Serine protease inhibitors decrease metastasis in prostate, breast, and ovarian cancers.

Targeted therapies for prostate, breast, and ovarian cancers are based on their activity against primary tumors rather than their anti-metastatic activity. Consequently, there is an urgent need for new agents targeting the metastatic process. Emerging evidence correlates in vitro and in vivo cancer invasion and metastasis with increased activity of the proteases mesotrypsin (prostate and breast cancer) and kallikrein 6 (KLK6; ovarian cancer). Thus, mesotrypsin and KLK6 are attractive putative targets for therapeutic intervention. As potential therapeutics for advanced metastatic prostate, breast, and ovarian cancers, we report novel mesotrypsin- and KLK6-based therapies, based on our previously developed mutants of the human amyloid ß-protein precursor Kunitz protease inhibitor domain (APPI). These mutants, designated APPI-3M (prostate and breast cancer) and APPI-4M (ovarian cancer), demonstrated significant accumulation in tumors and therapeutic efficacy in orthotopic preclinical models, with the advantages of long retention times in vivo, high affinity and favorable pharmacokinetic properties. The applicability of the APPIs, as a novel therapy and for imaging purposes, is supported by their good safety profile and their controlled and scalable manufacturability in bioreactors.

Acclimatization of a coral-dinoflagellate mutualism at a CO2 vent.

Ocean acidification caused by shifts in ocean carbonate chemistry resulting from increased atmospheric CO2 concentrations is threatening many calcifying organisms, including corals. Here we assessed autotrophy vs heterotrophy shifts in the Mediterranean zooxanthellate scleractinian coral Balanophyllia europaea acclimatized to low pH/high pCO2 conditions at a CO2 vent off Panarea Island (Italy). Dinoflagellate endosymbiont densities were higher at lowest pH Sites where changes in the distribution of distinct haplotypes of a host-specific symbiont species, Philozoon balanophyllum, were observed. An increase in symbiont C/N ratios was observed at low pH, likely as a result of increased C fixation by higher symbiont cell densities. δ13C values of the symbionts and host tissue reached similar values at the lowest pH Site, suggesting an increased influence of autotrophy with increasing acidification. Host tissue δ15N values of 0‰ strongly suggest that diazotroph N2 fixation is occurring within the coral tissue/mucus at the low pH Sites, likely explaining the decrease in host tissue C/N ratios with acidification. Overall, our findings show an acclimatization of this coral-dinoflagellate mutualism through trophic adjustment and symbiont haplotype differences with increasing acidification, highlighting that some corals are capable of acclimatizing to ocean acidification predicted under end-of-century scenarios.

Leaf coordination between petiole vascular development and water demand in response to elevated CO2 in tomato plants.

The rise in atmospheric CO2 has a profound impact on plants physiology and performance. Stomatal gas exchange such as reduction in water loss via transpiration and higher photosynthetic rates are among the key plant physiological traits altered by the increase of CO2. Water acquired in plant roots is transported via the xylem vessels to the shoots. Under conditions of elevated CO2, water flux decreases due to higher water use efficiency and a decline in stomatal conductance. However, the mechanism by which the shoot vascular development is affected under elevated CO2 is still largely unclear in herbaceous crops. In the current study, tomato plants were exposed to either 400 or 800 ppm of CO2 and were analyzed for growth, leaf area, gas exchange rate, and petiole anatomy. Elevated CO2 caused a reduction in metaxylem vessel diameter, which in turn, decreased leaf theatrical conductivity by 400% as compared with plants grown under ambient CO2. This work links anatomical changes in the petioles to the rise in atmospheric CO2 and water use. Plant water demand declined under elevated CO2, while photosynthesis increased. Thus, the decrease in leaf specific conductivity was attributed to lower water consumption in leaf gas exchange and, by extension, to higher leaf water use efficiency. As the global climate changes and water scarcity becomes more common, such anatomical alterations caused by elevated CO2 may affect plant response to water limitation. Further research on petiole anatomical alterations under conditions of combined climate change factors such as drought and heat with elevated CO2 may assist in clarifying the responses expected by future climate scenarios.

