CRISPR-Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames.

Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR-Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis-regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B. Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the 'dark' proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.

Antibodies to coagulase of Staphylococcus aureus crossreact to Efb and reveal different binding of shared fibrinogen binding repeats.

Staphylococcus aureus pathology is caused by a plethora of virulence factors able to combat multiple host defence mechanisms. Fibrinogen (Fg), a critical component in the host coagulation cascade, plays an important role in the pathogenesis of this bacterium, as it is the target of numerous staphylococcal virulence proteins. Amongst its secreted virulence factors, coagulase (Coa) and Extracellular fibrinogen-binding protein (Efb) share common Fg binding motives and have been described to form a Fg shield around staphylococcal cells, thereby allowing efficient bacterial spreading, phagocytosis escape and evasion of host immune system responses. Targeting these proteins with monoclonal antibodies thus represents a new therapeutic option against S. aureus. To this end, here we report the selection and characterization of fully human, sequence-defined, monoclonal antibodies selected against the C-terminal of coagulase. Given the functional homology between Coa and Efb, we also investigated if the generated antibodies bound the two virulence factors. Thirteen unique antibodies were isolated from naïve antibodies gene libraries by antibody phage display. As anticipated, most of the selected antibodies showed cross-recognition of these two proteins and among them, four were able to block the interaction between Coa/Efb and Fg. Furthermore, our monoclonal antibodies could interact with the two main Fg binding repeats present at the C-terminal of Coa and distinguish them, suggesting the presence of two functionally different Fg-binding epitopes.

Click display: a rapid and efficient in vitro protein display method for directed evolution.

We describe a novel method for in vitro protein display-click display-that does not depend on maintaining RNA integrity during biopanning and yields covalently linked protein-cDNA complexes from double-stranded input DNA within 2 h. The display is achieved in a one-pot format encompassing transcription, translation and reverse transcription reactions in series. Stable linkage between proteins and the encoding cDNA is mediated by a modified DNA linker-ML-generated via a click chemistry reaction between a puromycin-containing oligo and a cDNA synthesis primer. Biopanning of a click-displayed mock library coupled with next-generation sequencing analysis revealed >600-fold enrichment of target binders within a single round of panning. A synthetic library of Designed Ankyrin Repeat Proteins (DARPins) with ∼1012 individual members was generated using click display in a 25-µl reaction and six rounds of library panning against a model protein yielded a panel of nanomolar binders. This study establishes click display as a powerful tool for protein binder discovery/engineering and provides a convenient platform for in vitro biopanning selection even in RNase-rich environments such as on whole cells.

Prokaryotic Collagen-Like Proteins as Novel Biomaterials.

Collagens are the major structural component in animal extracellular matrices and are critical signaling molecules in various cell-matrix interactions. Its unique triple helical structure is enabled by tripeptide Gly-X-Y repeats. Understanding of sequence requirements for animal-derived collagen led to the discovery of prokaryotic collagen-like protein in the early 2000s. These prokaryotic collagen-like proteins are structurally similar to mammalian collagens in many ways. However, unlike the challenges associated with recombinant expression of mammalian collagens, these prokaryotic collagen-like proteins can be readily expressed in E. coli and are amenable to genetic modification. In this review article, we will first discuss the properties of mammalian collagen and provide a comparative analysis of mammalian collagen and prokaryotic collagen-like proteins. We will then review the use of prokaryotic collagen-like proteins to both study the biology of conventional collagen and develop a new biomaterial platform. Finally, we will describe the application of Scl2 protein, a streptococcal collagen-like protein, in thromboresistant coating for cardiovascular devices, scaffolds for bone regeneration, chronic wound dressing and matrices for cartilage regeneration.

The Collagen Receptor Discoidin Domain Receptor 1b Enhances Integrin ß1-Mediated Cell Migration by Interacting With Talin and Promoting Rac1 Activation.

