Structural basis of bacterial defense against g-type lysozyme-based innate immunity

Gram-negative bacteria can produce specific proteinaceous inhibitors to defend themselves against the lytic action of host lysozymes. So far, four different lysozyme inhibitor families have been identified. Here, we report the crystal structure of the Escherichia coli periplasmic lysozyme inhibitor of g-type lysozyme (PliG-Ec) in complex with Atlantic salmon g-type lysozyme (SalG) at a resolution of 0.95 Å, which is exceptionally high for a complex of two proteins. The structure reveals for the first time the mechanism of g-type lysozyme inhibition by the PliG family. The latter contains two specific conserved regions that are essential for its inhibitory activity. The inhibitory complex formation is based on a double ‘key-lock’ mechanism. The first key-lock element is formed by the insertion of two conserved PliG regions into the active site of the lysozyme. The second element is defined by a distinct pocket of PliG accommodating a lysozyme loop. Computational analysis indicates that this pocket represents a suitable site for small molecule binding, which opens an avenue for the development of novel antibacterial agents that suppress the inhibitory activity of PliG.     

Ablation of cyclase-associated protein 2 (CAP2) leads to cardiomyopathy

Cyclase-associated proteins are highly conserved proteins that have a role in the regulation of actin dynamics. Higher eukaryotes have two isoforms, CAP1 and CAP2. To study the in vivo function of CAP2, we generated mice in which the CAP2 gene was inactivated by a gene-trap approach. Mutant mice showed a decrease in body weight and had a decreased survival rate. Further, they developed a severe cardiac defect marked by dilated cardiomyopathy (DCM) associated with drastic reduction in basal heart rate and prolongations in atrial and ventricular conduction times. Moreover, CAP2-deficient myofibrils exhibited reduced cooperativity of calcium-regulated force development. At the microscopic level, we observed disarrayed sarcomeres with development of fibrosis. We analyzed CAP2’s role in actin assembly and found that it sequesters G-actin and efficiently fragments filaments. This activity resides completely in its WASP homology domain. Thus CAP2 is an essential component of the myocardial sarcomere and is essential for physiological functioning of the cardiac system, and a deficiency leads to DCM and various cardiac defects.     

Vaspin inhibits kallikrein 7 by serpin mechanism

The molecular target of the adipokine vaspin (visceral adipose tissue-derived serpin; serpinA12) and its mode of action are unknown. Here, we provide the vaspin crystal structure and identify human kallikrein 7 (hK7) as a first protease target of vaspin inhibited by classical serpin mechanism with high specificity in vitro. We detect vaspin–hK7 complexes in human plasma and find co-expression of both proteins in murine pancreatic β-cells. We further demonstrate that hK7 cleaves human insulin in the A- and B-chain. Vaspin treatment of isolated pancreatic islets leads to increased insulin concentration in the media upon glucose stimulation without influencing insulin secretion. By application of vaspin and generated inactive mutants, we find the significantly improved glucose tolerance in C57BL/6NTac and db/db mice treated with recombinant vaspin fully dependent on the vaspin serpin activity and not related to vaspin-mediated changes in insulin sensitivity as determined by euglycemic-hyperinsulinemic clamp studies. Improved glucose metabolism could be mediated by increased insulin plasma concentrations 150 min after a glucose challenge in db/db mice, supporting the hypothesis that vaspin may inhibit insulin degradation by hK7 in the circulation. In conclusion, we demonstrate the inhibitory serpin nature and the first protease target of the adipose tissue-derived serpin vaspin, and our findings suggest hK7 inhibition by vaspin as an underlying physiological mechanism for its compensatory actions on obesity-induced insulin resistance.     

The role of estrogen receptor α in the regulation of bone and growth plate cartilage

Estrogens are important endocrine regulators of skeletal growth and maintenance in both females and males. Studies have demonstrated that the estrogen receptor (ER)-α is the main mediator of these estrogenic effects in bone. Therefore, estrogen signaling via ERα is a target both for affecting longitudinal bone growth and bone remodeling. However, treatment with estradiol (E2) leads to an increased risk of side effects such as venous thromboembolism and breast cancer. Thus, an improved understanding of the signaling pathways of ERα will be essential in order to find better bone specific treatments with minimal adverse effects for different estrogen-related bone disorders. This review summarizes the recent data regarding the intracellular signaling mechanisms, in vivo, mediated by the ERα activation functions (AFs), AF-1 and AF-2, and the effect on bone, growth plate and other estrogen responsive tissues. In addition, we review the recent cell-specific ERα-deleted mouse models lacking ERα specifically in neuronal cells or growth plate cartilage. The newly characterized signaling pathways of estrogen, described in this review, provide a better understanding of the ERα signaling pathways, which may facilitate the design of new, bone-specific treatment strategies with minimal adverse effects.     

Anti-apoptotic mechanisms of HIV: lessons and novel approaches to curing HIV

Past efforts at curing infection with the human immunodeficiency virus (HIV) have been blocked by the resistance of some infected cells to viral cytopathic effects and the associated development of a latent viral reservoir. Furthermore, current efforts to clear the viral reservoir by means of reactivating latent virus are hampered by the lack of cell death in the newly productively infected cells. The purpose of this review is to describe the many anti-apoptotic mechanisms of HIV, as well as the current limitations in the field. Only by understanding how infected cells avoid HIV-induced cell death can an effective strategy to kill infected cells be developed.     

