Negatively charged phospholipids accelerate the membrane fusion activity of the plant-specific insert domain of an aspartic protease.

Various plants use antimicrobial proteins/peptides to resist phytopathogens. In the potato, Solanum tuberosum, the plant-specific insert (PSI) domain of an aspartic protease performs this role by disrupting phytopathogen plasma membranes. However, the mechanism by which PSI selects target membranes has not been elucidated. Here, we studied PSI-induced membrane fusion, focusing on the effects of lipid composition on fusion efficiency. Membrane fusion by the PSI involves an intermediate state whereby adjacent liposomes share their bilayers. We found that increasing the concentration of negatively charged phosphatidylserine (PS) phospholipids substantially accelerated PSI-mediated membrane fusion. NMR data demonstrated that PS did not affect the binding between the PSI and liposomes but had seminal effects on the dynamics of PSI interaction with liposomes. In PS-free liposomes, the PSI underwent significant motion, which was suppressed on PS-contained liposomes. Molecular dynamics simulations showed that the PSI binds to PS-containing membranes with a dominant angle ranging from -31° to 30°, with respect to the bilayer, and is closer to the membrane surfaces. In contrast, PSI is mobile and exhibits multiple topological states on the surface of PS-free membranes. Taken together, our data suggested that PS lipids limit the motion of the anchored PSI, bringing it closer to the membrane surface and efficiently bridging different liposomes to accelerate fusion. As most phytopathogens have a higher content of negatively charged lipids as compared with host cells, these results indicate that the PSI selectively targets negatively charged lipids, which likely represents a way of distinguishing the pathogen from the host.

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics.

The Solanum tuberosum plant specific insert (StPSI) has a defensive role in potato plants, with the requirements of acidic pH and anionic lipids. The StPSI contains a set of three highly conserved disulfide bonds that bridge the protein's helical domains. Removal of these bonds leads to enhanced membrane interactions. This work examined the effects of their sequential removal, both individually and in combination, using all-atom molecular dynamics to elucidate the role of disulfide linkages in maintaining overall protein tertiary structure. The tertiary structure was found to remain stable at both acidic (active) and neutral (inactive) pH despite the removal of disulfide linkages. The findings include how the dimer structure is stabilized and the impact on secondary structure on a residue-basis as a function of disulfide bond removal. The StPSI possesses an extensive network of inter-monomer hydrophobic interactions and intra-monomer hydrogen bonds, which is likely the key to the stability of the StPSI by stabilizing local secondary structure and the tertiary saposin-fold, leading to a robust association between monomers, regardless of the disulfide bond state. Removal of disulfide bonds did not significantly impact secondary structure, nor lead to quaternary structural changes. Instead, disulfide bond removal induces regions of amino acids with relatively higher or lower variation in secondary structure, relative to when all the disulfide bonds are intact. Although disulfide bonds are not required to preserve overall secondary structure, they may have an important role in maintaining a less plastic structure within plant cells in order to regulate membrane affinity or targeting.

Insights into the mechanism of membrane fusion induced by the plant defense element, plant-specific insert.

In plants, many natural defense mechanisms include cellular membrane fusion as a way to resist infection by external pathogens. Several plant proteins mediate membrane fusion, but the detailed mechanism by which they promote fusion is less clear. Understanding this process could provide valuable insights into these proteins' physiological functions and guide bioengineering applications (i.e. the design of antimicrobial proteins). The plant-specific insert (PSI) from Solanum tuberosum can help reduce certain pathogen attack via membrane fusion. To gain new insights into the process of PSI-induced membrane fusion, a combined approach of NMR, FRET, and in silico studies was used. Our results indicate that (i) under acidic conditions, the PSI experiences a monomer-dimer equilibrium, and the dimeric PSI induces membrane fusion below a certain critical pH; (ii) after fusion, the PSI resides in a highly dehydrated environment with limited solvent accessibility, suggesting its capability in reducing repulsive dehydration forces between liposomes to facilitate fusion; and (iii) as shown by molecular dynamics simulations, the PSI dimer can bind stably to membrane surfaces and can bridge liposomes in close proximity, a critical step for the membrane fusion. In summary, this study provides new and unique insights into the mechanisms by which the PSI and similar proteins induce membrane fusion.

pH dependent membrane binding of the Solanum tuberosum plant specific insert: An in silico study.