Integrins and discoidin domain receptors (DDRs) 1 and 2 promote cell adhesion and migration on both fibrillar and non fibrillar collagens. Collagen I contains DDR and integrin selective binding motifs; however, the relative contribution of these two receptors in regulating cell migration is unclear. DDR1 has five isoforms (DDR1a-e), with most cells expressing the DDR1a and DDR1b isoforms. We show that human embryonic kidney 293 cells expressing DDR1b migrate more than DDR1a expressing cells on DDR selective substrata as well as on collagen I in vitro. In addition, DDR1b expressing cells show increased lung colonization after tail vein injection in nude mice. DDR1a and DDR1b differ from each other by an extra 37 amino acids in the DDR1b cytoplasmic domain. Interestingly, these 37 amino acids contain an NPxY motif which is a central control module within the cytoplasmic domain of ß integrins and acts by binding scaffold proteins, including talin. Using purified recombinant DDR1 cytoplasmic tail proteins, we show that DDR1b directly binds talin with higher affinity than DDR1a. In cells, DDR1b, but not DDR1a, colocalizes with talin and integrin ß1 to focal adhesions and enhances integrin ß1-mediated cell migration. Moreover, we show that DDR1b promotes cell migration by enhancing Rac1 activation. Mechanistically DDR1b interacts with the GTPase-activating protein (GAP) Breakpoint cluster region protein (BCR) thus reducing its GAP activity and enhancing Rac activation. Our study identifies DDR1b as a major driver of cell migration and talin and BCR as key players in the interplay between integrins and DDR1b in regulating cell migration.

KRAS Inhibitor that Simultaneously Inhibits Nucleotide Exchange Activity and Effector Engagement.

We describe a small molecule ligand ACA-14 (2-hydroxy-5-{[(2-phenylcyclopropyl) carbonyl] amino} benzoic acid) as an initial lead for the development of direct inhibitors of KRAS, a notoriously difficult anticancer drug target. We show that the compound binds to KRAS near the switch regions with affinities in the low micromolar range and exerts different effects on KRAS interactions with binding partners. Specifically, ACA-14 impedes the interaction of KRAS with its effector Raf and reduces both intrinsic and SOS-mediated nucleotide exchange rates. Likely as a result of these effects, ACA-14 inhibits signal transduction through the MAPK pathway in cells expressing mutant KRAS and inhibits the growth of pancreatic and colon cancer cells harboring mutant KRAS. We thus propose compound ACA-14 as a useful initial lead for the development of broad-acting inhibitors that target multiple KRAS mutants and simultaneously deplete the fraction of GTP-loaded KRAS while abrogating the effector-binding ability of the already GTP-loaded fraction.

vhp Is a Fibrinogen-Binding Protein Related to vWbp in Staphylococcus aureus.

Staphylococcus aureus can target a variety of tissues, causing life-threatening infections. The basis for this diversity stems from the microorganism's ability to spread in the vascular system throughout the body. To survive in blood, S. aureus coats itself with a fibrinogen (Fg)/fibrin shield. The protective shield is assembled by the coordinated actions of a number of Fg-binding bacterial proteins that manipulate the host's blood coagulation system. Several of the Fg binders appear redundant, sharing similar functional motifs. This observation led us to screen for the presence of novel proteins with significant amino acid identities to von Willebrand factor-binding protein (vWbp), a key component in the shield assembly machinery. One identified protein showed significant sequence identity with the C-terminal region of vWbp, and we consequently named it vWbp homologous protein (vhp). The vhp gene lies within a cluster of genes that encode other virulence factors in S. aureus. Although each isolate only contains one copy of the vhp gene, S. aureus has at least three distinct alleles, vhpA, B, and C, that are present in the core genome. All three vhp isoforms bind Fg with high affinity, targeting a site located in the D fragment of Fg. We further identified an ∼79 amino acid-long, conserved segment within the C-terminal region of vWbp that shares high sequence identities (54 to 67%) with the vhps and binds soluble Fg with high affinity. Further analysis of this conserved motif and the intact vhps revealed intriguing differences in the Fg binding behavior, perhaps suggesting that these proteins have similar but discrete functions in the shield assembly. IMPORTANCE The life-threatening diseases caused by multidrug-resistant Staphylococcus aureus strains are a worldwide medical problem due to treatment limitations and the lack of an effective vaccine. The ability of S. aureus to coat itself with a protective fibrinogen (Fg)/fibrin shield allows the organism to survive in blood and to disseminate and cause invasive diseases. This process represents a promising target for novel antistaphylococcal treatment strategies but is incompletely understood. S. aureus expresses a number of Fg-binding proteins. Some of these proteins have apparently redundant functions. Proteins with similar functions often share a structural or functional motif with each other. In this study, we identified a protein homologous to the C-terminal of von Willebrand factor-binding protein (vWbp), a key contributor in the Fg shield assembly that also binds Fg. Further analysis allowed us to identify a common Fg-binding motif.

