Design of Nanohydrogels for Targeted Intracellular Drug Transport to the Trans-Golgi Network.

Nanohydrogels combine advantages of hydrogels and nanoparticles. In particular, they represent promising drug delivery systems. Nanogel synthesis by oxidative condensation of polyglycidol prepolymers, that are modified with thiol groups, results in crosslinking by disulfide bonds. Hereby, biomolecules like the antidiabetic peptide RS1-reg, derived from the regulatory protein RS1 of the Na+ -D-glucose cotransporter SGLT1, can be covalently bound by cysteine residues to the nanogel in a hydrophilic, stabilizing environment. After oral uptake, the acid-stable nanogels protect their loading during gastric passage from proteolytic degradation. Under alkaline conditions in small intestine the nanohydrogels become mucoadhesive, pass the intestinal mucosa and are taken up into small intestinal enterocytes by endocytosis. Using Caco-2 cells as a model for small intestinal enterocytes, by confocal laser scanning microscopy and structured illumination microscopy, the colocalization of fluorescent-labeled RS1-reg with markers of endosomes, lysosomes, and trans-Golgi-network after uptake with polyglycidol-based nanogels formed by precipitation polymerization is demonstrated. This indicates that RS1-reg follows the endosomal pathway. In the following, the design of bespoken nanohydrogels for specific targeting of RS1-reg to its site of action at the trans-Golgi network is described that might also represent a way of targeted transport for other drugs to their targets at the Golgi apparatus.

Cell-free expression tools to study co-translational folding of alpha helical membrane transporters.

Most helical membrane proteins fold co-translationally during unidirectional polypeptide elongation by the ribosome. Studies thus far, however, have largely focussed on refolding full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation offers opportunities to remedy this deficit in folding studies and has previously been used for membrane proteins. We exploit this cell-free approach to develop tools to probe co-translational folding. We show that two transporters from the ubiquitous Major Facilitator Superfamily can successfully insert into a synthetic bilayer without the need for translocon insertase apparatus that is essential in vivo. We also assess the cooperativity of domain insertion, by expressing the individual transporter domains cell-free. Furthermore, we manipulate the cell-free reaction to pause and re-start protein synthesis at specific points in the protein sequence. We find that full-length protein can still be made when stalling after the first N terminal helix has inserted into the bilayer. However, stalling after the first three helices have exited the ribosome cannot be successfully recovered. These three helices cannot insert stably when ribosome-bound during co-translational folding, as they require insertion of downstream helices.

Molecular cloning of SLC35D3 and analysis of its role during porcine intramuscular preadipocyte differentiation.

BACKGROUND: Solute carrier family 35 (SLC35) is one of a large number of membrane transporter protein families. Member D3 of this family is thought to be involved in adipose deposition and metabolic control. RESULTS: We obtained 2238 bp cDNA of porcine SLC35D3, it contains a 1272 bp ORF, encoding a 423 amino acid polypeptide, and a 966 bp 3' UTR. BLAST results revealed that the amino acid sequence of porcine SLC35D3 had the closest phylogenetic relationship with members of the genus Ovis aries. Further bioinformatics analysis showed that the SLC35D3 protein contains 8 transmembrane domains, and that there is no signal peptide structure. The secondary structure of the protein mainly contains 37.12% α-helixes, 7.8% in ß-folds, and 33.57% random coils. mRNA expression analysis showed that SLC35D3 is expressed in lung, liver, heart, spleen, kidney, longissimus dorsi muscle (LDM), leaf fat (LF), and subcutaneous adipose tissue (SAT). To examine the effects of SLC35D3 expression on fat synthesis and catabolism, SLC35D3-siRNA was transfected into cultured intramuscular adipocytes. SLC35D3 silenced cells showed increased expression of genes related to fat synthesis, and increased deposition of intramuscular fat (IMF), abundance of lipid droplets, and the level of free fatty acid (FFA) in the culture medium. In contrast, the siRNA decreased the expression genes involved in fat catabolism. CONCLUSIONS: Our results demonstrate that silenced SLC35D3 results in increased adipogenic processes in pig intramuscular adipocytes. These data represent the first exploration of SLC35D3 expression in swine, and provide valuable insights into the functions of SLC35D3 in adipocyte differentiation.

In silico analysis of the structural and functional implications of SLC19A1 R27H polymorphism.