Climbing Up and Down Binding Landscapes through Deep Mutational Scanning of Three Homologous Protein-Protein Complexes.

Protein-protein interactions (PPIs) have evolved to display binding affinities that can support their function. As such, cognate and noncognate PPIs could be highly similar structurally but exhibit huge differences in binding affinities. To understand this phenomenon, we study three homologous protease-inhibitor PPIs that span 9 orders of magnitude in binding affinity. Using state-of-the-art methodology that combines protein randomization, affinity sorting, deep sequencing, and data normalization, we report quantitative binding landscapes consisting of ΔΔGbind values for the three PPIs, gleaned from tens of thousands of single and double mutations. We show that binding landscapes of the three complexes are strikingly different and depend on the PPI evolutionary optimality. We observe different patterns of couplings between mutations for the three PPIs with negative and positive epistasis appearing most frequently at hot-spot and cold-spot positions, respectively. The evolutionary trends observed here are likely to be universal to other biological complexes in the cell.

Meta-analysis of drought and heat stress combination impact on crop yield and yield components.

Episodes of prolonged drought coupled with heat waves (i.e. drought and heat combination) can have a devastating impact on agricultural production and crop yield. It is therefore not surprising that improving tolerance to drought and heat combination has been a major goal for breeders and biotech companies. Although much is known about the physiological and molecular responses of vegetative tissues to a combination of drought and heat stress, less is known about the impact of this stress combination on yield and different yield components. Here, we used a meta-analysis approach to synthesize results from over 120 published case studies of crop responses to combined drought and heat stress. Our findings reveal that drought and heat stress combination significantly impacts yield by decreasing harvest index, shortening the life cycle of crops, and altering seed number, size and composition. Furthermore, these impacts are more severe when the stress combination is applied during the reproductive stage of plants. We further identify differences in how legumes and cereals respond to the stress combination and reveal that utilizing C3 or C4 metabolism may not provide an advantage to plants during stress combinations. Taken together our study highlights a need to focus future studies, as well as breeding efforts, on crop responses to drought and heat combination at the reproductive stage of different crop species.

The impact of water deficit and heat stress combination on the molecular response, physiology, and seed production of soybean.

A combination of drought and heat stress, occurring at the vegetative or reproductive growth phase of many different crops can have a devastating impact on yield. In soybean (Glycine max), a considerable effort has been made to develop genotypes with enhanced yield production under conditions of drought or heat stress. However, how these genotypes perform in terms of growth, physiological responses, and most importantly seed production, under conditions of drought and heat combination is mostly unknown. Here, we studied the impact of water deficit and heat stress combination on the physiology, seed production, and yield per plant of two soybean genotypes, Magellan and Plant Introduction (PI) 548313, that differ in their reproductive responses to heat stress. Our findings reveal that although PI 548313 produced more seeds than Magellan under conditions of heat stress, under conditions of water deficit, and heat stress combination its seed production decreased. Because the number of flowers and pollen germination of PI 548313 remained high under heat or water deficit and heat combination, the reduced seed production exhibited by PI 548313 under the stress combination could be a result of processes that occur at the stigma, ovaries and/or other parts of the flower following pollen germination.

Local auxin biosynthesis is required for root regeneration after wounding.

The root meristem can regenerate following removal of its stem-cell niche by recruitment of remnant cells from the stump. Regeneration is initiated by rapid accumulation of auxin near the injury site but the source of this auxin is unknown. Here, we show that auxin accumulation arises from the activity of multiple auxin biosynthetic sources that are newly specified near the cut site and that their continuous activity is required for the regeneration process. Auxin synthesis is highly localized while PIN-mediated transport is dispensable for auxin accumulation and tip regeneration. Roots lacking the activity of the regeneration competence factor ERF115, or that are dissected at a zone of low regeneration potential, fail to activate local auxin sources. Remarkably, restoring auxin supply is sufficient to confer regeneration capacity to these recalcitrant tissues. We suggest that regeneration competence relies on the ability to specify new local auxin sources in a precise temporal pattern.

Effects of Light Pollution on the Early Life Stages of the Most Abundant Northern Red Sea Coral.