Collagen Binding Proteins of Gram-Positive Pathogens.

Collagens are the primary structural components of mammalian extracellular matrices. In addition, collagens regulate tissue development, regeneration and host defense through interaction with specific cellular receptors. Their unique triple helix structure, which requires a glycine residue every third amino acid, is the defining structural feature of collagens. There are 28 genetically distinct collagens in humans. In addition, several other unrelated human proteins contain a collagen domain. Gram-positive bacteria of the genera Staphylococcus, Streptococcus, Enterococcus, and Bacillus express cell surface proteins that bind to collagen. These proteins of Gram-positive pathogens are modular proteins that can be classified into different structural families. This review will focus on the different structural families of collagen binding proteins of Gram-positive pathogen. We will describe how these proteins interact with the triple helix in collagens and other host proteins containing a collagenous domain and discuss how these interactions can contribute to the pathogenic processes.

Host-directed therapy in foals can enhance functional innate immunity and reduce severity of Rhodococcus equi pneumonia.

Pneumonia caused by the intracellular bacterium Rhodococcus equi is an important cause of disease and death in immunocompromised hosts, especially foals. Antibiotics are the standard of care for treating R. equi pneumonia in foals, and adjunctive therapies are needed. We tested whether nebulization with TLR agonists (PUL-042) in foals would improve innate immunity and reduce the severity and duration of pneumonia following R. equi infection. Neonatal foals (n = 48) were nebulized with either PUL-042 or vehicle, and their lung cells infected ex vivo. PUL-042 increased inflammatory cytokines in BAL fluid and alveolar macrophages after ex vivo infection with R. equi. Then, the in vivo effects of PUL-042 on clinical signs of pneumonia were examined in 22 additional foals after intrabronchial challenge with R. equi. Foals infected and nebulized with PUL-042 or vehicle alone had a shorter duration of clinical signs of pneumonia and smaller pulmonary lesions when compared to non-nebulized foals. Our results demonstrate that host-directed therapy can enhance neonatal immune responses against respiratory pathogens and reduce the duration and severity of R. equi pneumonia.

Host fibrinogen drives antimicrobial function in Staphylococcus aureus peritonitis through bacterial-mediated prothrombin activation.