In view of the documented association of solute carrier family 19 member 1 (SLC19A1) G80A (R27H) polymorphism with the risk for different types of cancers and systemic lupus erythematosus (SLE), we have reanalysed the case-control study on breast cancer to ascertain the conditions in which this polymorphic variant exerts the risk of breast cancer. Association statistics have revealed that this polymorphism exerts the risk for breast cancer under the conditions of low folate intake, and in the absence of well-documented protective polymorphism in cytosolic serine hydroxymethyltransferase. To substantiate this observation, we have developed a homology model of SLC19A1 using glycerol-3-phosphate transporter (d1pw4a) as a template where 73% of the residues were modelled at 90% confidence while 162 residues were modelled ab initio. The wild and mutant proteins shared same topology in S3, S5, S6, S7, S11 and S12 transmembrane domains. The topology varied at S1 (28-43 residue vs 28-44 residue), S2 (66-87 residue vs 69-87 residue), S4 (117-140 residue vs 117-139 residue), S8 (305-325 residue vs 305-324 residue), S9 (336-356 residue vs 336-355residue), and S10 (361-386 residue vs 361-385 residue) transmembrane domains between wild versus mutant proteins. S2 domain is shortened by three amino acid residues in the mutant while in other domains the difference corresponds to one amino acid residue. The 3DLigandSite analysis revealed that the metallic-ligand-binding sites at 273Trp, 277Asn, 379Leu, 439Phe and 442Leu are although unaffected, there is a loss of active sites corresponding to nonmetallic ligand binding. Tetrahydrofolate and methotrexate have lesser affinity towards the mutant protein than the wild protein. To conclude, the R27H polymorphism affects the secondary and tertiary structures of SLC19A1 with the significant loss in ligand-binding sites.

Direct visualization of the E. coli Sec translocase engaging precursor proteins in lipid bilayers.

Escherichia coli exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA2 primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF3. Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.

Structural and functional characterization of a maltose/maltodextrin ABC transporter comprising a single solute binding domain (MalE) fused to the transmembrane subunit MalF.

Canonical ATP-binding cassette import systems rely on extracellular substrate binding proteins (SBP) for function. In gram-negative bacteria, SBPs are usually freely diffusible in the periplasm and, where studied, exist in excess over their cognate transporters. However, in vitro studies with the maltose transporter of Escherichia coli (MalFGK2) have demonstrated that mechanistically one copy of its SBP (MalE) per transport complex is sufficient for activity. To address whether such a condition is physiologically relevant, we have characterized a homolog of the E. coli system from the gram-negative bacterium Bdellovibrio bacteriovorus which has a single copy of a maltose binding domain fused to the MalF subunit. Both transporters share substrate specificity for maltose and linear maltodextrins. Specific ATPase and transport activities of the B. bacteriovorus transporter were comparable to those of the E. coli system assayed at a 1:1 M ratio of MalE to the transport complex. While MalEEc was able to additionally increase ATPase activity of MalFGK2Bb, the isolated MalE domain of B. bacteriovorus failed to stimulate the E. coli system. Strikingly, interactions of the MalE domain with the transmembrane subunits during the transport cycle as studied by site-specific cross-linking were found to differ from those observed for E. coli MalE-FGK2.

Label-free cytosensing of cancer cells based on the interaction between protein and an electron-transfer carbohydrate-mimetic peptide.

We used an electron-transfer carbohydrate-mimetic peptide (YYYYC) to construct an electrochemical cytosensing system. Magnetic beads were modified with either asialofetuin (ASF) or soybean agglutinin (SBA) to evaluate the effect on cell sensing. Because SBA binds to the galactose residue that exists at the terminals of the carbohydrate chains in ASF, the target protein was accumulated on the protein magnetic beads. SBA is an example of N-acetylgalactosamine- and galactose-binding proteins that readily combine with YYYYC. When the peptides and protein-immobilized beads competed for a target protein, the peak current of the peptides changed according to the concentration of the protein at the 10-12 M level. Next, human myeloid leukemia cells (K562 cell) were measured using the peptide and the carbohydrate chains on the cell surface that recognize SBA. The electrode response was linear to the number of K562 cells and ranged from 1.0 × 102 to 5.0 × 103 cells mL-1. In addition, detection of a human liver cancer cell (HepG2 cell) was carried out using interactions with the peptide, the ASF receptors in HepG2 cells, and the carbohydrate chains of ASF. The peak currents were proportional and ranged between 5.0 × 101 and 1.5 × 103 cells mL-1. When the values estimated from an electrochemical process were compared with those obtained by ELISA, the results were within the acceptable range of measurement error.

d-Amino Acid Pseudopeptides as Potential Amyloid-Beta Aggregation Inhibitors.

