Mechanisms and cellular functions of intramembrane proteases

The turn of the millennium coincided with the branding of a fundamentally different class of enzyme - proteases that reside immersed inside the membrane. This new field was the convergence of completely separate lines of research focused on cholesterol homeostasis, Alzheimer's disease, and developmental genetics. None intended their ultimate path, but soon became a richly-integrated fabric for an entirely new field: regulated intramembrane proteolysis. Our aim in this Special Issue is to focus on the ancient and nearly ubiquitous enzymes that catalyze this unexpected yet important reaction. The pace of progress has been dramatic, resulting in a rapidly-expanding universe of known cellular functions, and a paradigm shift in the biochemical understanding of these once heretical enzymes. More recently, the first therapeutic successes have been attained by targeting an intramembrane protease. We consider these advances and identify oncoming opportunities in four parts: growing spectra of cellular roles, insights into biochemical mechanisms, therapeutic strategies, and newly-emerging topics. Recent studies also expose challenges for the future, including non-linear relationships between substrate identification and physiological functions, and the need for potent and specific, not broad-class, inhibitors.     

Swelling pressure of the inervertebral disc: influence of proteoglycan and collagen contents

In this study we have considered how equilibrium water content of the human nucleus pulposus varies with applied pressure for discs of various spinal levels and of various ages. In all cases hydration decreased as pressure increased but the level of equilibrium hydration depended on the relative amounts of collagen and PG in the tissue. Provided we accounted for the exclusion of PGs from the intra-fibrillar space, the swelling pressure curve and the osmotic pressure curve of equivalent PGs were found to coincide. The result implies that under physiological hydrations the mechanical forces exerted by the collagen network of the nucleus are insignificant and that the osmotic pressure of the proteoglycans is balanced by the applied pressure arising from body weight and muscle and ligament tension alone. Since aged discs often have a low proteoglycan to collagen ratio, their equilibrium hydration also tends to be low. Moreover a far larger proportion of the total water is associated with the collagen than in the younger disc.     

The Internal Organization of Mycobacterial Partition Assembly: Does the DNA Wrap a Protein Core?

Before cell division in many bacteria, the ParBs spread on a large segment of DNA encompassing the origin-proximal parS site(s) to form the partition assembly that participates in chromosome segregation. Little is known about the structural organization of chromosomal partition assembly. We report solution X-ray and neutron scattering data characterizing the size parameters and internal organization of a nucleoprotein assembly formed by the mycobacterial chromosomal ParB and a 120-meric DNA containing a parS-encompassing region from the mycobacterial genome. The cross-sectional radii of gyration and linear mass density describing the rod-like ParB-DNA assembly were determined from solution scattering. A “DNA outside, protein inside” mode of partition assembly organization consistent with the neutron scattering hydrogen/deuterium contrast variation data is discussed. In this organization, the high scattering DNA is positioned towards the outer region of the partition assembly. The new results presented here provide a basis for understanding how ParBs organize the parS-proximal chromosome, thus setting the stage for further interactions with the DNA condensins, the origin tethering factors and the ParA.     

The Configurational Space of Rocksalt‐Type Oxides for High‐Capacity Lithium Battery Electrodes

A unifying theory is presented to explain the lithium exchange capacity of rocksalt‐like structures with any degree of cation ordering, and how lithium percolation properties can be used as a guideline for the development of novel high‐capacity electrode materials is demonstrated. The lithium percolation properties of the three most common lithium metal oxide phases, the layered α‐NaFeO₂ structure, the spinel‐like LT‐LiCoO₂ structure, and the γ‐LiFeO₂ structure, are demonstrated and a strong dependence of the percolation thresholds on the cation ordering and the lithium content is observed. The poor performance of γ‐LiFeO₂‐type structures is explained by their lack of percolation of good Li migration channels. The spinel‐like structure exhibits excellent percolation properties that are robust with respect to off‐stoichiometry and some amount of cation disorder. The layered structure is unique, as it possesses two different types of lithium diffusion channels, one of which is, however, strongly dependent on the lattice parameters, and therefore very sensitive to disorder. In general it is found that a critical Li‐excess concentration exists at which Li percolation occurs, although the amount of Li excess needed depends on the partial cation ordering. In fully cation‐disordered materials, macroscopic lithium diffusion is enabled by ≈10% excess lithium.     

