Characterization of the immune response to collagen-like proteins Scl1 and Scl2 of serotype M1 and M28 group A Streptococcus

Group A Streptococcus (GAS) infections may trigger autoimmune sequelae thought to involve streptococcal antibodies cross-reactive to human antigens. Here, the antigenicity of the streptococcal collagen-like (CL) proteins, Scl1 and Scl2, that exhibit structural similarity to human collagen, was analyzed. Antibodies to Scl1.1 protein were detected in human sera from healthy individuals previously infected with M1 GAS and from patients with various GAS infections, as well as in sera from mice infected with M1 GAS. Linear B-cell epitope mapping identified immunoreactive peptides corresponding to the CL region of Scl1.1. Humoral responses to Scl1.28 and Scl2.28 were also detected in pediatric patients and mice infected with M28 GAS.     

An Engineered α1 Integrin-binding Collagenous Sequence

Collagen is an extracellular matrix structural component that can regulate cellular processes through its interaction with the integrins, α1β1, α2β1, α10β1, and α11β1. Collagen-like proteins have been identified in a number of bacterial species. Here, we used Scl2 from Streptococcus pyogenes serotype M28 strain MGAS6274 as a backbone for the introduction of discrete integrin-binding sequences. The introduced sequences GLPGER, GFPGER, or GFPGEN did not affect triple helix stability of the Scl (Streptococcal collagen-like) protein. Using ELISA and surface plasmon resonance, we determined that Scl2_GLPGER and Scl2_GFPGER bound to recombinant human α1 and α2 I-domains in a metal ion-dependent manner and without a requirement for hydroxyproline. We predicted a novel and selective integrin-binding sequence, GFPGEN, through the use of computer modeling and demonstrated that Scl2_GFPGEN shows specificity toward the α1 I-domain and does not bind the α2 I-domain. Using C2C12 cells, we determined that intact integrins interact with the modified Scl2 proteins with the same selectivity as recombinant I-domains. These modified Scl2 proteins also acted as cell attachment substrates for fibroblast, endothelial, and smooth muscle cells. However, the modified Scl2 proteins were unable to aggregate platelets. These results indicate that Scl2 is a suitable backbone for the introduction of mammalian integrin-binding sequences, and these sequences may be manipulated to individually target α1β1 and α2β1.     

Net sodium fluxes change significantly at anatomically distinct root zones of rice (Oryza sativa L.) seedlings

Casparian bands of endodermis and exodermis play crucial roles in blocking apoplastic movement of ions and water into the stele of roots through the cortex. These apoplastic barriers differ considerably in structure and function along the developing root. The present study assessed net Na⁺ fluxes in anatomically distinct root zones of rice seedlings and analyzed parts of individual roots showing different Na⁺ uptake. The results indicated that anatomically distinct root zones contributed differently to the overall uptake of Na⁺. The average Na⁺ uptake in root zones in which Casparian bands of the endo- and exo-dermis were interrupted by initiating lateral root primordia (root zone III) was significantly greater than that at the root apex, where Casparian bands were not yet formed (root zone I), or in the region where endo- and exo-dermis with Casparian bands were well developed (root zone II). The measurement of net Na⁺ fluxes using a non-invasive scanning ion-selective electrode technique (SIET) demonstrated that net Na⁺ flux varied significantly in different positions along developing rice roots, and a net Na⁺ influx was obvious at the base of young lateral root primordia. Since sodium fluxes changed significantly along developing roots of rice seedlings, we suggest that the significantly distinct net Na⁺ flux profile may be attributed to different apoplastic permeability due to lateral root primordia development for non-selective apoplastic bypass of ions along the apoplast.     

Protein-protein interactions of hCsl4p with other human exosome subunits.

