Nuclear transport of the nitric oxide synthase interacting protein (NOSIP)

Proteins that need to actively enter the nucleus are thought to be transported by a specific nuclear transport receptor (NTR), depending on the type of the nuclear localization signal (NLS). Some cargoes are reported to be transported by several NTRs, like Histones, HIV-1-Rev or FUS. In a recent proteomic screen for importin 13 cargoes, Lipin 1 and NOSIP were found as potential import cargoes. However, both cargoes had been suggested to be imported by importin α/β. In this study the role of importin 13 and other NTRs in the nuclear import of Lipin 1 and NOSIP was analyzed. Lipin 1 has a dual function as transcriptional coactivator and as phosphatidate phosphatase. For both functions, the subcellular localization is important. Lipin 1 directly interacted with importin 13 and importin α/β. Further, importin α/β was confirmed as the preferred NTR for Lipin 1. However, importin 13 could partially rescue an importin β knockdown, suggesting that importin 13 is involved in the nuclear import of Lipin 1 as well, perhaps under specific conditions or in specific cell types. Moreover, the region comprising amino acids 398-414 was found to contain a CRM1-dependent NES. The best characterized function of NOSIP is the regulation of eNOS activity by translocating the membrane bound enzyme to the cytoskeleton, specifically in the G2 phase of the cell cycle. For this, NOSIP itself has to translocate from the nucleus to the cytoplasm. The strong nuclear accumulation of NOSIP was shown to depend on active nuclear import, whereas export depends only on passive diffusion. The cytoplasmic enrichment of NOSIP seemed to be regulated through phosphorylation. A phosphomimic mutant of NOSIP was enriched in the cytoplasm, but the nuclear transport was not affected, pointing to a retention of NOSIP through binding to a cytoplasmic binding partner. Moreover, NOSIP specifically interacted with multiple NTRs in a RanGTP-dependent manner. In competition assays, transportin 1 was able to replace all other NTRs from binding to NOSIP. In addition, knockdown experiments showed that transportin 1 is the major NTR for NOSIP. Interestingly, NOISP binds transportin 1 in an unusual binding-mode. The binding-site of NOSIP on transportin 1 was mapped to the N-terminal arch using crosslinking combined with mass spectrometry and interaction studies. This is in contrast to typical PY-NLS or RG/RGG-motif containing transportin 1 cargoes, which bind to the C-terminal arch of transportin 1. This N-terminal binding to NTRs was also observed for importin β and importin 13. No specific region or NLS like sequence of NOSIP could be identified. Instead, using different NOSIP fragments for binding assay and localization studies in cells, showed that several regions are important for the nuclear localization of NOSIP, suggesting that folded domains of NOSIP function as nuclear localization signals.2023-05-3

DNA Renaturation at the Water-Phenol Interface

We study DNA adsorption and renaturation in a water-phenol two-phase system, with or without shaking. In very dilute solutions, single-stranded DNA is adsorbed at the interface in a salt-dependent manner. At high salt concentrations the adsorption is irreversible. The adsorption of the single-stranded DNA is specific to phenol and relies on stacking and hydrogen bonding. We establish the interfacial nature of a DNA renaturation at a high salt concentration. In the absence of shaking, this reaction involves an efficient surface diffusion of the single-stranded DNA chains. In the presence of a vigorous shaking, the bimolecular rate of the reaction exceeds the Smoluchowski limit for a three-dimensional diffusion-controlled reaction. DNA renaturation in these conditions is known as the Phenol Emulsion Reassociation Technique or PERT. Our results establish the interfacial nature of PERT. A comparison of this interfacial reaction with other approaches shows that PERT is the most efficient technique and reveals similarities between PERT and the renaturation performed by single-stranded nucleic acid binding proteins. Our results lead to a better understanding of the partitioning of nucleic acids in two-phase systems, and should help design improved extraction procedures for damaged nucleic acids. We present arguments in favor of a role of phenol and water-phenol interface in prebiotic chemistry. The most efficient renaturation reactions (in the presence of condensing agents or with PERT) occur in heterogeneous systems. This reveals the limitations of homogeneous approaches to the biochemistry of nucleic acids. We propose a heterogeneous approach to overcome the limitations of the homogeneous viewpoint