Identification of Rabbit Oviductal Fluid Proteins Involved in Pre‐Fertilization Processes by Quantitative Proteomics

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable‐isotope dimethyl labeling prior to nanoLC‐MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N‐glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.     

Artifact‐free objective‐type multicolor total internal reflection fluorescence microscopy with light‐emitting diode light sources—Part I

Total internal reflection fluorescence excitation (TIRF) microscopy allows the selective observation of fluorescent molecules in immediate proximity to an interface between different refractive indices. Objective‐type or prism‐less TIRF excitation is typically achieved with laser light sources. We here propose a simple, yet optically advantageous light‐emitting diode (LED)‐based implementation of objective‐type TIRF (LED‐TIRF). The proposed LED‐TIRF condenser is affordable and easy to set up at any epifluorescence microscope to perform multicolor TIRF and/or combined TIRF‐epifluorescence imaging with even illumination of the entire field of view. Electrical control of LED light sources replaces mechanical shutters or optical modulators. LED‐TIRF microscopy eliminates safety burdens that are associated with laser sources, offers favorable instrument lifetime and stability without active cooling. The non‐coherent light source and the type of projection eliminate interference fringing and local scattering artifacts that are associated with conventional laser‐TIRF. Unlike azimuthal spinning laser‐TIRF, LED‐TIRF does not require synchronization between beam rotation and the camera and can be monitored with either global or rolling shutter cameras. Typical implementations, such as live cell multicolor imaging in TIRF and epifluorescence of imaging of short‐lived, localized translocation events of a Ca²⁺‐sensitive protein kinase C α fusion protein are demonstrated.     

Lead optimization and efficacy evaluation of quinazoline-based BET family inhibitors for potential treatment of cancer and inflammatory diseases

Extensive optimization of quinazoline-based lead 8 is described. The structure-activity relationship studies indicate the S-configuration is preferred for the phenylmorpholine substitution. Together with incorporation of a (2-hydroxyl-2-methylpropyl)pyrazole moiety at the 2-position leads to analogs with comparable potency and marked improvement in the pharmacokinetic profile over our previously reported lead compounds. Further in vivo efficacy studies in Kasumi-1 xenograft mouse model demonstrates that the selected inhibitors are well tolerated and highly efficacious in the inhibition of tumor growth. Additionally, the representative analog 19 also demonstrated significant improvement of arthritis severity in a collagen-induced arthritis (CIA) mouse model. These results indicate potential use of these quinazoline-based BET inhibitors for treatment of cancer and inflammatory diseases. A brief discussion of the co-crystallized structure of 19 with BRD4 (BD1) is also highlighted.     

A meta-analysis of the interactive effects of UV and drought on plants

Interactions between climate change and UV penetration in the biosphere are resulting in the exposure of plants to new combinations of UV radiation and drought. In theory, the impacts of combinations of UV and drought may be additive, synergistic or antagonistic. Lack of understanding of the impacts of combined treatments creates substantial uncertainties that hamper predictions of future ecological change. Here, we compiled information from 52 publications and analysed the relative impacts of UV and/or drought. Both UV and drought have substantial negative effects on biomass accumulation, plant height, photosynthesis, leaf area and stomatal conductance and transpiration, while increasing stress-associated symptoms such as MDA accumulation and reactive-oxygen-species content. Contents of proline, flavonoids, antioxidants and anthocyanins, associated with plant acclimation, are upregulated both under enhanced UV and drought. In plants exposed to both UV and drought, increases in plant defense responses are less-than-additive, and so are the damage and growth retardation. Less-than-additive effects were observed across field, glasshouse and growth-chamber studies, indicating similar physiological response mechanisms. Induction of a degree of cross-resistance seems the most likely interpretation of the observed less-than-additive responses. The data show that in future climates, the impacts of increases in drought exposure may be lessened by naturally high UV regimes.