Spiders spin high-performance silks through the expression and assembly of tissue-restricted fibroin proteins. Spider silks are composite protein biopolymers that have complex microstructures. Retrieval of cDNAs and genomic DNAs encoding silk fibroins has revealed an association between the protein sequences and structure-property relationships. However, before spider silks can be subject to genetic engineering for commercial applications, the complete protein sequences and their functions, as well as the details of the spinning mechanism, will require additional progress and collaborative efforts in the areas of biochemistry, molecular biology and material science. Novel approaches to reveal additional molecular constituents embedded in the spider fibers, as well as cloning strategies to manipulate the genes for expression, will continue to be important aspects of spider biology research. Here we summarize the molecular characteristics of the different spider fibroins, the mechanical properties and assembly process of spidroins and the advances in protein expression systems used for recombinant silk production. We also highlight different technical approaches being used to elucidate the molecular constituents of silk fibers.

Araneoid spiders use specialized abdominal glands to manufacture up to seven different protein-based silks/glues that have diverse physical properties. The fibroin sequences that encode egg case fibers (cover silk for the egg case sac) and the secondary structure of these threads have not been previously determined. In this study, MALDI tandem TOF mass spectrometry (MS/MS) and reverse genetics were used to isolate the first egg case fibroin, named tubuliform spidroin 1 (TuSp1), from the black widow spider, Latrodectus hesperus. Real-time quantitative PCR analysis demonstrates TuSp1 is selectively expressed in the tubuliform gland. Analysis of the amino acid composition of raw egg case silk closely aligns with the predicted amino acid composition from the primary sequence of TuSp1, which supports the assertion that TuSp1 represents a major component of egg case fibers. TuSp1 is composed of highly homogeneous repeats that are 184 amino acids in length. The long stretches of polyalanine and glycine-alanine subrepeats, which account for the crystalline regions of minor ampullate and major ampullate fibers, are very poorly represented in TuSp1. However, polyserine blocks and short polyalanine stretches were highly iterated within the primary sequence, and (13)C NMR spectroscopy demonstrated that the majority of alanine was found in a beta-sheet structure in post-spun egg case silk. The TuSp1 repeat unit does not display substantial sequence similarity to any previously described fibroin genes or proteins, suggesting that TuSp1 is a highly divergent member of the spider silk gene family.

Somatic hypermutation (SHM) diversifies the variable region of Ig genes and underlies the process of affinity maturation, in which B lymphocytes producing high affinity antibodies are generated and selected. SHM is triggered in activated B cells by deamination of deoxycytosine residues mediated by activation-induced deaminase (AID). While mistargeting of SHM and AID results in mutations and DNA damage in many non-Ig genes, they act preferentially at Ig loci. The mechanisms responsible for preferential targeting of SHM and AID activity to Ig loci are poorly understood. Using an assay involving a SHM reporter cassette inserted into the Ig light chain locus (IgL) of chicken DT40 B cells, we have identified a 1.9 kb DIVAC (diversificationactivator) element derived from chicken IgL that supports high levels of AID-dependent mutation activity. Systematic deletion analysis reveals that targeting activity is spread throughout much of the sequence and identifies two core regions that are particularly critical for function: a 200 bp region within the IgL enhancer, and a 350 bp 3′ element. Chromatin immunoprecipitation experiments demonstrate that while DIVAC does not alter levels of several epigenetic marks in the mutation cassette, it does increase levels of serine-5 phosphorylated RNA polymerase II in the mutation target region, consistent with an effect on transcriptional elongation/pausing. We propose that multiple, dispersed DNA elements collaborate to recruit and activate the mutational machinery at Ig gene variable regions during SHM.

Spider silk proteins are well-known for their extraordinary mechanical properties, displaying remarkable strength and toughness. In this study, matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF) mass spectrometry (MS/MS) and reverse genetics were used to isolate a new cDNA sequence that encodes for a protein assembled into egg case silk from the black widow spider, Latrodectus hesperus. Analysis of the primary sequence of this protein reveals approximately 52% identity to the egg case protein 1 (ECP-1) fibroin-like family member. On the basis of the similarity in the primary sequence and expression pattern, we have named this factor egg case protein 2 (ECP-2). Alignments of ECP-1 and ECP-2 demonstrate highly conserved N termini, with 16 Cys residues found within the first 153 amino acids. Traditional ensemble repeats found within reported fibroins were poorly represented in the primary sequence of ECP-2, but scattered blocks of polyalanine were present, along with a C terminus rich in GA repeats. Reverse transcription quantitative PCR analysis showed that ECP-2 is predominantly expressed in the tubuliform gland. Relative to ECP-1, ECP-2 mRNA levels were determined to be >2-fold higher. MALDI MS/MS analysis of peptide fragments generated from the large-diameter core fiber after enzymatic digestion and acid hydrolysis demonstrated the presence of a fiber that is trimeric in nature, containing tubuliform spidroin 1 (TuSp1), ECP-1, and ECP-2. We also report an additional primary sequence for TuSp1, demonstrating that TuSp1 contains two Cys residues within a nonrepetitive N-terminal region. In combination with the distinctive protein architectures of ECP-1 and ECP-2, along with their co-localization with TuSp1 in the core fiber, our findings suggest that ECP-1 and ECP-2 play important structural roles in the egg case silk fiber.