The growth in human population along coastal areas is exposing marine environments to increasing anthropogenic light sources. Despite the potential effects of this modern phenomenon, very few studies have examined its implications for corals. Here, we present a long-term study of coral early life stages under light pollution conditions at night. Coral larvae were collected from Stylophora pistillata colonies, and then settled and grown under experimental conditions of two different common city lighting methods (fluorescent or LED). Effects of the artificial lighting on the coral settlement success, survivorship, growth rate, photosynthetic efficiency, and calcification rate were examined over a period of one year. The control exhibited ~30% higher settlement success compared to the two light treatments, while under the light treatments corals showed higher survivorship, growth, and calcification rates. In addition, an indication of damage to the photosynthetic system was found in the light-polluted corals, which was reflected in their photosynthesis efficiency parameters: i.e., lower maximum light utilization coefficient (α), lower maximum potential photosynthetic rate (Pmax), and lower photosynthetic maximal quantum yield (Fv/Fm). Our findings provide evidence of the potential adverse effects of artificial lighting methods on the natural environment of coral reefs. We conclude that the use of the LED lighting method has high interference potential for the early life stages of corals.

CO2 and nitrogen interaction alters root anatomy, morphology, nitrogen partitioning and photosynthetic acclimation of tomato plants.

MAIN CONCLUSION: Nitrogen and CO2 supply interactively regulate whole plant nitrogen partitioning and root anatomical and morphological development in tomato plants. Nitrogen (N) and carbon (C) are the key elements in plant growth and constitute the majority of plant dry matter. Growing at CO2 enrichment has the potential to stimulate the growth of C3 plants, however, growth is often limited by N availability. Thus, the interactive effects of CO2 under different N fertilization rates can affect growth, acclimation to elevated CO2, and yield. However, the majority of research in this field has focused on shoot traits, while neglecting plants' hidden half-the roots. We hypothesize that elevated CO2 and low N effects on transpiration will interactively affect root vascular development and plant N partitioning. Here we studied the effects of elevated CO2 and N concentrations on greenhouse-grown tomato plants, a C3 crop. Our main objective was to determine in what manner the N fertilization rate and elevated CO2 affected root development and nitrogen partitioning among plant organs. Our results indicate that N interacting with the CO2 level affects the development of the root system in terms of the length, anatomy, and partitioning of the N concentration between the roots and shoot. Both CO2 and N concentrations were found to affect xylem size in an opposite manner, elevated CO2 found to repressed, whereas ample N stimulated xylem development. We found that under limiting N and eCO2, the N% increase in the root, while it decreased in the shoot. Under eCO2, the root system size increased with a coordinated decrease in root xylem area. We suggest that tomato root response to elevated CO2 depends on N fertilization rates, and that a decrease in xylem size is a possible underlying response that limits nitrogen allocation from the root into the shoot. Additionally, the greater abundance of root amino acids suggests increased root nitrogen metabolism at eCO2 conditions with ample N.

Photoacclimation and induction of light-enhanced calcification in the mesophotic coral Euphyllia paradivisa.

Corals and their photosymbionts experience inherent changes in light along depth gradients, leading them to have evolved several well-investigated photoacclimation strategies. As coral calcification is influenced by light (a process described as LEC-'light-enhanced calcification'), studies have sought to determine the link between photosynthesis and calcification, but many puzzling aspects still persist. Here, we examine the physiology of Euphyllia paradivisa, a coral species found at a wide range of depths but that is strictly mesophotic in the Red Sea; and also examines the coupling between photosynthesis and LEC by investigating the response of the coral under several controlled light regimes during a long-term experiment. E. paradivisa specimens were collected from 40 to 50 m depth and incubated under three light conditions for a period of 1 year: full-spectrum shallow-water light (approx. 3 m, e.g. shallow-light treatment); blue deep-water light (approx. 40 m, e.g. mesophotic-light treatment) or total darkness (e.g. dark treatment). Net photosynthesis remained similar in the shallow-light-treated corals compared to the mesophotic-light-treated corals, under both low and high light. However, calcification increased dramatically with increasing light intensity in the shallow-light-treated corals, suggesting a decoupling between these processes. Photoacclimation to shallow-water conditions was indicated by enhanced respiration, a higher density of zooxanthellae per polyp and lower chlorophyll a content per cell. The dark-treated corals became completely bleached but did not lower their metabolism below that of the mesophotic-light-treated corals. No Symbiodinium clade shift was found following the year-long light treatments. We conclude that E. paradivisa, and its original symbiont clade, can adapt to various light conditions by controlling its metabolic rate and growth energy investment, and consequently induce LEC.