A causative factor for neurotoxicity associated with Alzheimer's disease is the aggregation of the amyloid-ß (Aß) peptide into soluble oligomers. Two all d-amino acid pseudo-peptides, SGB1 and SGD1, were designed to stop the aggregation. Molecular dynamics (MD) simulations have been carried out to study the interaction of the pseudo-peptides with both Aß13⁻23 (the core recognition site of Aß) and full-length Aß1⁻42. Umbrella sampling MD calculations have been used to estimate the free energy of binding, ∆G, of these peptides to Aß13⁻23. The highest ∆Gbinding is found for SGB1. Each of the pseudo-peptides was also docked to Aß1⁻42 and subjected up to seven microseconds of all atom molecular dynamics simulations. The resulting structures lend insight into how the dynamics of Aß1⁻42 are altered by complexation with the pseudo-peptides and confirmed that SGB1 may be a better candidate for developing into a drug to prevent Alzheimer's disease.

AXER is an ATP/ADP exchanger in the membrane of the endoplasmic reticulum.

To fulfill its role in protein biogenesis, the endoplasmic reticulum (ER) depends on the Hsp70-type molecular chaperone BiP, which requires a constant ATP supply. However, the carrier that catalyzes ATP uptake into the ER was unknown. Here, we report that our screen of gene expression datasets for member(s) of the family of solute carriers that are co-expressed with BiP and are ER membrane proteins identifies SLC35B1 as a potential candidate. Heterologous expression of SLC35B1 in E. coli reveals that SLC35B1 is highly specific for ATP and ADP and acts in antiport mode. Moreover, depletion of SLC35B1 from HeLa cells reduces ER ATP levels and, as a consequence, BiP activity. Thus, human SLC35B1 may provide ATP to the ER and was named AXER (ATP/ADP exchanger in the ER membrane). Furthermore, we propose an ER to cytosol low energy response regulatory axis (termed lowER) that appears as central for maintaining ER ATP supply.

Clec16a, Nrdp1, and USP8 Form a Ubiquitin-Dependent Tripartite Complex That Regulates ß-Cell Mitophagy.

Mitophagy is a cellular quality-control pathway, which is essential for elimination of unhealthy mitochondria. While mitophagy is critical to pancreatic ß-cell function, the posttranslational signals governing ß-cell mitochondrial turnover are unknown. Here, we report that ubiquitination is essential for the assembly of a mitophagy regulatory complex, comprised of the E3 ligase Nrdp1, the deubiquitinase enzyme USP8, and Clec16a, a mediator of ß-cell mitophagy with unclear function. We discover that the diabetes gene Clec16a encodes an E3 ligase, which promotes nondegradative ubiquitin conjugates to direct its mitophagy effectors and stabilize the Clec16a-Nrdp1-USP8 complex. Inhibition of the Clec16a pathway by the chemotherapeutic lenalidomide, a selective ubiquitin ligase inhibitor associated with new-onset diabetes, impairs ß-cell mitophagy, oxygen consumption, and insulin secretion. Indeed, patients treated with lenalidomide develop compromised ß-cell function. Moreover, the ß-cell Clec16a-Nrdp1-USP8 mitophagy complex is destabilized and dysfunctional after lenalidomide treatment as well as after glucolipotoxic stress. Thus, the Clec16a-Nrdp1-USP8 complex relies on ubiquitin signals to promote mitophagy and maintain mitochondrial quality control necessary for optimal ß-cell function.

Critical Temperature of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine Monolayers and Its Possible Biological Relevance.

Because transmembrane proteins (TMPs) can be obtained with sufficient purity for X-ray diffraction studies more frequently than decades ago, their mechanisms of action may now be elucidated. One of the pending issues is the actual interplay between transmembrane proteins and membrane lipids. There is strong evidence of the involvement of specific lipids with some membrane proteins, such as the potassium crystallographically sited activation channel (KcsA) of Streptomyces lividans and the secondary transporter of lactose LacY of Escherichia coli, the activities of which are associated with the presence of anionic phospholipids such as the phosphatidylglycerol (PG) and phosphatidyethanolamine (PE), respectively. Other proteins such as the large conductance mechanosensitive channel (MscL) of E. coli seem to depend on the adaptation of specific phospholipids to the irregular surface of the integral membrane protein. In this work we investigated the lateral compressibility of two homoacid phosphatidylethanolamines (one with both acyl chains unsaturated (DOPE), the other with the acyl chains saturated (DPPE)) and the heteroacid phosphatidyletanolamine (POPE) and their mixtures with POPG. The liquid expanded (LE) to liquid condensed (LC) transition was observed in POPE at a temperature below its critical temperature (Tc = 36 °C). Because Tc lies below the physiological temperature, the occurrence of this phase transition may have something to do with the functioning of LacY. This magnitude is discussed within the context of the experiments carried out at temperatures below the Tc of POPE at which the activity of Lac Y and other TMPs are frequently studied.