The blood-clotting protein fibrinogen has been implicated in host defense following Staphylococcus aureus infection, but precise mechanisms of host protection and pathogen clearance remain undefined. Peritonitis caused by staphylococci species is a complication for patients with cirrhosis, indwelling catheters, or undergoing peritoneal dialysis. Here, we sought to characterize possible mechanisms of fibrin(ogen)-mediated antimicrobial responses. Wild-type (WT) (Fib+) mice rapidly cleared S. aureus following intraperitoneal infection with elimination of ∼99% of an initial inoculum within 15 min. In contrast, fibrinogen-deficient (Fib-) mice failed to clear the microbe. The genotype-dependent disparity in early clearance resulted in a significant difference in host mortality whereby Fib+ mice uniformly survived whereas Fib- mice exhibited high mortality rates within 24 h. Fibrin(ogen)-mediated bacterial clearance was dependent on (pro)thrombin procoagulant function, supporting a suspected role for fibrin polymerization in this mechanism. Unexpectedly, the primary host initiator of coagulation, tissue factor, was found to be dispensable for this antimicrobial activity. Rather, the bacteria-derived prothrombin activator vWbp was identified as the source of the thrombin-generating potential underlying fibrin(ogen)-dependent bacterial clearance. Mice failed to eliminate S. aureus deficient in vWbp, but clearance of these same microbes in WT mice was restored if active thrombin was administered to the peritoneal cavity. These studies establish that the thrombin/fibrinogen axis is fundamental to host antimicrobial defense, offer a possible explanation for the clinical observation that coagulase-negative staphylococci are a highly prominent infectious agent in peritonitis, and suggest caution against anticoagulants in individuals susceptible to peritoneal infections.

A type VII secretion system of Streptococcus gallolyticus subsp. gallolyticus contributes to gut colonization and the development of colon tumors.

Streptococcus gallolyticus subspecies gallolyticus (Sgg) has a strong clinical association with colorectal cancer (CRC) and actively promotes the development of colon tumors. However, the molecular determinants involved in Sgg pathogenicity in the gut are unknown. Bacterial type VII secretion systems (T7SS) mediate pathogen interactions with their host and are important for virulence in pathogenic mycobacteria and Staphylococcus aureus. Through genome analysis, we identified a locus in Sgg strain TX20005 that encodes a putative type VII secretion system (designated as SggT7SST05). We showed that core genes within the SggT7SST05 locus are expressed in vitro and in the colon of mice. Western blot analysis showed that SggEsxA, a protein predicted to be a T7SS secretion substrate, is detected in the bacterial culture supernatant, indicating that this SggT7SST05 is functional. Deletion of SggT7SST05 (TX20005Δesx) resulted in impaired bacterial adherence to HT29 cells and abolished the ability of Sgg to stimulate HT29 cell proliferation. Analysis of bacterial culture supernatants suggest that SggT7SST05-secreted factors are responsible for the pro-proliferative activity of Sgg, whereas Sgg adherence to host cells requires both SggT7SST05-secreted and bacterial surface-associated factors. In a murine gut colonization model, TX20005Δesx showed significantly reduced colonization compared to the parent strain. Furthermore, in a mouse model of CRC, mice exposed to TX20005 had a significantly higher tumor burden compared to saline-treated mice, whereas those exposed to TX20005Δesx did not. Examination of the Sgg load in the colon in the CRC model suggests that SggT7SST05-mediated activities are directly involved in the promotion of colon tumors. Taken together, these results reveal SggT7SST05 as a previously unrecognized pathogenicity determinant for Sgg colonization of the colon and promotion of colon tumors.

Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group A Streptococcus.

Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIP-seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP-seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.

Staphylococcus epidermidis MSCRAMM SesJ Is Encoded in Composite Islands.