The Solanum tuberosum plant-specific insert (StPSI) has been shown to possess potent antimicrobial activity against both human and plant pathogens. Furthermore, in vitro, the StPSI is capable of fusing phospholipid vesicles, provided the conditions of net anionic vesicle charge and acidic pH are met. Constant pH replica-exchange simulations indicate several acidic residues on the dimer have highly perturbed pKas (<3.0; E15, D28, E85 & E100) due to involvement in salt bridges. After setting the pH of the system to either 3.0 or 7.4, all-atom simulations provided details of the effect of pH on secondary structural elements, particularly in the previously unresolved crystallographic structure of the loop section. Coarse-grained dimer-bilayer simulations demonstrated that at pH 7.4, the dimer had no affinity for neutral or anionic membranes over the course of 1 µs simulations. Conversely, at pH 3.0 two binding modes were observed. Mode 1 is mediated primarily via strong N-terminal interactions on one monomer only, whereas in mode 2, N- and C-terminal residues of one monomer and numerous polar and basic residues on the second monomer, particularly in the third helix, participate in membrane interactions. Mode 2 was accompanied by re-orientation of the dimer to a more vertical position with respect to helices 1 and 4, positioning the dimer for membrane interactions. These results offer the first examination at near-atomic resolution of residues mediating the StPSI-membrane interactions, and allow for the postulation of a possible fusion mechanism.

Deciphering the mechanism of potent peptidomimetic inhibitors targeting plasmepsins - biochemical and structural insights.

Malaria is a deadly disease killing worldwide hundreds of thousands people each year and the responsible parasite has acquired resistance to the available drug combinations. The four vacuolar plasmepsins (PMs) in Plasmodium falciparum involved in hemoglobin (Hb) catabolism represent promising targets to combat drug resistance. High antimalarial activities can be achieved by developing a single drug that would simultaneously target all the vacuolar PMs. We have demonstrated for the first time the use of soluble recombinant plasmepsin II (PMII) for structure-guided drug discovery with KNI inhibitors. Compounds used in this study (KNI-10742, 10743, 10395, 10333, and 10343) exhibit nanomolar inhibition against PMII and are also effective in blocking the activities of PMI and PMIV with the low nanomolar Ki values. The high-resolution crystal structures of PMII-KNI inhibitor complexes reveal interesting features modulating their differential potency. Important individual characteristics of the inhibitors and their importance for potency have been established. The alkylamino analog, KNI-10743, shows intrinsic flexibility at the P2 position that potentiates its interactions with Asp132, Leu133, and Ser134. The phenylacetyl tripeptides, KNI-10333 and KNI-10343, accommodate different ρ-substituents at the P3 phenylacetyl ring that determine the orientation of the ring, thus creating novel hydrogen-bonding contacts. KNI-10743 and KNI-10333 possess significant antimalarial activity, block Hb degradation inside the food vacuole, and show no cytotoxicity on human cells; thus, they can be considered as promising candidates for further optimization. Based on our structural data, novel KNI derivatives with improved antimalarial activity could be designed for potential clinical use. DATABASE: Structural data are available in the PDB under the accession numbers 5YIE, 5YIB, 5YID, 5YIC, and 5YIA.

Protein Structure Insights into the Bilayer Interactions of the Saposin-Like Domain of Solanum tuberosum Aspartic Protease.

Many plant aspartic proteases contain a saposin-like domain whose principal functions are intracellular sorting and host defence. Its structure is characterised by helical segments cross-linked by three highly conserved cystines. The present study on the saposin-like domain of Solanum tuberosum aspartic protease revealed that acidification from inactive to active conditions causes dimerisation and a strand-to-helix secondary structure transition independent of bilayer interaction. Bilayer fusion was shown to occur under reducing conditions yielding a faster shift to larger vesicle sizes relative to native conditions, implying that a lower level structural motif might be bilayer-active. Characterisation of peptide sequences based on the domain's secondary structural regions showed helix-3 to be active (~4% of the full domain's activity), and mutation of its sole positively charged residue resulted in loss of activity and disordering of structure. Also, the peptides' respective circular dichroism spectra suggested that native folding within the full domain is dependent on surrounding structure. Overall, the present study reveals that the aspartic protease saposin-like domain active structure is an open saposin fold dimer whose formation is pH-dependent, and that a bilayer-active motif shared among non-saposin membrane-active proteins including certain plant defence proteins is nested within an overall structure essential for native functionality.