Disulfide engineering of human Kunitz-type serine protease inhibitors enhances proteolytic stability and target affinity toward mesotrypsin.

Serine protease inhibitors of the Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family are ubiquitous biological regulators of proteolysis. These small proteins are resistant to proteolysis, but can be slowly cleaved within the protease-binding loop by target proteases, thereby compromising their activity. For the human protease mesotrypsin, this cleavage is especially rapid. Here, we aimed to stabilize the Kunitz domain structure against proteolysis through disulfide engineering. Substitution within the Kunitz inhibitor domain of the amyloid precursor protein (APPI) that incorporated a new disulfide bond between residues 17 and 34 reduced proteolysis by mesotrypsin 74-fold. Similar disulfide engineering of tissue factor pathway inhibitor-1 Kunitz domain 1 (KD1TFPI1) and bikunin Kunitz domain 2 (KD2bikunin) likewise stabilized these inhibitors against mesotrypsin proteolysis 17- and 6.6-fold, respectively. Crystal structures of disulfide-engineered APPI and KD1TFPI1 variants in a complex with mesotrypsin at 1.5 and 2.0 Å resolution, respectively, confirmed the formation of well-ordered disulfide bonds positioned to stabilize the binding loop. Long all-atom molecular dynamics simulations of disulfide-engineered Kunitz domains and their complexes with mesotrypsin revealed conformational stabilization of the primed side of the inhibitor-binding loop by the engineered disulfide, along with global suppression of conformational dynamics in the Kunitz domain. Our findings suggest that the Cys-17-Cys-34 disulfide slows proteolysis by dampening conformational fluctuations in the binding loop and minimizing motion at the enzyme-inhibitor interface. The generalizable approach developed here for the stabilization against proteolysis of Kunitz domains, which can serve as important scaffolds for therapeutics, may thus find applications in drug development.

Synergistic effects of abiotic stresses in plants: a case study of nitrogen limitation and saturating light intensity in Arabidopsis thaliana.

Under natural conditions, plants are regularly exposed to combinations of stress factors. A common example is the conjunction between nitrogen (N) deficiency and excess light. The combined effect of stress factors is often ignored in studies using controlled conditions, possibly resulting in misleading conclusions. To address this issue, the present study examined the physiological behavior of Arabidopsis thaliana under the effect of varying nitrogen levels and light intensities. The joint influence of low N and excess light had an adverse effect on plant growth, chlorophyll and anthocyanin concentrations, photochemical capacity and the abundance of proteins involved in carbon assimilation and antioxidative metabolism. In contrast, no adverse physiological responses were observed for plants under either nitrogen limitation or high light (HL) intensity conditions (i.e. single stress). The underlying mechanisms for the increased growth in conditions of HL and sufficient nitrogen were a combination of chlorophyll accumulation and an increased number of proteins involved in C3 carbon assimilation, amino acids biosynthesis and chloroplast development. In contrast, combined stress conditions shifts plants from growth to survival by displaying anthocyanin accumulation and an increased number of proteins involved in catabolism of lipids and amino acids as energy substrates. Ultimately switching plants development from growth to survival. Our results suggest that an assessment of the physiological response to the combined effect of multiple stresses cannot be directly extrapolated from the physiological response to a single stress. Specifically, the synergistic interaction between N deficiency and saturating light in Arabidopsis plants could not have been modeled via only one of the stress factors.

Mapping protein selectivity landscapes using multi-target selective screening and next-generation sequencing of combinatorial libraries.

