The molecular scaffold Gab2 is a crucial component of RANK signaling and osteoclastogenesis

Morphogenesis and remodeling of bone involve synthesis of bone matrix by osteoblasts and coordinate resorption of bone by osteoclasts. Defective bone remodeling caused by altered osteoclast activity underlies a multitude of osteopenic disorders. Receptor activator of NF-kappaB (RANK) and its ligand RANKL have been identified as essential factors involved in osteoclast development and bone remodeling, but their mechanism and interacting factors have not been fully characterized. Here we report that the molecular adapter Grb-2-associated binder-2 (Gab2) associates with RANK and mediates RANK-induced activation of NF-kappaB, Akt and Jnk. Inactivation of the gene encoding Gab2 in mice results in osteopetrosis and decreased bone resorption as a result of defective osteoclast differentiation. We also show that Gab2 has a crucial role in the differentiation of human progenitor cells into osteoclasts. We have thus identified a new, key regulatory scaffold molecule, Gab2, that controls select RANK signaling pathways and is essential for osteoclastogenesis and bone homeostasis.     

Improved absorption of caseinophosphopeptide-bound iron: role of alkaline phosphatase

Hydrolysis of proteins could lessen their inhibiting effect on the poor absorption of cow's milk iron (Fe), which is responsible for the high incidence of Fe deficiency worldwide. When bound to Fe, caseinophosphopeptides (CPP) derived from milk proteins resist luminal digestion, enhance Fe solubility and could improve its bioavailability; brush border enzyme alkaline phosphatase activity could influence iron absorption by releasing free Fe; this study assessed its role in the absorption of CPP-bound Fe. Rat duodenal loops were perfused with Fe gluconate or Fe bound to the CPP of beta casein [beta-CN (1-25)], with or without the addition of an inhibitor of alkaline phosphatase, Na2WO4. The uptake of Fe-beta-CN (1-25) was greater than Fe gluconate. Na2WO4 enhanced the uptake of Fe-beta-CN (1-25) and not of Fe gluconate. So the release of free, insoluble Fe, by alkaline phosphatase seems to be prevented by providing Fe in the Fe-beta-CN (1-25) complex form. Its good disappearance rate makes beta-CN (1-25)-bound Fe a candidate for food fortification.     

Invasive recordings from the human brain: clinical insights and beyond

Although non-invasive methods such as functional magnetic resonance imaging, electroencephalograms and magnetoencephalograms provide most of the current data about the human brain, their resolution is insufficient to show physiological processes at the cellular level. Clinical approaches sometimes allow invasive recordings to be taken from the human brain, mainly in patients with epilepsy or with movement disorders, and such recordings can sample neural activity at spatial scales ranging from single cells to distributed cell assemblies. In addition to their clinical relevance, these recordings can provide unique insights into brain functions such as movement control, perception, memory, language and even consciousness.     

Targeting IL-15 receptor-bearing cells with an antagonist mutant IL-15/Fc protein prevents disease development and progression in murine collagen-induced arthritis

It has been suggested that the inflammatory cytokine IL-15 plays an important role in the development of several autoimmune diseases, including rheumatoid arthritis. We have generated a unique lytic and antagonistic IL-15 mutant/Fcgamma2a fusion protein (CRB-15) that targets the IL-15R. In the present study we examined the effects of targeting the IL-15R on the prevention and treatment of collagen-induced arthritis (CIA) in mice and probed the possible mechanisms of action of this IL-15 mutant/Fcgamma2a protein. Upon immunization with type II collagen, DBA/1 mice develop severe articular inflammation and destruction. Treatment of DBA/1 mice with a brief course of CRB-15 at the time of type II collagen challenge markedly inhibited the incidence and severity of arthritis. Moreover, in animals with ongoing established arthritis, treatment with CRB-15 effectively blocked disease progression compared with that in control-treated animals. The therapeutic effect of CRB-15 on either disease development or disease progression is remarkably stable, because withdrawal of treatment did not lead to disease relapse. A detailed analysis revealed that treatment with CRB-15 decreased synovitis in the joints; reduced bone erosion and cartilage destruction; reduced in situ production of the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-17; and decreased the responder frequency of autoreactive T cells. Our study suggests that the effective targeting of IL-15R-triggered events with CRB-15 can be of therapeutic importance in the treatment of rheumatoid arthritis.     

Inhibition of protein-protein interactions: the discovery of druglike beta-catenin inhibitors by combining virtual and biophysical screening

The interaction between beta-catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 A(2)), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain "hot spots," small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4-binding surface on beta-catenin contains a well-defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to beta-catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4-competitive compounds with the tightest binder having a K(D) of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein-protein interactions.     

WT p53, but not tumor-derived mutants, bind to Bcl2 via the DNA binding domain and induce mitochondrial permeabilization

The induction of apoptosis by p53 in response to cellular stress is its most conserved function and crucial for p53 tumor suppression. We recently reported that p53 directly induces oligomerization of the BH1,2,3 effector protein Bak, leading to outer mitochondrial membrane permeabilization (OMMP) with release of apoptotic activator proteins. One important mechanism by which p53 achieves OMMP is by forming an inhibitory complex with the anti-apoptotic BclXL protein. In contrast, the p53 complex with the Bcl2 homolog has not been interrogated. Here we have undertaken a detailed characterization of the p53-Bcl2 interaction using structural, biophysical, and mutational analyses. We have identified the p53 DNA binding domain as the binding interface for Bcl2 using solution NMR. The affinity of the p53-Bcl2 complex was determined by surface plasmon resonance analysis (BIAcore) to have a dominant component KD 535 +/- 24 nm. Moreover, in contrast to wild type p53, endogenous missense mutants of p53 are unable to form complexes with endogenous Bcl2 in human cancer cells. Functionally, these mutants are all completely or strongly compromised in mediating OMMP, as measured by cytochrome c release from isolated mitochondria. These data implicate p53-Bcl2 complexes in contributing to the direct mitochondrial p53 pathway of apoptosis and further support the notion that the DNA binding domain of p53 is a dual function domain, mediating both its transactivation function and its direct mitochondrial apoptotic function.     

Differential induction of connexins 26 and 30 in skin tumors and their adjacent epidermis.

Gap junctions (GJs) have been shown to play a role in tumor progression including a variety of keratinocyte-derived and non-keratinocyte-derived skin tumors. Here we show that the synthesis of the GJ proteins connexin 26 and connexin 30 (Cx26 and Cx30) is induced in keratinocyte-derived epithelial skin tumors whereas there is either no change or a downregulation of Cx43. Cx26, Cx30, and Cx43 are absent in non-epithelial skin tumors. Further, Cx26 and Cx30 are induced in the epidermis adjacent to malignant melanoma but absent in the epidermis adjacent to benign non-epithelial skin lesions (melanocytic nevi and angioma). The keratinocyte-derived skin tumors are very heterogeneous regarding the Cx26/Cx30 pattern in the epidermis at the periphery of the tumors. We did not observe any difference in the localization of the very similar proteins Cx26 and Cx30 but a variation in intensity of immunoreactivity. As the staining patterns of Cx26 and Cx30 antibodies are not identical to those of CK6, a marker for hyperproliferation, and CK17, a marker for trauma, we discuss that the induction of these gap junctional proteins exceeds a reflection of reactive hyperproliferative or traumatized epidermis. We further discuss the putative roles of these gap junctional proteins in tumor progression.