Pull-and-Paste of Single Transmembrane Proteins.

How complex cytoplasmic membrane proteins insert and fold into cellular membranes is not fully understood. One problem is the lack of suitable approaches that allow investigating the process by which polypeptides insert and fold into membranes. Here, we introduce a method to mechanically unfold and extract a single polytopic α-helical membrane protein, the lactose permease (LacY), from a phospholipid membrane, transport the fully unfolded polypeptide to another membrane and insert and refold the polypeptide into the native structure. Insertion and refolding of LacY is facilitated by the transmembrane chaperone/insertase YidC in the absence of the SecYEG translocon. Insertion into the membrane occurs in a stepwise, stochastic manner employing multiple coexisting pathways to complete the folding process. We anticipate that our approach will provide new means of studying the insertion and folding of membrane proteins and to mechanically reconstitute membrane proteins at high spatial precision and stoichiometric control, thus allowing the functional programming of synthetic and biological membranes.

Identification of residues critical for proton-coupled glutathione translocation in the yeast glutathione transporter, Hgt1p.

The proton gradient acts as the driving force for the transport of many metabolites across fungal and plant plasma membranes. Identifying the mechanism of proton relay is critical for understanding the mechanism of transport mediated by these transporters. We investigated two strategies for identifying residues critical for proton-dependent substrate transport in the yeast glutathione transporter, Hgt1p, a member of the poorly understood oligopeptide transporter family of transporters. In the first strategy, we tried to identify the pH-independent mutants that could grow at higher pH when dependant on glutathione transport. Screening a library of 269 alanine mutants of the transmembrane domains (TMDs) along with a random mutagenesis strategy yielded two residues (E135K on the cusp of TMD2 and N710S on TMD12) that permitted growth on glutathione at pH 8.0. Further analysis revealed that these residues were not involved in proton symport even though they conferred better transport at a higher pH. The second strategy involved a knowledge-driven approach, targeting 31 potential residues based on charge, conservation and location. Mutation of these residues followed by functional and biochemical characterization revealed E177A, Y193A, D335A, Y374A, H445A and R554A as being defective in proton transport. Further analysis enabled possible roles of these residues to be assigned in proton relay. The implications of these findings in relation to Hgt1p and the suitability of these strategic approaches for identifying such residues are discussed.

Tunable GLUT-Hexose Binding and Transport via Modulation of Hexose C-3 Hydrogen-Bonding Capabilities.

The importance of the hydrogen bonding interactions in the GLUT-hexose binding process (GLUT=hexose transporter) has been demonstrated by studying the binding of structurally modified d-fructose analogues to GLUTs, and in one case its transport into cells. The presence of a hydrogen bond donor at the C-3 position of 2,5-anhydro-d-mannitol derivatives is essential for effective binding to GLUT5 and transport into tumor cells. Surprisingly, installation of a group that can function only as a hydrogen bond acceptor at C-3 resulted in selective recognition by GLUT1 rather than GLUT5. A fluorescently labelled analogue clearly showed GLUT-mediated transport and low efflux properties of the probe. This study reveals that a single positional modification of a 2,5-anhydro-d-mannitol derivative is sufficient to switch its binding preference from GLUT5 to GLUT1, and uncovers general scaffolds that are suitable for the potential selective delivery of molecular payloads into tumor cells via GLUT transport machinery.

An Asymmetric Conformational Change in LacY.

Galactoside/H+ symport by the lactose permease of Escherichia coli (LacY) involves reciprocal opening and closing of periplasmic and cytoplasmic cavities so that sugar- and H+-binding sites become alternatively accessible to either side of the membrane. After reconstitution into proteoliposomes, LacY with the periplasmic cavity sealed by cross-linking paired-Cys residues does not bind sugar from the periplasmic side. However, reduction of the S-S bond restores opening of the periplasmic cavity and galactoside binding. Furthermore, nanobodies that stabilize the double-Cys mutant in a periplasmic-open conformation and allow free access of galactoside to the binding site do so only after reduction of the S-S bond. In contrast, when cross-linked LacY is solubilized in detergent, galactoside binding is observed, indicating that the cytoplasmic cavity is patent. Sugar binding from the cytoplasmic side exhibits nonlinear stopped-flow kinetics, and analysis reveals a two-step process in which a conformational change precedes binding. Because the cytoplasmic cavity is spontaneously closing and opening in the symporter with a sealed periplasmic cavity, it is apparent that an asymmetrical conformational transition controls access of sugar to the binding site.

