Distinct Roles of the Phosphatidate Phosphatases Lipin 1 and 2 during Adipogenesis and Lipid Droplet Biogenesis in 3T3-L1 Cells

Lipins are evolutionarily conserved Mg²⁺-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis.     

Novel alkenal/one reductase activity of yeast NADPH:quinone reductase Zta1p. Prospect of the functional role for the ζ-crystallin family

ζ-Crystallins are a Zn(2+)-lacking enzyme group with quinone reductase activity, which belongs to the medium-chain dehydrogenase/reductase superfamily. It has been recently observed that human ζ-crystallin is capable of reducing the α,β-double bond of alkenals and alkenones. Here we report that this activity is also shared by the homologous Zta1p enzyme from Saccharomyces cerevisiae. While the two enzymes show similar substrate specificity, human ζ-crystallin exhibits higher activity with lipid peroxidation products and Zta1p is more active with cinnamaldehyde. The presence of Zta1p has an in vivo protective effect on yeast strains exposed to the toxic substrate 3-penten-2-one. Analysis of ZTA1 gene expression indicates an induction under different types of cellular stress, including ethanol and dimethylsulfoxide exposure and by reaching the stationary growth phase. The role of Zta1p in the yeast adaptation to some stress types and the general functional significance of ζ-crystallins are discussed.     

Beyond proteinuria: VDR activation reduces renal inflammation in experimental diabetic nephropathy

Local inflammation is thought to contribute to the progression of diabetic nephropathy. The vitamin D receptor (VDR) activator paricalcitol has an antiproteinuric effect in human diabetic nephropathy at high doses. We have explored potential anti-inflammatory effects of VDR activator doses that do not modulate proteinuria in an experimental model of diabetic nephropathy to gain insights into potential benefits of VDR activators in those patients whose proteinuria is not decreased by this therapy. The effect of calcitriol and paricalcitol on renal function, albuminuria, and renal inflammation was explored in a rat experimental model of diabetes induced by streptozotocin. Modulation of the expression of mediators of inflammation by these drugs was explored in cultured podocytes. At the doses used, neither calcitriol nor paricalcitol significantly modified renal function or reduced albuminuria in experimental diabetes. However, both drugs reduced the total kidney mRNA expression of IL-6, monocyte chemoattractant protein (MCP)-1, and IL-18. Immunohistochemistry showed that calcitriol and paricalcitol reduced MCP-1 and IL-6 in podocytes and tubular cells as well as glomerular infiltration by macrophages, glomerular cell NF-κB activation, apoptosis, and extracellular matrix deposition. In cultured podocytes, paricalcitol and calcitriol at concentrations in the physiological and clinically significant range prevented the increase in MCP-1, IL-6, renin, and fibronectin mRNA expression and the secretion of MCP-1 to the culture media induced by high glucose. In conclusion, in experimental diabetic nephropathy VDR activation has local renal anti-inflammatory effects that can be observed even when proteinuria is not decreased. This may be ascribed to decreased inflammatory responses of intrinsic renal cells, including podocytes, to high glucose.     

Specific degradation of histones H1 and H3

It is shown that acid treated histones H1 and H3 are susceptible to specific degradation by an associated acid resistant protease. Dialysis against distilled water (pH 5.5–6) of the acid treated histones enhances proteolysis. On the other hand, no degradation is observed in nucleohistone either in the presence of Ca⁺⁺ or Na⁺⁺ ions. The conditions required to avoid degradation during nucleohistone and histone manipulation are described.     

Lys6-modified ubiquitin inhibits ubiquitin-dependent protein degradation

Ubiquitin plays essential roles in various cellular processes; therefore, it is of keen interest to study the structure-function relationship of ubiquitin itself. We investigated the modification of Lys(6) of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, of the 7 Lys residues in ubiquitin, Lys(6) was the most readily labeled with sulfosuccinimidobiotin. Lys(6)-biotinylated ubiquitin was incorporated into high molecular mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, Lys(6)-biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with Lys(6)-biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of Lys(6) had similar phenotypes as Lys(6)-biotinylated ubiquitin. Lys(6) mutant ubiquitins (K6A, K6R, and K6W) also inhibited ATP-dependent proteolysis and caused accumulation of ubiquitin conjugates. Conjugates formed with K6W mutant ubiquitin were also resistant to proteasomal degradation. The dominant-negative effect of Lys(6)-modified ubiquitin was further demonstrated in intact cells. Overexpression of K6W mutant ubiquitin resulted in accumulation of intracellular ubiquitin conjugates, stabilization of typical substrates for ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that Lys(6)-modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.     

Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter

We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state.The amino acid/polyamine/organocation (APC)² superfamily comprises about 250 members that occur in all phyla from prokaryotes to higher eukaryotes. These membrane proteins function as solute/cation symporters or solute/solute antiporters (1). One APC subfamily is established by l-amino acid transporters (LATs), which correspond to the light subunits of eukaryotic heteromeric amino acid transporters (2, 3). Heteromeric amino acid transporters are composed of a light subunit that provides transport activity and a disulfide-linked heavy subunit that shows responsibility for plasma membrane targeting. Genetic defects in light and heavy subunits cause a number of inherited human diseases. Mutations in the light as well as the heavy subunit of system b^0,+ lead to cystinuria (4, 5), whereas mutations in the light subunit y⁺LAT1 cause lysinuric protein intolerance (6, 7). Another light subunit, xCT that mediates cysteine uptake and glutamate efflux (8, 9), is involved in vivo in cocaine relapse (10) and maintenance of the plasma redox balance (11). LAT1, the light subunit of system L, is overexpressed in certain primary human tumors. It transports essential neutral amino acids with long, branched, or aromatic side chains required by tumor cells to support their unabated growth (12). Therefore, amino acid transporters like LAT1 are attractive anticancer drug targets.So far a high resolution structure of a eukaryotic LAT family member is not available. However, studies on xCT revealed a membrane topology of 12 transmembrane helices (TMHs) with cytosolic N and C termini and a re-entrant loop structure between TMHs II and III (13). The identified first prokaryotic member of the LAT family, SteT from Bacillus subtilis, is a serine/threonine antiporter, which shows high sequence identity (∼30%) to the light subunits of eukaryotic heteromeric amino acid transporters. Moreover SteT exhibits a similar putative membrane topology and sequential mode of obligate exchange (14). Thus, SteT is an excellent model for studying the structure-function relationship of LAT family members.According to current models, transport proteins undergo functionally related conformational changes. Transporters alternate between two conformations to expose their binding sites to the cytoplasmic and extracellular side (15–22). However, prior to conformational changes substrates have to be recognized and bound. If substrates are amino acids, three main features can be used for specific selection and binding: (i) the negatively charged α-carboxyl group, (ii) the positively charged α-amino group, and (iii) the electrostatic, hydrophobic, or spatial properties of the side chain (22–24). α-Carboxyl and α- amino groups of l-amino acids possess similar structural and chemical characteristics (except for proline); however, their side chains differ in shape, size, and electrostatic properties. Combinations of these features are assumed to establish different interactions within the side chain binding pocket, which determines the substrate specificity of the transporter. The two main substrates of SteT, l-serine and l-threonine, differ by only one methylene group in their side chain; thus they have similar properties. Additionally SteT transports aromatic l-amino acids (Trp, Tyr, and Phe) albeit less efficiently (14).Since its invention, the atomic force microscope (AFM) (25) has evolved from a surface imaging device to a versatile tool for studying interactions of manifold biological systems (26–31). Introduced to characterize interactions between receptor-ligand complexes (32, 33) and complementary DNA strands (34), AFM-based single molecule force spectroscopy (SMFS) has been exploited to explore antibody-antigen recognition (35) and unfolding and refolding of soluble proteins (29, 36) and to probe the adhesion of living cells at molecular resolution (37). Applied to membrane proteins, SMFS uses the AFM stylus to exert a mechanical pulling force to the terminal end of a protein that is embedded and anchored by the lipid membrane (see Fig. 1A) (38). Sufficiently high stretching forces initiate sequential unfolding of the membrane protein with each step indicating the unfolding of a structural segment (39). Recording the applied force over pulling distance results in a force-distance (F-D) curve in which individual force peaks represent the rupture of intra- and intermolecular interactions. The height of a force peak measures the strength of an interaction with piconewton accuracy, and the pulling distance, at which the force peak occurs, allows the interaction within the membrane protein structure to be located (38).Open in a separate windowFIGURE 1.SMFS of SteT. A, pushing the AFM stylus onto the proteoliposomes promotes contacting single transporters to the stylus. This molecular link allows exertion of a mechanical pulling force that initiates stepwise unfolding of SteT. During the experiments, sample and cantilever are immersed in buffer solution. B, F-D curves recorded while unfolding single substrate-free SteT molecules. C, superimpositions of F-D curves recorded while unfolding SteT in buffer lacking any substrate (top) and supplemented with 5 mm l-serine (middle) or 5 mm l-threonine (bottom). Superimpositions are represented as density plots, each calculated from 60 F-D curves. Gray