Staphylococcus epidermidis is a leading cause of nosocomial infections in patients with a compromised immune system and/or an implanted medical device. Seventy to 90% of S. epidermidis clinical isolates are methicillin resistant and carry the mecA gene, present in a mobile genetic element (MGE) called the staphylococcal cassette chromosome mec (SCCmec) element. Along with the presence of antibiotic and heavy metal resistance genes, MGEs can also contain genes encoding secreted or cell wall-anchored virulence factors. In our earlier studies of S. epidermidis clinical isolates, we discovered S. epidermidis surface protein J (SesJ), a prototype of a recently discovered subfamily of the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) group. MSCRAMMs are major virulence factors of pathogenic Gram-positive bacteria. Here, we report that the sesJ gene is always accompanied by two glycosyltransferase genes, gtfA and gtfB, and is present in two MGEs, called the arginine catabolic mobile element (ACME) and the staphylococcal cassette chromosome (SCC) element. The presence of the sesJ gene was associated with the left-hand direct repeat DR_B or DR_E. When inserted via DR_E, the sesJ gene was encoded in the SCC element. When inserted via DR_B, the sesJ gene was accompanied by the genes for the type 1 restriction modification system and was encoded in the ACME. Additionally, the SCC element and ACME carry different isoforms of the SesJ protein. To date, the genes encoding MSCRAMMs have been seen to be located in the bacterial core genome. Here, we report the presence of an MSCRAMM in an MGE in S. epidermidis clinical isolates.IMPORTANCES. epidermidis is an opportunistic bacterium that has established itself as a successful nosocomial pathogen. The modern era of novel therapeutics and medical devices has extended the longevity of human life, but at the same time, we also witness the evolution of pathogens to adapt to newly available niches in the host. Increasing antibiotic resistance among pathogens provides an example of such pathogen adaptation. With limited opportunities to modify the core genome, most of the adaptation occurs by acquiring new genes, such as virulence factors and antibiotic resistance determinants present in MGEs. In this study, we describe that the sesJ gene, encoding a recently discovered cell wall-anchored protein in S. epidermidis, is present in both ACME and the SCC element. The presence of virulence factors in MGEs can influence the virulence potential of a specific strain. Therefore, it is critical to study the virulence factors found in MGEs in emerging pathogenic bacteria or strains to understand the mechanisms used by these bacteria to cause infections.

Host-specialized fibrinogen-binding by a bacterial surface protein promotes biofilm formation and innate immune evasion.

Fibrinogen is an essential part of the blood coagulation cascade and a major component of the extracellular matrix in mammals. The interface between fibrinogen and bacterial pathogens is an important determinant of the outcome of infection. Here, we demonstrate that a canine host-restricted skin pathogen, Staphylococcus pseudintermedius, produces a cell wall-associated protein (SpsL) that has evolved the capacity for high strength binding to canine fibrinogen, with reduced binding to fibrinogen of other mammalian species including humans. Binding occurs via the surface-expressed N2N3 subdomains, of the SpsL A-domain, to multiple sites in the fibrinogen α-chain C-domain by a mechanism analogous to the classical dock, lock, and latch binding model. Host-specific binding is dependent on a tandem repeat region of the fibrinogen α-chain, a region highly divergent between mammals. Of note, we discovered that the tandem repeat region is also polymorphic in different canine breeds suggesting a potential influence on canine host susceptibility to S. pseudintermedius infection. Importantly, the strong host-specific fibrinogen-binding interaction of SpsL to canine fibrinogen is essential for bacterial aggregation and biofilm formation, and promotes resistance to neutrophil phagocytosis, suggesting a key role for the interaction during pathogenesis. Taken together, we have dissected a bacterial surface protein-ligand interaction resulting from the co-evolution of host and pathogen that promotes host-specific innate immune evasion and may contribute to its host-restricted ecology.

The Complex Fibrinogen Interactions of the Staphylococcus aureus Coagulases.