Background: Spiders extrude adhesive glues to form connection joints that mediate web construction and prey wrapping. Results: DNA microarray analysis and mass spectrometry reveal new protein glue constituents that comprise connection joints. Conclusion: Spider glue proteins represent a diverse group of polypeptides with distinct molecular architectures. Significance: Learning how spider glues mediate the fusion of fibers is crucial for understanding adhesion mechanisms in biology.

Spiders produce multiple types of silk that exhibit diverse mechanical properties and biological functions. Most molecular studies of spider silk have focused on fibroins from dragline silk and capture silk, two important silk types involved in the survival of the spider. In our studies we have focused on the characterization of egg case silk, a third silk fiber produced by the black widow spider, Latrodectus hesperus. Analysis of the physical structure of egg case silk using scanning electron microscopy demonstrates the presence of small and large diameter fibers. By using the strong protein denaturant 8 M guanidine hydrochloride to solubilize the fibers, we demonstrated by SDS-PAGE and protein silver staining that an abundant component of egg case silk is a 100-kDa protein doublet. Combining matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry and reverse genetics, we have isolated a novel gene called ecp-1, which encodes for one of the protein components of the 100-kDa species. BLAST searches of the NCBInr protein data base using the primary sequence of ECP-1 revealed similarity to fibroins from spiders and silkworms, which mapped to two distinct regions within the ECP-1. These regions contained the conserved repetitive fibroin motifs poly(Ala) and poly(Gly-Ala), but surprisingly, no larger ensemble repeats could be identified within the primary sequence of ECP-1. Consistent with silk gland-restricted patterns of expression for fibroins, ECP-1 was demonstrated to be predominantly produced in the tubuliform gland, with lower levels detected in the major and minor ampullate glands. ECP-1 monomeric units were also shown to assemble into higher aggregate structures through the formation of disulfide bonds via a unique cysteine-rich N-terminal region. Collectively, our findings provide new insight into the components of egg case silk and identify a new class of silk proteins with distinctive molecular features relative to traditional members of the spider silk gene family.

Cell-autonomous immunity relies on the rapid detection of invasive pathogens by host proteins. Guanylate binding proteins (GBPs) have emerged as key mediators of vertebrate immune defense through their ability to recognize a diverse array of intracellular pathogens and pathogen-containing cellular compartments. Human and mouse GBPs have been shown to target distinct groups of microbes, although the molecular determinants of pathogen specificity remain unclear. We show that rapid diversification of a C-terminal polybasic motif (PBM) in primate GBPs controls recognition of the model cytosolic bacterial pathogen Shigella flexneri. By swapping this membrane-binding motif between primate GBP orthologs, we found that the ability to target S. flexneri has been enhanced and lost in specific lineages of New World primates. Single substitutions in rapidly evolving sites of the GBP1 PBM are sufficient to abolish or restore bacterial detection abilities, illustrating a role for epistasis in the evolution of pathogen recognition. We further demonstrate that the squirrel monkey GBP2 C-terminal domain recently gained the ability to target S. flexneri through a stepwise process of convergent evolution. These findings reveal a mechanism by which accelerated evolution of a PBM shifts GBP target specificity and aid in resolving the molecular basis of GBP function in cell-autonomous immune defense. IMPORTANCE Many infectious diseases are caused by microbes that enter and survive within host cells. Guanylate binding proteins (GBPs) are a group of immune proteins which recognize and inhibit a variety of intracellular pathogenic microbes. We discovered that a short sequence within GBPs required for the detection of bacteria, the polybasic motif (PBM), has been rapidly evolving between primate species. By swapping PBMs between primate GBP1 genes, we were able to show that specific sequences can both reduce and improve the ability of GBP1 to target intracellular bacteria. We also show that the ability to envelop bacteria has independently evolved in GBP2 of South American monkeys. Taking the results together, this report illustrates how primate GBPs have adapted to defend against infectious pathogens.

Lung Protective Mechanical Ventilation (MV) of critically ill adults and children is lifesaving but it may decrease diaphragm contraction and promote Ventilator Induced Diaphragm Dysfunction (VIDD). An ideal MV strategy would balance lung and diaphragm protection. Building off a Phase I pilot study, we are conducting a Phase II controlled clinical trial that seeks to understand the evolution of VIDD in critically ill children and test whether a novel computer-based approach (Real-time Effort Driven ventilator management (REDvent)) can balance lung and diaphragm protective ventilation to reduce time on MV. REDvent systematically adjusts PEEP, FiO 2 , inspiratory pressure, tidal volume and rate, and uses real-time measures from esophageal manometry to target normal levels of patient effort of breathing. This trial targets 276 children with pulmonary parenchymal disease. Patients are randomized to REDvent vs. usual care for the acute phase of MV (intubation to first Spontaneous Breathing Trial (SBT)). Patients in either group who fail their first SBT will be randomized to REDvent vs usual care for weaning phase management (interval from first SBT to passing SBT). The primary clinical outcome is length of weaning, with several mechanistic outcomes. Upon completion, this study will provide important information on the pathogenesis and timing of VIDD during MV in children and whether this computerized protocol targeting lung and diaphragm protection can lead to improvement in intermediate clinical outcomes. This will form the basis for a larger, Phase III multi-center study, powered for key clinical outcomes such as 28day ventilator free days. Clinical Trials Registration: NCT03266016