José M. González

Role of A-type lamins in signaling, transcription, and chromatin organization

A-type lamins (lamins A and C), encoded by the LMNA gene, are major protein constituents of the mammalian nuclear lamina, a complex structure that acts as a scaffold for protein complexes that regulate nuclear structure and functions. Interest in these proteins has increased in recent years with the discovery that LMNA mutations cause a variety of human diseases termed laminopathies, including progeroid syndromes and disorders that primarily affect striated muscle, adipose, bone, and neuronal tissues. In this review, we discuss recent research supporting the concept that lamin A/C and associated nuclear envelope proteins regulate gene expression in health and disease through interplay with signal transduction pathways, transcription factors, and chromatin-associated proteins.

Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope

Sequestration of c-Fos at the nuclear envelope (NE) through interaction with A-type lamins suppresses AP-1–dependent transcription. We show here that c-Fos accumulation within the extraction-resistant nuclear fraction (ERNF) and its interaction with lamin A are reduced and enhanced by gain-of and loss-of ERK1/2 activity, respectively. Moreover, hindering ERK1/2-dependent phosphorylation of c-Fos attenuates its release from the ERNF induced by serum and promotes its interaction with lamin A. Accordingly, serum stimulation rapidly releases preexisting c-Fos from the NE via ERK1/2-dependent phosphorylation, leading to a fast activation of AP-1 before de novo c-Fos synthesis. Moreover, lamin A–null cells exhibit increased AP-1 activity and reduced levels of c-Fos phosphorylation. We also find that active ERK1/2 interacts with lamin A and colocalizes with c-Fos and A-type lamins at the NE. Thus, NE-bound ERK1/2 functions as a molecular switch for rapid mitogen-dependent AP-1 activation through phosphorylation-induced release of preexisting c-Fos from its inhibitory interaction with lamin A/C.

Complete genome sequence of transmissible gastroenteritis coronavirus PUR46-MAD clone and evolution of the purdue virus cluster.

The complete sequence (28580u2009nt) of the PUR46-MAD clone of the Purdue cluster of transmissible gastroenteritis coronavirus (TGEV) has been determined and compared with members of this cluster and other coronaviruses. The computing distances among their S gene sequences resulted in the grouping of these coronaviruses into four clusters, one of them exclusively formed by the Purdue viruses. Three new potential sequence motifs with homology to the α-subunit of the polymerase-associated nucleocapsid phosphoprotein of rinderpest virus, the Bowman–Birk type of proteinase inhibitors, and the metallothionein superfamily of cysteine rich chelating proteins have been identified. Comparison of the TGEV polymerase sequence with that of other RNA viruses revealed high sequence homology with the A–E domains of the palm subdomain of nucleic acid polymerases.

Intermolecular Reaction of Internal Alkynes and Imines: Propargyl Tosylates as Key Partners in a Gold-Catalyzed [4 + 1] Unusual Cyclization Leading to Cyclopent-2-enimines

Propargyl tosylates react with N-tosylaldimines to afford cyclopent-2-enimines in a gold-catalyzed process that involves a deep reorganization of both substrates. The formal [4 + 1] cyclization is initiated by a 1,2-migration of the tosylate that eventually generates a substituted 1,3-diene. Subsequent interaction with the imine launches a series of reaction steps prior to a Nazarov-like cyclization to yield the final product.

Scoring docking models with evolutionary information

We have developed methods for the extraction of evolutionary information from multiple sequence alignments for use in the study of the evolution of protein interaction networks and in the prediction of protein interaction. For Rounds 3, 4, and 5 of the CAPRI experiment, we used scores derived from the analysis of multiple sequence alignments to submit predictions for 7 of the 12 targets. Our docking models were generated with Hex and GRAMM, but all our predictions were selected using methods based on multiple sequence alignments and on the available experimental evidence. With this approach, we were able to predict acceptable level models for 4 of the targets, and for a fifth target, we located the residues involved in the binding surface. Here we detail our successes and highlight several of the limitations and problems that we faced while dealing with particular docking cases. Proteins 2005;60:275–280.