The two coagulases, von Willebrand factor binding protein (vWbp) and Coagulase (Coa), are critical virulence factors in several animal models of invasive Staphylococcus aureus (S. aureus) infections. These proteins are part of an intricate system of proteins that S. aureus uses to assemble a fibrinogen (Fg)/fibrin protective shield surrounding itself. This shield allows the microorganism to evade clearance by the host phagocytic cells. The coagulases can non-proteolytically activate the zymogen prothrombin to convert Fg to fibrin and promote the Fg/fibrin shield formation. The coagulases also bind directly to Fg and the interaction between Coa and Fg has been previously characterized in some detail. However, the mechanism(s) by which vWbp interacts with Fg remains unclear. Here, we show that vWbp and Coa have distinct interactions with Fg, despite being structurally similar. Coa binds with a significantly higher affinity to soluble Fg than to Fg coated on a plastic surface, whereas vWbp demonstrates no preference between the two forms of Fg. The two coagulases appear to target different sites on Fg, as they do not compete with each other in binding to Fg. Similar to Coa, both the N- and C-terminal halves of vWbp (vWbp-N, vWbp-C, respectively) harbor Fg-binding activities. The higher affinity Fg-binding activity resides in vWbp-N; whereas, the C-terminal region of Coa encompasses the major Fg-binding activity. Peptides constituting the previously identified Coa/Efb1 Fg-binding motif fail to inhibit vWbp-C from binding to Fg, indicating that vWbp-C lacks a functional homolog to this motif. Interestingly, the N-terminal prothrombin-binding domains of both coagulases recognize the Fg ß-chain, but they appear to interact with different sequence motifs in the host protein. Collectively, our data provide insight into the complex interactions between Fg and the S. aureus coagulases.

Elucidation of Endothelial Cell Hemostatic Regulation with Integrin-Targeting Hydrogels.

Despite advances in the development of materials for cardiovascular devices, current strategies generally lack the thromboresistance of the native endothelium both in terms of efficacy and longevity. To harness this innate hemostatic regulation and improve long-term hemocompatibility, biohybrid devices are designed to promote endothelialization. Much of the research effort to date has focused on the use of extracellular matrix (ECM)-mimics and coatings to promote endothelial cell adhesion and migration with less attention given to the effect of the supported ECM binding events on hemostatic regulation. In this study, we developed integrin-targeted hydrogels to investigate the individual and combined effects of integrin binding events supported by many ECM-based coatings (α1ß1, α2ß1, α5ß1, αvß3). Targeted endothelial cell integrin interactions were first confirmed with antibody blocking studies and then correlated with gene expression of hemostatic regulators and a functional assay of platelet attachment and activation. Surfaces that targeted integrins α1ß1 and α2ß1 resulted in an endothelial cell layer that exhibited a thromboresistant phenotype with an associated reduction in platelet attachment and activation. It is anticipated that identification of specific integrins that promote endothelial cell adhesion as well as thromboresistance will enable the design of cardiovascular materials with improved long-term hemocompatibility.

Influence of collagen-based integrin α1 and α2 mediated signaling on human mesenchymal stem cell osteogenesis in three dimensional contexts.

Collagen I interactions with integrins α1 and α2 are known to support human mesenchymal stem cell (hMSC) osteogenesis. Nonetheless, elucidating the relative impact of specific integrin interactions has proven challenging, in part due to the complexity of native collagen. In the present work, we employed two collagen-mimetic proteins-Scl2-2 and Scl2-3- to compare the osteogenic effects of integrin α1 versus α2 signaling. Scl2-2 and Scl2-3 were both derived from Scl2-1, a triple helical protein lacking known cell adhesion, cytokine binding, and matrix metalloproteinase sites. However, Scl2-2 and Scl2-3 were each engineered to display distinct collagen-based cell adhesion motifs: GFPGER (binding integrins α1 and α2 ) or GFPGEN (binding only integrin α1 ), respectively. hMSCs were cultured within poly(ethylene glycol) (PEG) hydrogels containing either Scl2-2 or Scl2-3 for 2 weeks. PEG-Scl2-2 gels were associated with increased hMSC osterix expression, osteopontin production, and calcium deposition relative to PEG-Scl2-3 gels. These data indicate that integrin α2 signaling may have an increased osteogenic effect relative to integrin α1 . Since p38 is activated by integrin α2 but not by integrin α1 , hMSCs were further cultured in PEG-Scl2-2 hydrogels in the presence of a p38 inhibitor. Results suggest that p38 activity may play a key role in collagen-supported hMSC osteogenesis. This knowledge can be used toward the rational design of scaffolds which intrinsically promote hMSC osteogenesis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2594-2604, 2018.