An insight into the orphan nucleotide sugar transporter SLC35A4.

SLC35A4 has been classified in the SLC35A subfamily based on amino acid sequence homology. Most of the proteins belonging to the SLC35 family act as transporters of nucleotide sugars. In this study, the subcellular localization of endogenous SLC35A4 was determined via immunofluorescence staining, and it was demonstrated that SLC35A4 localizes mainly to the Golgi apparatus. In silico topology prediction suggests that SLC35A4 has an uneven number of transmembrane domains and its N-terminus is directed towards the Golgi lumen. However, an experimental assay refuted this prediction: SLC35A4 has an even number of transmembrane regions with both termini facing the cytosol. In vivo interaction analysis using the FLIM-FRET approach revealed that SLC35A4 neither forms homomers nor associates with other members of the SLC35A subfamily except SLC35A5. Additional assays demonstrated that endogenous SLC35A4 is 10 to 40nm proximal to SLC35A2 and SLC35A3. To determine SLC35A4 function SLC35A4 knock-out cells were generated with the CRISPR-Cas9 approach. Although no significant changes in glycosylation were observed, the introduced mutation influenced the subcellular distribution of the SLC35A2/SLC35A3 complexes. Additional FLIM-FRET experiments revealed that overexpression of SLC35A4-BFP together with SLC35A3 and the SLC35A2-Golgi splice variant negatively affects the interaction between the two latter proteins. The results presented here strongly indicate a modulatory role for SLC35A4 in intracellular trafficking of SLC35A2/SLC35A3 complexes.

Kinetics and Structure of a Cold-Adapted Hetero-Octameric ATP Phosphoribosyltransferase.

Adenosine 5'-triphosphate phosphoribosyltransferase (ATPPRT) catalyzes the first step in histidine biosynthesis, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to generate N1-(5-phospho-ß-d-ribosyl)-ATP and inorganic pyrophosphate. The enzyme is allosterically inhibited by histidine. Two forms of ATPPRT, encoded by the hisG gene, exist in nature, depending on the species. The long form, HisGL, is a single polypeptide chain with catalytic and regulatory domains. The short form, HisGS, lacks a regulatory domain and cannot bind histidine. HisGS instead is found in complex with a regulatory protein, HisZ, constituting the ATPPRT holoenzyme. HisZ triggers HisGS catalytic activity while rendering it sensitive to allosteric inhibition by histidine. Until recently, HisGS was thought to be catalytically inactive without HisZ. Here, recombinant HisGS and HisZ from the psychrophilic bacterium Psychrobacter arcticus were independently overexpressed and purified. The crystal structure of P. arcticus ATPPRT was determined at 2.34 Å resolution, revealing an equimolar HisGS-HisZ hetero-octamer. Steady-state kinetics indicate that both the ATPPRT holoenzyme and HisGS are catalytically active. Surprisingly, HisZ confers only a modest 2-4-fold increase in kcat. Reaction profiles for both enzymes cannot be distinguished by 31P nuclear magnetic resonance, indicating that the same reaction is catalyzed. The temperature dependence of kcat shows deviation from Arrhenius behavior at 308 K with the holoenzyme. Interestingly, such deviation is detected only at 313 K with HisGS. Thermal denaturation by CD spectroscopy resulted in Tm's of 312 and 316 K for HisZ and HisGS, respectively, suggesting that HisZ renders the ATPPRT complex more thermolabile. This is the first characterization of a psychrophilic ATPPRT.

Investigating cell-substrate and cell-cell interactions by means of single-cell-probe force spectroscopy.

Cell adhesion forces are typically a mixture of specific and nonspecific cell-substrate and cell-cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell-cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 µg/mL (FN20) and 100 µg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech. 80:124-130, 2017. © 2016 Wiley Periodicals, Inc.

Toward high-resolution computational design of the structure and function of helical membrane proteins.

The computational design of α-helical membrane proteins is still in its infancy but has already made great progress. De novo design allows stable, specific and active minimal oligomeric systems to be obtained. Computational reengineering can improve the stability and function of naturally occurring membrane proteins. Currently, the major hurdle for the field is the experimental characterization of the designs. The emergence of new structural methods for membrane proteins will accelerate progress.