lines represent WLC curves with a persistence length of 0.4 nm and contour length (in amino acids) as indicated by the numbers next to the lines. The contour lengths were obtained from the Gaussian fits shown in D. F-D curves were obtained at room temperature at a pulling velocity of 654 nm/s in buffer solution (150 mm NaCl, 20 mm Tris-HCl, pH 8.0, substrate as indicated). D, frequency of force peaks detected at different positions of the stretched polypeptide. Every force peak detected in individual F-D curves (B) was fitted using the WLC model with the contour length of the stretched polypeptide as the only fitting parameter. The frequency at which the force peaks appeared is plotted in the histogram: substrate-free, n = 132; 5 mm l-serine, n = 128; and 5 mm l-threonine, n = 127. The bin size of the histograms is 3 aa and reflects the accuracy of fitting the WLC model (55) to individual force peaks. Error bars representing the S.E. were calculated using S.E. = (p(1 − p)/n)^0.5 where p is the probability and n is the total number of F-D curves. The width of each force peak distribution is given by the experimental noise, conformational variability of the structural segments, and fitting accuracy of the force peaks (53, 99–102). The gray solid curve represents the sum of seven Gaussian fits to the seven main peaks from the histograms and superimpositions (C). Numbers next to peaks denote peak positions (measured in amino acids) obtained from Gaussian fits.Besides quantification and localization of molecular interactions in membrane proteins, SMFS provides information about their energy landscape. For that purpose, the interactions of membrane proteins are probed over a range of different time scales by dynamic force spectroscopy (DFS). Bell (40) and Evans and co-worker (41, 42) provided the most commonly used theoretical framework to analyze DFS data. Their model describes the deformation of the energy landscape by an externally applied force, F. Such force-induced deformations reduce the energy barriers that separate bound and unbound states (see Fig. 2). Consequently transition rates over such energy barriers are force-dependent. Probing the interactions at different pulling velocities and thus at different force loading rates, r_f, leads to a so-called dynamic force spectrum in which the most probable force, F*, of rupture is plotted versus the logarithm of r_f. In these dynamic force spectra, each linear regime represents an energy barrier. Energy barriers located closer to the bound state are probed at higher pulling velocities because the energy barriers located further from the bound state are suppressed by increasingly applied forces (see Fig. 2) (41). The slope of each linear regime measures the distance from the ground state to the transition state, whereas extrapolation of a linear regime to zero force provides the rate constant of crossing the corresponding barrier in the absence of any load. These two parameters allow an estimate of the rigidity of the probed structure (43, 44).Open in a separate windowFIGURE 2.Energy landscape tilted by force. Schematic representation of the free energy profile along the reaction coordinate and applied force according to the Bell-Evans theory (40–42). The potential along the reaction coordinate (vector of force) in the absence of force (black curve) exhibits two energy barriers separating the folded from the unfolded state. Application of an external force, F, changes the thermal likelihood of reaching the top of the energy barrier(s). Although for a sharp barrier the position, x_u, of the energy barrier relative to the folded state is not changed, the thermally averaged projection of the energy profile along the pulling direction is tilted by the mechanical energy (−F·cos θ)x (long-dashed line). This tilt decreases the energy barriers (short-dashed curve). Consequently the relevant energy barrier that has to be overcome is the outermost barrier. At slow pulling velocities, the thermal contribution is higher, and therefore, the mechanical energy required to overcome the barrier is smaller. With increasing pulling velocities, the barriers are further lowered. At some velocity, the height of the outer barrier will be lower than that of the inner barrier (short-dashed curve), which then becomes the relevant energy barrier to be overcome. Each energy barrier manifests as a linear regime in dynamic force spectra (Fig. 3).In this study, we applied SMFS to characterize molecular interactions that stabilize SteT in the absence and in the presence of its substrates, l-serine and l-threonine. We used DFS to characterize how substrate binding changes the energy landscape and the mechanical properties of the antiporter. It was observed that the structural regions stabilized within SteT did not depend on substrate binding. However, substrate binding dynamically changed the energy landscape of these structures. In the absence of substrate all structural regions within SteT were stabilized by a narrow inner energy barrier and co-stabilized by a second outer energy barrier. The unique properties of these energy barriers restricted the conformation of SteT thereby trapping the antiporter in a kinetically instable and mechanically rigid conformation. In contrast, substrate binding sets SteT into a different energy minimum that significantly increased the kinetic stability and conformational flexibility of the antiporter.