Methyl Complexes of the Transition Metals

Organometallic chemistry can be considered as a wide area of knowledge that combines concepts of classic organic chemistry, that is, based essentially on carbon, with molecular inorganic chemistry, especially with coordination compounds. Transition-metal methyl complexes probably represent the simplest and most fundamental way to view how these two major areas of chemistry combine and merge into novel species with intriguing features in terms of reactivity, structure, and bonding. Citing more than 500 bibliographic references, this review aims to offer a concise view of recent advances in the field of transition-metal complexes containing M-CH fragments. Taking into account the impressive amount of data that are continuously provided by organometallic chemists in this area, this review is mainly focused on results of the last five years. After a panoramic overview on M-CH compounds of Groups 3 to 11, which includes the most recent landmark findings in this area, two further sections are dedicated to methyl-bridged complexes and reactivity.Ministerio de Ciencia e Innovación Projects CTQ2010–15833, CTQ2013-45011 - P and Consolider - Ingenio 2010 CSD2007 - 00006Junta de Andalucía FQM - 119, Projects P09 - FQM - 5117 and FQM - 2126EU 7th Framework Program, Marie Skłodowska - Curie actions C OFUND – Agreement nº 26722

Kinetics and thermodynamics of DNA hybridization on gold nanoparticles

Hybridization of single-stranded DNA immobilized on the surface of gold nanoparticles (GNPs) into double stranded DNA and its subsequent dissociation into ssDNA were investigated. Melting curves and rates of dissociation and hybridization were measured using fluorescence detection based on hybridization-induced fluorescence change. Two distribution functions, namely the state distribution and the rate distribution, were proposed in order to take interfacial heterogeneity into account and to quantitatively analyze the data. Reaction and activation enthalpies and entropies of DNA hybridization and dissociation on GNPs were derived and compared with the same quantities in solution. Our results show that the interaction between GNPs and DNA reduces the energetic barrier and accelerates the dissociation of adhered DNA. At low surface densities of ssDNA adhered to GNP surface, the primary reaction pathway is that ssDNA in solution first adsorbs onto the GNP, and then diffuses along the surface until hybridizing with an immobilized DNA. We also found that the secondary structure of a DNA hairpin inhibits the interaction between GNPs and DNA and enhances the stability of the DNA hairpin adhered to GNPs

Defective Olfactomedin-2 connects adipocyte dysfunction to obesity

Olfactomedin-2 (OLFM2) is a pleiotropic glycoprotein emerging as a regulator of energy homeostasis. We here show the expression of OLFM2 to be adipocyte-specific and inversely associated with obesity. OLFM2 levels increase during adipogenesis and are suppressed in inflamed adipocytes. Functionally, OLFM2 deficiency impairs adipocyte differentiation, while its over-production enhances the adipogenic transformation of fat cell progenitors. Loss and gain of function experiments revealed that OLFM2 modulates key metabolic and structural pathways, including PPAR signaling, citrate cycle, fatty acid degradation, axon guidance and focal adhesion in 3T3 cell lines and primary human adipocytes. On the molecular level, OLFM2 deficiency in differentiated adipocytes predominantly downregulates genes involved in cell cycle. Extending these findings in vivo, both whole-body Olfm2 knockout and adipose-specific Olfm2 depletion in mice resulted in impaired adipose cell cycle gene expression, with the latter also displaying fat mass accretion and metabolic dysfunction. Collectively, our results underscore a critical role for OLFM2 in adipocyte biology, and support a causative link between reduced adipose OLFM2 and the pathophysiology of obesity

Analysis of In-Vivo LacR-Mediated Gene Repression Based on the Mechanics of DNA Looping

Interactions of E. coli lac repressor (LacR) with a pair of operator sites on the same DNA molecule can lead to the formation of looped nucleoprotein complexes both in vitro and in vivo. As a major paradigm for loop-mediated gene regulation, parameters such as operator affinity and spacing, repressor concentration, and DNA bending induced by specific or non-specific DNA-binding proteins (e.g., HU), have been examined extensively. However, a complete and rigorous model that integrates all of these aspects in a systematic and quantitative treatment of experimental data has not been available. Applying our recent statistical-mechanical theory for DNA looping, we calculated repression as a function of operator spacing (58–156 bp) from first principles and obtained excellent agreement with independent sets of in-vivo data. The results suggest that a linear extended, as opposed to a closed v-shaped, LacR conformation is the dominant form of the tetramer in vivo. Moreover, loop-mediated repression in wild-type E. coli strains is facilitated by decreased DNA rigidity and high levels of flexibility in the LacR tetramer. In contrast, repression data for strains lacking HU gave a near-normal value of the DNA persistence length. These findings underscore the importance of both protein conformation and elasticity in the formation of small DNA loops widely observed in vivo, and demonstrate the utility of quantitatively analyzing gene regulation based on the mechanics of nucleoprotein complexes

mRNA Secondary Structures Fold Sequentially But Exchange Rapidly In Vivo

Self-cleavage assays of RNA folding reveal that mRNA structures fold sequentially in vitro and in vivo, but exchange between adjacent structures is much faster in vivo than it is in vitro