Cross-cultural adaptation and validation of the Spanish version of the Calgary Depression Scale for Schizophrenia

BACKGROUNDnThe Calgary Depression Scale for Schizophrenia (CDSS) is a valid tool to assess depression in schizophrenics and has been translated, adapted, and validated to be used in different non-English languages. Therefore, it may be predicted that a Spanish version of this scale will be also a valid instrument to assess symptoms of depression in patients with schizophrenia.nnnOBJECTIVEnWe determined the validity of the Spanish version of the Calgary scale (CDSS-S).nnnMETHODSnOutpatients and inpatients (n=93) diagnosed as having schizophrenia by DSM-IV criteria confirmed by SCID-IV interview were included. The Positive and Negative Syndrome Scale (PANSS), Hamilton Depression Rating Scale (HDRS-17 and HDRS-21 items), Montgomery-Asberg Depression Rating Scale (MADRS), Extrapyramidal Symptoms Rating Scale (ESRS), and Barnes Acathisia Rating Scale were administered by a first rater, whereas the CDSS-S was assessed by a second independent rater.nnnRESULTSnThe internal consistency (Cronbachs alpha 0.83) and the interrater reliability (>0.73 intraclass correlation coefficient [ICC] for single items and 0.92 for total score) were good. The test-retest reliability was high (ICC of 0.89). The scale showed a good construct validity with statistically significant correlations with HDRS-17, HDRS-21, MADRS, and G6 item (depression) of PANSS. The CDSS showed no correlation with the positive subscale of PANSS and a weak correlation with the negative subscale, general psychopathology subscale, and total score of PANSS. A cut point of five showed 94.7% sensitivity, 86.5% specificity, and 70% and 98% positive and negative predictive values, respectively.nnnCONCLUSIONSnThe Spanish version of CDSS is a valid instrument to assess depressive episodes for stabilized and acute patients with schizophrenia.

Coronavirus Derived Expression Systems

n Abstractn n Both helper dependent expression systems, based on two components, and single genomes constructed by targeted recombination, or by using infectious cDNA clones, have been developed. The sequences that regulate transcription have been characterized mainly using helper dependent expression systems and it will now be possible to validate them using single genomes. The genome of coronaviruses has been engineered by modification of the infectious cDNA leading to an efficient (>20 μg ml−1) and stable (>20 passages) expression of the foreign gene. The possibility of engineering the tissue and species tropism to target expression to different organs and animal species, including humans, increases the potential of coronaviruses as vectors. Thus, coronaviruses are promising virus vectors for vaccine development and, possibly, for gene therapy.n n

Capillary HPLC-ICPMS and Tyrosine Iodination for the Absolute Quantification of Peptides Using Generic Standards

The validity of using tyrosine iodination chemistry for the absolute and generic quantification of peptides by capillary high-performance liquid chromatography (capHPLC) coupled to inductively coupled plasma mass spectrometry (ICPMS) is investigated in detail. In this approach, two iodine atoms are specifically bioconjugated to the meta positions of the aromatic ring of every tyrosine residue. Characterization studies by capHPLC with parallel ICPMS and electrospray ionization tandem mass spectrometry (ESIMS/MS) detection clearly showed that such labeling iodination reaction affords one to obtain most accurate peptide determinations (after translation of the picomoles of iodine, quantified by ICPMS in each chromatographic peak, into picomoles of the corresponding labeled peptide). It is demonstrated that only, but every, tyrosine residue present in the peptide is completely diiodinated. The excellent detection limits for iodine using ICPMS allowed robust and highly sensitive tyrosine-containing peptide quantification (480 pM, 480 amol absolute). Derivatization is easily accomplished in a water/acetonitrile solution in only 2 min. Moreover, since the signal in ICPMS is completely independent from the chemical species containing the detected element, any iodine-containing standard (e.g., iodobenzoic acid) could be used as internal standard for the absolute quantification of every iodine-labeled tyrosine-containing peptide separated and detected along the gradient. The approach was optimized for tyrosine labeling and then validated by application to the absolute quantification of the three standard peptides present in the only reference material for peptide quantity (NIST 8327) commercially available. Identification of the species quantified by ICPMS was carried out by parallel capHPLC-ESI quadrupole time-of-flight (Q/TOF) analysis and corresponded, as expected, to the diiodinated peptides. The collision-induced dissociation (CID) spectra obtained demonstrated unequivocally the specific and complete derivatization of the tyrosine residues. The obtained quantitative results closely matched the reference values reported by the National Institute of Standards and Technology (NIST). In terms of precision, the relative standard deviation was as low as 3% RSD. Finally the approach was tested for the absolute quantification of proteins using a model standard protein (beta-casein). Results agreed again with the value specified showing that this labeling reaction is compatible with tryptic digestion.