Randomized controlled trial assessing the efficacy of a reusable fish-shaped iron ingot to increase hemoglobin concentration in anemic, rural Cambodian women.

Background: Anemia affects 45% of women of childbearing age in Cambodia. Iron supplementation is recommended in populations in which anemia prevalence is high. However, there are issues of cost, distribution, and adherence. A potential alternative is a reusable fish-shaped iron ingot, which, when added to the cooking pot, leaches iron into the fluid in which it is prepared.Objective: We sought to determine whether there was a difference in hemoglobin concentrations in rural Cambodian anemic women (aged 18-49 y) who cooked with the iron ingot or consumed a daily iron supplement compared with a control after 1 y.Design: In Preah Vihear, 340 women with mild or moderate anemia were randomly assigned to 1) an iron-ingot group, 2) an iron-supplement (18 mg/d) group, or 3) a nonplacebo control group. A venous blood sample was taken at baseline and at 6 and 12 mo. Blood was analyzed for hemoglobin, serum ferritin, and serum transferrin receptor. Hemoglobin electrophoresis was used to detect structural hemoglobin variants.Results: Anemia prevalence was 44% with the use of a portable hemoglobinometer during screening. At baseline, prevalence of iron deficiency was 9% on the basis of a low serum ferritin concentration. There was no significant difference in mean hemoglobin concentrations between the iron-ingot group (115 g/L; 95% CI: 113, 118 g/L; P = 0.850) or iron-supplement group (115 g/L; 95% CI: 113, 117 g/L; P = 0.998) compared with the control group (115 g/L; 95% CI: 113, 117 g/L) at 12 mo. Serum ferritin was significantly higher in the iron-supplement group (73 µg/L; 95% CI: 64, 82 µg/L; P = 0.002) than in the control group at 6 mo; however, this significance was not maintained at 12 mo (73 µg/L; 95% CI: 58, 91 µg/L; P = 0.176).Conclusions: Neither the iron ingot nor iron supplements increased hemoglobin concentrations in this population at 6 or 12 mo. We do not recommend the use of the fish-shaped iron ingot in Cambodia or in countries where the prevalence of iron deficiency is low and genetic hemoglobin disorders are high. This trial was registered at clinicaltrials.gov as NCT02341586.

Comparative structure-function characterization of the saposin-like domains from potato, barley, cardoon and Arabidopsis aspartic proteases.

The present study characterized the aspartic protease saposin-like domains of four plant species, Solanum tuberosum (potato), Hordeum vulgare L. (barley), Cynara cardunculus L. (cardoon; artichoke thistle) and Arabidopsis thaliana, in terms of bilayer disruption and fusion, and structure pH-dependence. Comparison of the recombinant saposin-like domains revealed that each induced leakage of bilayer vesicles composed of a simple phospholipid mixture with relative rates Arabidopsis>barley>cardoon>potato. When compared for leakage of bilayer composed of a vacuole-like phospholipid mixture, leakage was approximately five times higher for potato saposin-like domain compared to the others. In terms of fusogenic activity, distinctions between particle size profiles were noted among the four proteins, particularly for potato saposin-like domain. Bilayer fusion assays in reducing conditions resulted in altered fusion profiles except in the case of cardoon saposin-like domain which was virtually unchanged. Secondary structure profiles were similar across all four proteins under different pH conditions, although cardoon saposin-like domain appeared to have higher overall helix structure. Furthermore, increases in Trp emission upon protein-bilayer interactions suggested that protein structure rearrangements equilibrated with half-times ranging from 52 to 120s, with cardoon saposin-like domain significantly slower than the other three species. Overall, the present findings serve as a foundation for future studies seeking to delineate protein structural features and motifs in protein-bilayer interactions based upon variability in plant aspartic protease saposin-like domain structures.

Physicochemical properties and in vitro starch digestibility of potato starch/protein blends.