Characterizing the binding selectivity landscape of interacting proteins is crucial both for elucidating the underlying mechanisms of their interaction and for developing selective inhibitors. However, current mapping methods are laborious and cannot provide a sufficiently comprehensive description of the landscape. Here, we introduce a novel and efficient strategy for comprehensively mapping the binding landscape of proteins using a combination of experimental multi-target selective library screening and in silico next-generation sequencing analysis. We map the binding landscape of a non-selective trypsin inhibitor, the amyloid protein precursor inhibitor (APPI), to each of the four human serine proteases (kallikrein-6, mesotrypsin, and anionic and cationic trypsins). We then use this map to dissect and improve the affinity and selectivity of APPI variants toward each of the four proteases. Our strategy can be used as a platform for the development of a new generation of target-selective probes and therapeutic agents based on selective protein-protein interactions.

A potent, proteolysis-resistant inhibitor of kallikrein-related peptidase 6 (KLK6) for cancer therapy, developed by combinatorial engineering.

Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry-based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPIM17L,I18F,S19F,F34V (APPI-4M), an APPI variant with a KLK6 inhibition constant (Ki ) of 160 pm and a turnover time of 10 days. To the best of our knowledge, APPI-4M is the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPIWT). We further demonstrate that APPI-4M acts as a functional inhibitor in a cell-based model of KLK6-dependent breast cancer invasion. Finally, the crystal structures of the APPIWT/KLK6 and APPI-4M/KLK6 complexes revealed the structural and mechanistic bases for the improved KLK6 binding and proteolytic resistance of APPI-4M. We anticipate that APPI-4M will have substantial translational potential as both imaging agent and therapeutic.

The effects of elevated CO2 and nitrogen nutrition on root dynamics.

Ambient CO2 concentration is currently 400 µmol mol-1, and projections forecast an increase up to 970 µmol mol-1 by century's end. Elevated CO2 can stimulate C3 plant growth, whereas nitrogen is the main nutrient plants acquire from soils and often limits growth. Plants primarily obtain two nitrogen sources from the soil, ammonium (NH4+) and nitrate (NO3-). At elevated CO2 levels, plant growth and nitrogen metabolism is affected by the nitrogen source. Most research has focused on shoot traits, while neglecting the plants' hidden half, the root. We studied the effects of elevated CO2 and nitrogen source on hydroponically grown tomato plants, a C3 model and crop plant. Our main objective was to determine how the nitrogen source and elevated CO2 affect root development. Our results indicate they affect development in terms of the size and anatomy of different root orders. Specifically, root xylem development was found sensitive to the nitrogen source, whereas NO3--supplied plants displayed greater xylem development compared to their NH4+ counterparts, and also to a lesser extent, to elevated CO2, which we found inhibits this development. Additionally, elevated CO2 decreased root respiration in different root orders exclusively in plants supplied with NH4+as the sole nitrogen source.

Pre-equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases.

High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid ß-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPIP13W/M17G/I18F/F34V, with up to 30-fold greater specificity relative to the parental APPIM17G/I18F/F34V protein, and 6500- to 230 000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins.

Converting a broad matrix metalloproteinase family inhibitor into a specific inhibitor of MMP-9 and MMP-14.

MMP-14 and MMP-9 are two well-established cancer targets for which no specific clinically relevant inhibitor is available. Using a powerful combination of computational design and yeast surface display technology, we engineered such an inhibitor starting from a nonspecific MMP inhibitor, N-TIMP2. The engineered purified N-TIMP2 variants showed enhanced specificity toward MMP-14 and MMP-9 relative to a panel of off-target MMPs. MMP-specific N-TIMP2 sequence signatures were obtained that could be understood from the structural perspective of MMP/N-TIMP2 interactions. Our MMP-9 inhibitor exhibited 1000-fold preference for MMP-9 vs. MMP-14, which is likely to translate into significant differences under physiological conditions. Our results provide new insights regarding evolution of promiscuous proteins and optimization strategies for design of inhibitors with single-target specificities.

Development of High Affinity and High Specificity Inhibitors of Matrix Metalloproteinase 14 through Computational Design and Directed Evolution.

Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (Ki ) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays ∼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an in vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.