Collagen-Mimetic Proteins with Tunable Integrin Binding Sites for Vascular Graft Coatings.

Achieving graft endothelialization following implantation continues to be a challenge in the development of "off-the-shelf," small-caliber, arterial prostheses. Coating grafts with biomolecules to support the retention, migration, and differentiation of adherent endothelial precursor cells (EPCs) is a promising approach toward improving graft endothelialization. Designer Collagen Scl2-2 with 1 integrin binding site per strand (DC2-1X) is a Streptococcus pyogenes-derived, collagen-like protein that has previously been evaluated as a graft coating due to its ability to resist platelet aggregation and to promote attachment and migration of "late outgrowth" EPCs (EOCs). However, these prior assessments were performed in the absence of physiological shear. In addition, although DC2-1X coatings supported increased migration rates relative to native collagen coatings, EOC attachment and spreading remained inferior to collagen controls at all DC2-1X concentrations assayed. Thus, the objectives of the present work were the following: (1) to improve EOC attachment on DC2 coatings by modulating the number and spacing of DC2 integrin binding sites (IBS) and (2) to evaluate the retention, migration, and differentiation of adherent EOCs under physiological shear stress. Using single point mutations, three novel DC2 variants were generated containing either two IBS (DC2-2X) or three IBS (DC2-3X1 and DC2-3X2) per strand. After initial evaluation of the potential of each DC2 variant to support increased EOC attachment relative to DC2-1X, DC2-2X and DC2-3X1 coatings were further assessed under physiological shear for their capacity to promote EOC retention, migration, and differentiation relative to DC2-1X and collagen controls. An increase in the number of IBS from 1 to 3 significantly improved EOC retention on DC2 coatings while also supporting increased average migration rates. Moreover, EOCs on DC2-3X1 coatings showed increased gene-level expression of intermediate endothelial cell differentiation markers relative to collagen. Overall, the current results suggest that DC2-3X1 warrants further investigation as a vascular graft coating.

Amino acid polymorphisms in the fibronectin-binding repeats of fibronectin-binding protein A affect bond strength and fibronectin conformation.

The Staphylococcus aureus cell surface contains cell wall-anchored proteins such as fibronectin-binding protein A (FnBPA) that bind to host ligands (e.g. fibronectin; Fn) present in the extracellular matrix of tissue or coatings on cardiac implants. Recent clinical studies have found a correlation between cardiovascular infections caused by S. aureus and nonsynonymous SNPs in FnBPA. Atomic force microscopy (AFM), surface plasmon resonance (SPR), and molecular simulations were used to investigate interactions between Fn and each of eight 20-mer peptide variants containing amino acids Ala, Asn, Gln, His, Ile, and Lys at positions equivalent to 782 and/or 786 in Fn-binding repeat-9 of FnBPA. Experimentally measured bond lifetimes (1/koff) and dissociation constants (Kd = koff/kon), determined by mechanically dissociating the Fn·peptide complex at loading rates relevant to the cardiovascular system, varied from the lowest-affinity H782A/K786A peptide (0.011 s, 747 µm) to the highest-affinity H782Q/K786N peptide (0.192 s, 15.7 µm). These atomic force microscopy results tracked remarkably well to metadynamics simulations in which peptide detachment was defined solely by the free-energy landscape. Simulations and SPR experiments suggested that an Fn conformational change may enhance the stability of the binding complex for peptides with K786I or H782Q/K786I (Kdapp = 0.2-0.5 µm, as determined by SPR) compared with the lowest-affinity double-alanine peptide (Kdapp = 3.8 µm). Together, these findings demonstrate that amino acid substitutions in Fn-binding repeat-9 can significantly affect bond strength and influence the conformation of Fn upon binding. They provide a mechanistic explanation for the observation of nonsynonymous SNPs in fnbA among clinical isolates of S. aureus that cause endovascular infections.