This study aimed to investigate effects of starch-protein interactions on physicochemical properties and in vitro starch digestibility of composite potato starch/protein blends (0, 5, 10, or 15% protein) during processing (cooking, cooling and reheating). The effect on recrystallization and short-range ordering in starch was studied by light microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy. The results show that protein in the blend proportionally restricted starch granule swelling during cooking and facilitated amylopectin recrystallization during cold-storage. The facilitating effect of protein diminished with increasing blend ratio. Resistant starch content in the processed blends was positively correlated to intensity ratio of 1053/1035cm(-1) in FTIR spectra arising from slow retrogradation of amylopectin (r(2)>0.88, P≤0.05), whose formation was favored by the presence of protein in the blends and further enhanced by cooling of cooked blends. As a conclusion, starch-protein interaction reduced starch digestibility of the processed blends.

Evaluation of nutritional profiles of starch and dry matter from early potato varieties and its estimated glycemic impact.

To identify healthier potatoes with respect to starch profiles, fourteen early varieties were evaluated for their dietary fiber, total starch, rapidly digestible (RDS), slowly digestible (SDS), and resistant (RS) starch for nutrition and with regard to estimated glycemic index (eGI) and glycemic load (eGL). While all these profiles were highly dependent on the potato variety, eleven out of fourteen varieties were classified as low GL foods (p<0.05). A strong positive correlation was observed with eGI and RDS (r=0.975-1.00, 0.96-1.00 and 0.962-0.997 for uncooked, cooked and retrograded varieties, respectively), whereas a strong negative correlation was observed between eGI and RS (r=-0.985 to -0.998, -0.96 to -1.00 and -0.983 to -0.999 for uncooked, cooked and retrograded varieties respectively, p<0.05). For the cultivars examined, the present study identified RDS and RS as major starch factors contributing to eGI.

A molecular modeling approach to understand the structure and conformation relationship of (GlcpA)Xylan.

The structure and conformation relationships of a heteropolysaccharide (GlcpA)Xylan in terms of various molecular weights, Xylp/GlcpA ratio and the distribution of GlcpA along xylan chain were investigated using computer modeling. The adiabatic contour maps of xylobiose, XylpXylp(GlcpA) and (GlcpA)XylpXylp(GlcpA) indicated that the insertion of the side group (GlcpA) influenced the accessible conformational space of xylobiose molecule. RIS-Metropolis Monte Carlo method indicated that insertion of GlcpA side chain induced a lowering effect of the calculated chain extension at low GlcpA:Xylp ratio (GlcpA:Xylp = 1:3). The chain, however, became extended when the ratio of GlcpA:Xylp above 2/3. It was also shown that the spatial extension of the polymer chains was dependent on the distribution of side chain: the random distribution demonstrated the most flexible structure compared to block and alternative distribution. The present studies provide a unique insight into the dependence of both side chain ratio and distribution on the stiffness and flexibility of various (GlcpA)Xylan molecules.

In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease.

The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.

Genotype by environment interaction effects on starch content and digestibility in potato (Solanum tuberosum L.).

Biochemically, starch is composed of amylose and amylopectin but can also be defined by its digestibility rates within the human intestinal tract, i.e., rapidly digested (RDS), slowly digested (SDS), or resistant (RS). The relative ratio of these starch components is the main contributor to differences in the glycemic index (GI) of carbohydrate sources. This study evaluated the digestible starch profile of 12 potato genotypes comprising elite breeding lines and commercial varieties in six environments, with the optimal profile defined as low RDS and high SDS. Genotype by environment interaction (GEI) analysis found significant (p = 0.05) genotypic and environmental effects for all digestibility rate components; however, interaction effects were only significant for SDS. Optimal starch profiles were identified for two genotypes, CV96044-3 and Goldrush. The desirable starch profile in these potato cultivars can be exploited in breeding programs for the improvement of starch profile and other important characteristics such as high yields and disease resistance.

Structural characterization of a low-molecular-weight heteropolysaccharide (glucomannan) isolated from Artemisia sphaerocephala Krasch.