Some surprising facts about (the problem of) surprising facts: (from the Dusseldorf Conference,February 2011)

A common intuition about evidence is that if data x have been used to construct a hypothesis H, then x should not be used again in support of H. It is no surprise that x fits H, if H was deliberately constructed to accord with x. The question of when and why we should avoid such “double-counting” continues to be debated in philosophy and statistics. It arises as a prohibition against data mining, hunting for significance, tuning on the signal, and ad hoc hypotheses, and as a preference for predesignated hypotheses and “surprising” predictions. I have argued that it is the severity or probativeness of the test—or lack of it—that should determine whether a double-use of data is admissible. I examine a number of surprising ambiguities and unexpected facts that continue to bedevil this debate.     

The FHIT gene product: tumor suppressor and genome “caretaker”

The FHIT gene at FRA3B is one of the earliest and most frequently altered genes in the majority of human cancers. It was recently discovered that the FHIT gene is not the most fragile locus in epithelial cells, the cell of origin for most Fhit-negative cancers, eroding support for past claims that deletions at this locus are simply passenger events that are carried along in expanding cancer clones, due to extreme vulnerability to DNA damage rather than to loss of FHIT function. Indeed, recent reports have reconfirmed FHIT as a tumor suppressor gene with roles in apoptosis and prevention of the epithelial–mesenchymal transition. Other recent works have identified a novel role for the FHIT gene product, Fhit, as a genome “caretaker.” Loss of this caretaker function leads to nucleotide imbalance, spontaneous replication stress, and DNA breaks. Because Fhit loss-induced DNA damage is “checkpoint blind,” cells accumulate further DNA damage during subsequent cell cycles, accruing global genome instability that could facilitate oncogenic mutation acquisition and expedite clonal expansion. Loss of Fhit activity therefore induces a mutator phenotype. Evidence for FHIT as a mutator gene is discussed in light of these recent investigations of Fhit loss and subsequent genome instability.     

A tale of tails: deciphering the contribution of terminal tails to the biochemical properties of two Dps proteins from Streptomyces coelicolor

Dps proteins are members of an extensive family of proteins that oxidise and deposit iron in the form of ferric oxide, and are also able to bind DNA. Ferroxidation centres are formed at the interface of anti-parallel dimers, which further assemble into dodecameric nanocages with a hollow core where ferric oxide is deposited. Streptomyces coelicolor encodes three Dps-like proteins (DpsA, B and C). Despite sharing the conserved four-helix bundle organisation observed in members of the Dps family, they display significant differences in the length of terminal extensions, or tails. DpsA possess both N- and C-terminal tails of different lengths, and their removal affects quaternary structure assembly to varying degrees. DpsC quaternary structure, on the other hand, is heavily dependent on its N-terminal tail as its removal abolishes correct protein folding. Analysis of the crystal structure of dodecamers from both proteins revealed remarkable differences in the position of tails and interface surface area; and provides insight to explain the differences in biochemical behaviour observed while comparing DpsA and DpsC.     

Platelet-derived growth factor receptor beta identifies mesenchymal stem cells with enhanced engraftment to tissue injury and pro-angiogenic property

Mesenchymal stem cells (MSCs) are heterogeneous likely consisting of subpopulations with various therapeutic potentials. Here we attempted to acquire a subset of MSCs with enhanced effect in wound healing. We found that human placental MSCs expressing platelet-derived growth factor (PDGF) receptor (PDGFR)-β exhibited greater proliferation rates and generated more colony-forming unit-fibroblast (CFU-F), compared to PDGFR-β^? MSCs. Notably, PDGFR-β⁺ MSCs expressed higher levels of pro-angiogenic factors such as Ang1, Ang2, VEGF, bFGF and PDGF. When 10⁶ GFP-expressing MSCs were topically applied into excisional wounds in mice, PDGFR-β⁺ MSCs actively incorporated into the wound tissue, resulting in enhanced engraftment (3.92 ± 0.31 × 10⁵ remained in wound by 7 days) and accelerated wound closure; meanwhile, PDGFR-β^? MSCs tended to remain on the top of the wound bed with significantly fewer cells (2.46 ± 0.26 × 10⁵) engrafted into the wound, suggesting enhanced chemotactic migration and engraftment of PDGFR-β⁺ MSCs into the wound. Real-Time PCR and immunostain analyses revealed that the expression of PDGF-B was upregulated after wounding; transwell migration assay showed that PDGFR-β⁺ MSCs migrated eightfold more than PDGFR-β^? MSCs toward PDGF-BB. Intriguingly, PDGFR-β⁺ MSC-treated wounds showed significantly enhanced angiogenesis compared to PDGFR-β^? MSC- or vehicle-treated wounds. Thus, our results indicate that PDGFR-β identifies a subset of MSCs with enhanced chemotactic migration to wound injury and effect in promoting angiogenesis and wound healing, implying a greater therapeutic potential for certain diseases.