The exosome is a complex of 3'-->5' exoribonucleases, which functions in a variety of cellular processes, all requiring the processing or degradation of RNA. We demonstrate that the two human proteins hCsl4p and hRrp42p, which have been identified on the basis of their sequence homology with Saccharomyces cerevisiae proteins, are associated with the human exosome. By mammalian two-hybrid and GST pull-down assays, we show that the hCsl4p protein interacts directly with two other exosome proteins, hRrp42p and hRrp46p. Mutants of hCsl4p that fail to interact with either hRrp42p or hRrp46p are also not able to associate with exosome complexes in vivo. These results indicate that the association of hCsl4p with the exosome is mediated by protein-protein interactions with hRrp42p and hRrp46p.     

Protein-protein interactions between human exosome components support the assembly of RNase PH-type subunits into a six-membered PNPase-like ring

The exosome is a complex of 3'-->5' exoribonucleases, which functions in a variety of cellular processes, all requiring the processing or degradation of RNA. Here we present a model for the assembly of the six human RNase PH-like exosome subunits into a hexameric ring structure. In part, this structure is on the basis of the evolutionarily related bacterial degradosome, the core of which consists of three copies of the PNPase protein, each containing two RNase PH domains. In our model three additional exosome subunits, which contain S1 RNA-binding domains, are positioned on the outer surface of this ring. Evidence for this model was obtained by the identification of protein-protein interactions between individual exosome subunits in a mammalian two-hybrid system. In addition, the results of co-immunoprecipitation assays indicate that at least two copies of hRrp4p and hRrp41p are associated with a single exosome, suggesting that at least two of these ring structures are present in this complex. Finally, the identification of a human gene encoding the putative human counterpart of the bacterial PNPase protein is described, which suggests that the exosome is not the eukaryotic equivalent of the bacterial degradosome, although they do share similar functional activities.     

Update on Wnt signaling in bone cell biology and bone disease

For more than a decade, Wnt signaling pathways have been the focus of intense research activity in bone biology laboratories because of their importance in skeletal development, bone mass maintenance, and therapeutic potential for regenerative medicine. It is evident that even subtle alterations in the intensity, amplitude, location, and duration of Wnt signaling pathways affects skeletal development, as well as bone remodeling, regeneration, and repair during a lifespan. Here we review recent advances and discrepancies in how Wnt/Lrp5 signaling regulates osteoblasts and osteocytes, introduce new players in Wnt signaling pathways that have important roles in bone development, discuss emerging areas such as the role of Wnt signaling in osteoclastogenesis, and summarize progress made in translating basic studies to clinical therapeutics and diagnostics centered around inhibiting Wnt pathway antagonists, such as sclerostin, Dkk1 and Sfrp1. Emphasis is placed on the plethora of genetic studies in mouse models and genome wide association studies that reveal the requirement for and crucial roles of Wnt pathway components during skeletal development and disease.     

Temporal regulation of Cre-recombinase activity in Scl-positive neurons of the central nervous system

The Cre/LoxP system provides a powerful tool to investigate gene function in vivo. This system requires Cre-recombinase expressing mouse lines that permit control of gene recombination in a tissue-specific and time-dependent manner. To allow spatio-temporal gene deletion in specific central nervous system (CNS) neuronal populations, we generated mice with a tamoxifen-inducible Cre (Cre-ER(T)) transgene under control of the Scl/Tal1 neural promoter/enhancer -0.9E3 (-0.9E3CreER(T) transgenic mice). Using Cre-reporter mice we have shown that tamoxifen-mediated Cre-ER(T) recombination in -0.9E3CreER(T) mice recapitulated the anticipated expression pattern of Scl in the caudal thalamus, midbrain, hindbrain, and spinal cord. Cre-mediated recombination was also effectively induced during embryogenesis and marked the same population of neurons as observed in the adult. Additionally, we identified a tamoxifen-independent constitutively active -0.9E3CreER(T) mouse line that will be useful for gene deletion during early neurogenesis. These -0.9E3CreER(T) mice will provide tools to investigate the role of neuronal genes in the developing and mature CNS. CNS.