Using 60% (w/v) ammonium sulfate precipitation, a heteropolysaccharide (designated 60S), with relatively low molecular weight (38.7kDa), was isolated from the seeds of Artemisiasphaerocephala Krasch. The structural properties of 60S were elucidated by partial acid hydrolysis, methylation analysis, 1D and 2D NMR spectroscopy, and MALDI-TOF-MS. The results of the partial acid hydrolysis and methylation analysis indicated that the main chain of 60S consisted of (1→4)-linked d-Manp and (1→4)-linked d-Glcp in a molar ratio of 1:1.3. Over half of the glucosyl residues in the main chain were branched at the O-6 position. The terminal sugar residues were mainly composed of T-Araf, T-Arap, T-Galp, T-GlcpA, and T-Glcp. Besides, 3-Araf and 2-Galp were also observed in comparable amounts. Based on all the aforementioned results and the data obtained by 1D and 2D NMR spectroscopy as well as MALDI-TOF-MS, a structure of 60S is proposed as follows: R could be one or some of -(3-α-Araf)(n)-(A), T-α-Galp(B), T-α-Glcp(C), T-Araf(H) or T-Arap.

Structure and mechanism of the saposin-like domain of a plant aspartic protease.

Many plant aspartic proteases contain an additional sequence of ~100 amino acids termed the plant-specific insert, which is involved in host defense and vacuolar targeting. Similar to all saposin-like proteins, the plant-specific insert functions via protein-membrane interactions; however, the structural basis for such interactions has not been studied, and the nature of plant-specific insert-mediated membrane disruption has not been characterized. In the present study, the crystal structure of the saposin-like domain of potato aspartic protease was resolved at a resolution of 1.9 Å, revealing an open V-shaped configuration similar to the open structure of human saposin C. Notably, vesicle disruption activity followed Michaelis-Menten-like kinetics, a finding not previously reported for saposin-like proteins including plant-specific inserts. Circular dichroism data suggested that secondary structure was pH-dependent in a fashion similar to influenza A hemagglutinin fusion peptide. Membrane effects characterized by atomic force microscopy and light scattering indicated bilayer solubilization as well as fusogenic activity. Taken together, the present study is the first report to elucidate the membrane interaction mechanism of plant saposin-like domains whereby pH-dependent membrane interactions resulted in bilayer fusogenic activity that probably arose from a viral type pH-dependent helix-kink-helix motif at the plant-specific insert N terminus.

Interactions of vitamin D3 with bovine beta-lactoglobulin A and beta-casein.

It is of nutritional significance to fortify processed dairy products (e.g., cheese, yogurt, and ice cream) with vitamin D3; however, the inherent complexity of these foods may influence the stability and bioavailability of this nutrient. In the present study, the interactions of vitamin D3 with beta-lactoglobulin A and beta-casein were investigated under various environmental conditions (i.e., pH and ionic strength) using fluorescence and circular dichroism spectroscopic techniques. The results indicated that vitamin D3 was bound to both beta-lactoglobulin A and beta-casein depending on the solution conditions. The apparent dissociation constants ranged from 0.02 to 0.29 microM for beta-lactoglobulin A, whereas the beta-casein apparent dissociation constants ranged from 0.06 to 0.26 microM. The apparent mole ratios were also comparable, i.e., 0.51-2.04 and 1.16-2.05 mol of vitamin D3 were bound per mole of beta-lactoglobulin A and beta-casein, respectively. It was concluded that these interactions may strongly influence vitamin D3 stability and, hence, bioavailability in processed dairy products.

Changes in compositional parameters of tubers of potato (Solanum tuberosum) during low-temperature storage and their relationship to chip processing quality.

A four-year study of a number of compositional parameters of potato tubers during low-temperature storage was conducted to examine the compositional differences between cold-tolerant (low sugar-accumulating) and cold-sensitive (high sugar-accumulating) tubers in relation to potato chip processing quality. Compositional parameters analyzed included sucrose, reducing sugars, nitrogen, protein, ascorbic acid, and dry matter content. Pearson correlation analysis of the data illustrated that chip color was most closely correlated with reducing sugar concentration. Multiple regression analysis revealed that the relative contribution of each parameter to chip color varied greatly among the cultivars and selections evaluated and from season to season. This analysis demonstrates that the quantitative relationships between the measured compositional parameters and chip color were not sufficient to provide a general predictive index of chip color quality for tubers processed directly from low-temperature storage.