Coupled motions direct electrons along human microsomal P450 Chains

Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR) to all microsomal cytochrome P450s (CYPs). Our hypothesis is that tight coupling of motion with enzyme chemistry can signal "ready and waiting" states for electron transfer from CPR to downstream CYPs and support vectorial electron transfer across complex redox chains. We developed a novel approach to study the time-dependence of dynamical change during catalysis that reports on the changing conformational states of CPR. FRET was linked to stopped-flow studies of electron transfer in CPR that contains donor-acceptor fluorophores on the enzyme surface. Open and closed states of CPR were correlated with key steps in the catalytic cycle which demonstrated how redox chemistry and NADPH binding drive successive opening and closing of the enzyme. Specifically, we provide evidence that reduction of the flavin moieties in CPR induces CPR opening, whereas ligand binding induces CPR closing. A dynamic reaction cycle was created in which CPR optimizes internal electron transfer between flavin cofactors by adopting closed states and signals "ready and waiting" conformations to partner CYP enzymes by adopting more open states. This complex, temporal control of enzyme motion is used to catalyze directional electron transfer from NADPH→FAD→FMN→heme, thereby facilitating all microsomal P450-catalysed reactions. Motions critical to the broader biological functions of CPR are tightly coupled to enzyme chemistry in the human NADPH-CPR-CYP redox chain. That redox chemistry alone is sufficient to drive functionally necessary, large-scale conformational change is remarkable. Rather than relying on stochastic conformational sampling, our study highlights a need for tight coupling of motion to enzyme chemistry to give vectorial electron transfer along complex redox chains.     

Structure and function of intercellular junctions in human cervical and vaginal mucosal epithelia

The mucosal epithelium is a major portal for microbial invasion. Mucosal barrier integrity is maintained by the physical interactions of intercellular junctional molecules on opposing epithelial cells. The epithelial mucosa in the female reproductive tract provides the first line of defense against sexually transmitted pathogenic bacteria and viruses, but little is known concerning the structure and molecular composition of epithelial junctions at this site. In the present study, the distribution of tight, adherens, and desmosomal junctions were imaged in the human endocervix (columnar epithelium) and ectocervix (stratified squamous epithelium) by electron microscopy, and permeability was assessed by tracking the penetration of fluorescent immunoglobulin G (IgG). To further define the molecular structure of the intercellular junctions, select junctional molecules were localized in the endocervical, ectocervical, and vaginal epithelium by fluorescent immunohistology. The columnar epithelial cells of the endocervix were joined by tight junctions that excluded apically applied fluorescent IgG. In contrast, the most apical layers of the ectocervical stratified squamous epithelium did not contain classical cell-cell adhesions and were permeable to IgG. The suprabasal and basal epithelial layers in ectocervical and vaginal tissue contained the most robust adhesions; molecules characteristic of exclusionary junctions were detected three to four cellular layers below the luminal surface and extended to the basement membrane. These data indicate that the uppermost epithelial layers of the ectocervix and vagina constitute a unique microenvironment; their lack of tight junctions and permeability to large-molecular-weight immunological mediators suggest that this region is an important battlefront in host defense against microbial pathogens.     

Allosteric Mechanism Controls Traffic in the Chaperone/Usher Pathway

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Highlights? Allosteric mechanism controls traffic in the chaperone/usher pathway ? Usher-binding site in free chaperone is conformationally locked ? Subunit binding opens the lock in the chaperone, allowing usher targeting     

Postnatal differentiation of the gametogenic and endocrine functions of the testis in the tree-shrew (Tupaia belangeri)

Summary Testicular development was studied in Tupaia belangeri (tree-shrew) from birth to sexual maturity. At birth the seminiferous cords contained peripheral supporting cells and centrally located gonocytes. Large foetal Leydig cells were prominent in the interstitium. The mitotic index of the gonocytes was low at birth and rose to peak levels at Day 20, following the regression of the foetal generation of Leydig cells, and during the nadir in circulating testosterone concentrations. Mitotic activity returned to low levels at Day 30 in association with the reappearance of differentiated Leydig cells and the first signs of increased androgenesis. The negative temporal relationship between mitogenesis and androgenic function suggests that the proliferation of the gonocytes does not require, and may be inhibited by, high titres of androgens. Post-mitotic development of the gonocytes occurred during a period of rising testosterone levels, and the first appearance of spermatogonia coincided with peak testosterone levels. This indicates that androgens may be specifically involved in the initiation of spermatogenesis. Spermatogenesis progressed to completion during a phase of declining testosterone levels. The precise temporal correlations established during post-natal development suggest that the tree-shrew is a suitable animal model for studies on the endocrine control of the initiation of spermatogenesis in primates.     

