservation with other HLH proteins, including Drosophila melanogaster achaete and scute, as well as three HLH proteins identified by gene prediction programs. The expression pattern of SGSF was restricted to a subset of silk-producing glands, which include the tubuliform and major ampullate glands. SGSF was capable of binding an E-box element as a heterodimer with the E protein, E47, but was unable to bind this motif as a homodimer. SGSF was demonstrated to be a nuclear transcription factor capable of attenuating the transactivation of E47 homodimers in mammalian cells. SGSF represents the first example of a silk gland-restricted bHLH protein, and its expression pattern suggests that SGSF plays a role in regulating differentiation of cells in the spider that control silk gland formation or egg case silk gene expression.