A-type lamins and Hutchinson-Gilford progeria syndrome: pathogenesis and therapy

Lamin A and lamin C (A-type lamins, both encoded by the LMNA gene) are major components of the mammalian nuclear lamina, a complex proteinaceous structure that acts as a scaffold for protein complexes that regulate nuclear structure and function. Abnormal accumulation of farnesylated-progerin, a mutant form of prelamin A, plays a key role in the pathogenesis of the Hutchinson-Gilford progeria syndrome (HGPS), a devastating disorder that causes the death of affected children at an average age of 13.5 years, predominantly from premature atherosclerosis and myocardial infarction or stroke. Remarkably, progerin is also present in normal cells and appears to progressively accumulate during aging of non-HGPS cells. Therefore, understanding how this mutant form of lamin A provokes HGPS may shed significant insight into physiological aging. In this review, we discuss recent advances into the pathogenic mechanisms underlying HGPS, the main murine models of the disease, and the therapeutic strategies developed in cellular and animal models with the aim of reducing the accumulation of farnesylated-progerin, as well as their use in clinical trials of HGPS.

Iodoarylation Reactions of Allenes: Inter‐ and Intramolecular Processes

Polar addition reactions to allenes are well established and various processes involving this mechanistic pathway are known. The reaction of arenes with electrophiles is a conceptually powerful method to functionalize C H bonds. Therefore, the feasibility of using carbon-based electrophiles, in reactions with arenes, was soon recognized as an entry for C C bond formation. The Friedel–Crafts alkylation of arenes with alkenes is a relevant example. Nevertheless, the related alkenylation reaction involving alkynes was more sluggishly developed and only recent advances involving metal-catalyzed transformations have made this target practicable. The reaction of arenes with the transitory species generated upon interaction of iodonium ions with alkenes or alkynes represents a powerful route for preparing cyclic compounds involving related C C bondforming processes. Moreover, examples of Friedel–Crafts arylation reactions of allenes are rare. Recent advances involve formally related processes using precious metal catalysts and are mainly intramolecular transformations. Herein, we report the first solid evidence on the use of iodonium ions as the trigger for an unprecedented b iodoallylation of a C H bond of an arene by reaction with an allene (Scheme 1). Both, intraand intermolecular processes are presented. The iodonium approach would complement the requirements of the powerful carbometallation reaction of allenes. The intermolecular process requires a substituted arene, whereas in the intramolecular process the assembled skeleton is endowed with further functionality. Interestingly, the application of this technology to ring elaboration enables a direct entry into the 2-iodo-1,4-dihydronaphthalene core, an elusive and valuable building block for crosscoupling chemistry that will also be highlighted. The iodonium-promoted carbocyclization was first tested on 2,3-butadienyl benzene (1 a), a demanding model substrate considering that the participation of an electronically activated arene is mandatory in most allene hydroarylation reactions. Several iodinating reagents were tested in an effort to synthesize 2-iodo-1,4-dihydronaphthalene (2 a) through a straightforward iodoarylation strategy. Some key experiments are outlined in Table 1.