The physico-chemical characterization of a boiling stable antifreeze protein from a perennial grass (Lolium perenne)

We have characterized a cold-induced, boiling stable antifreeze protein. This highly active ice recrystallization inhibition protein shows a much lower thermal hysteresis effect and displays binding behavior that is uncharacteristic of any AFP from fish or insects. Ice-binding studies show it binds to the (1 0 1 0) plane of ice and FTIR studies reveal that it has an unusual type of highly beta-sheeted secondary structure. Ice-binding studies of both glycosylated and nonglycosylated expressed forms indicate that it adsorbs to ice through the protein backbone. These results are discussed in light of the currently proposed mechanisms of AFP action.     

RNF121 Inhibits Angiogenic Growth Factor Signaling by Restricting Cell Surface Expression of VEGFR‐2

Ligand stimulation promotes downregulation of RTKs, a mechanism by which RTKs, through the ubiquitination pathway are removed from the cell surface, causing a temporary termination of RTK signaling. The molecular mechanisms governing RTK trafficking and maturation in the endoplasmic reticulum (ER)/Golgi compartments are poorly understood. Vascular endothelial growth factor receptor‐2 (VEGFR‐2) is a prototypic RTK that plays a critical role in physiologic and pathologic angiogenesis. Here we demonstrate that Ring Finger Protein 121 (RNF121), an ER ubiquitin E3 ligase, is expressed in endothelial cells and regulates maturation of VEGFR‐2. RNF121 recognizes newly synthesized VEGFR‐2 in the ER and controls its trafficking and maturation. Over‐expression of RNF121 promoted ubiquitination of VEGFR‐2, inhibited its maturation and resulted a significantly reduced VEGFR‐2 presence at the cell surface. Conversely, the shRNA‐mediated knockdown of RNF121 in primary endothelial cells reduced VEGFR‐2 ubiquitination and increased its cell surface level. The RING Finger domain of RNF121 is required for its activity toward VEGFR‐2, as its deletion significantly reduced the effect of RNF121 on VEGFR‐2. Additionally, RNF121 inhibited VEGF‐induced endothelial cell proliferation and angiogenesis. Taken together, these data identify RNF121 as a key determinant of angiogenic signaling that restricts VEGFR‐2 cell surface presence and its angiogenic signaling.      

Organization of interstitial tissue in the testis of the salamander Necturus maculosus (Caudata: Proteidae)

In Necturus maculosus the organization of the interstitial tissue varies according to the stage of spermatogenesis. Leydig cells at various stages of differentiation and myoid cells are always present in this tissue. The Leydig cells are undifferentiated at all phases of germ cell activity and only hypertrophy following spermiation and degeneration of Sertoli cells. These Leydig cells are structurally analogous to mammalian Leydig cells. They do not form part of the lamina propria of the seminiferous lobules and hence cannot be referred to as lobule-boundary cells previously described in the urodele testis (Lofts, '74). When the Leydig cells hypertrophy, numerous unmyelinated axons appear in the interstitial tissue. These axons, often devoid of Schwann-cell cytoplasm, occur in close proximity to Leydig cells. Because the levels of both Substance P and neurotensin increased in the testis of Necturus maculosus as Leydig cells differentiated, we concluded that these neural elements may regulate Leydig-cell function locally, through the release of neuropeptides.     

Individual optimization: Mechanisms shaping the optimal reaction norm

In general, optimal reaction norms in heterogeneous populations can be obtained only by iterative numerical procedures (McNamara, 1991; Kawecki and Stearns, 1993). We consider two particular, but biologically plausible and analytically tractable cases of individual optimization to gain insight into the mechanisms which shape the optimal reaction norm of fecundity in relation to an environmental variable or an individual trait. In the first case, we assume that the quality of the environment (e.g. food abundance) or the quality of the individual (e.g. body size) is fixed during its entire life; it may also be a heritable individual trait. In the second case, individual quality is assumed to change randomly such that the probability distribution of quality in the next year is the same for the parent and for her offspring. For these two cases, we obtain analytical expressions for the shape of the optimal reaction norm, which are heuristically interpretable in terms of underlying selective mechanisms. It is shown that better quality may reduce the optimal fecundity. This outcome is particularly likely if better quality increases a fecundity-independent factor of parental survival in a long-lived species with fixed quality. This revised version was published online in July 2006 with corrections to the Cover Date.