Mandibuloacral dysplasia is caused by a mutation in LMNA-encoding lamin A/C

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive disorder, characterized by postnatal growth retardation, craniofacial anomalies, skeletal malformations, and mottled cutaneous pigmentation. The LMNA gene encoding two nuclear envelope proteins (lamins A and C [lamin A/C]) maps to chromosome 1q21 and has been associated with five distinct pathologies, including Dunnigan-type familial partial lipodystrophy, a condition that is characterized by subcutaneous fat loss and is invariably associated with insulin resistance and diabetes. Since patients with MAD frequently have partial lipodystrophy and insulin resistance, we hypothesized that the disease may be caused by mutations in the LMNA gene. We analyzed five consanguineous Italian families and demonstrated linkage of MAD to chromosome 1q21, by use of homozygosity mapping. We then sequenced the LMNA gene and identified a homozygous missense mutation (R527H) that was shared by all affected patients. Patient skin fibroblasts showed nuclei that presented abnormal lamin A/C distribution and a dysmorphic envelope, thus demonstrating the pathogenic effect of the R527H LMNA mutation.     

Unraveling the organization of the internal nuclear matrix: RNA-dependent anchoring of NuMA to a lamin scaffold

Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.     

Structural dynamics of myoglobin: ligand migration among protein cavities studied by Fourier transform infrared/temperature derivative spectroscopy.

Fourier transform infrared (FTIR) spectroscopy in the CO stretch bands combined with temperature derivative spectroscopy (TDS) was used to characterize intermediate states obtained by photolysis of two sperm whale mutant myoglobins, YQR (L29(B10)Y, H64(E7)Q, T67(E10)R) and YQRF (with an additional I107(G8)F replacement). Both mutants assume two different bound-state conformations, A(0) and A(3), which can be distinguished by their different CO bands near 1965 and 1933 cm(-1). They most likely originate from different conformations of the Gln-64 side chain. Within each A substate, a number of photoproduct states have been characterized on the basis of the temperature dependence of recombination in TDS experiments. Different locations and orientations of the ligand within the protein can be distinguished by the infrared spectra of the photolyzed CO. Recombination from the primary docking site, B, near the heme dominates below 50 K. Above 60 K, ligand rebinding occurs predominantly from a secondary docking site, C', in which the CO is trapped in the Xe4 cavity on the distal side, as shown by crystallography of photolyzed YQR and L29W myoglobin CO. Another kinetic state (C") has been identified from which rebinding occurs around 130 K. Moreover, a population appearing above the solvent glass transition at approximately 180 K (D state) is assigned to rebinding from the Xe1 cavity, as suggested by the photoproduct structure of the L29W sperm whale myoglobin mutant. For both the YQR and YQRF mutants, rebinding from the B sites near the heme differs for the two A substates, supporting the view that the return of the ligand from the C', C", and D states is not governed by the recombination barrier at the heme iron but rather by migration to the active site. Comparison of YQR and YQRF shows that access to the Xe4 site (C') is severely restricted by introduction of the bulky Phe side chain at position 107.     

Glycerol-induced formation of the molten globule from acid-denatured cytochrome c: implication for hierarchical folding

At high concentration (98% or higher, v/v), glycerol induces collapse of acid-denatured cytochrome c into a compact state, the G_U state, showing a molten globule character. The G_U state possesses a nativelike -helix structure but a tertiary conformation less packed with respect to the native state. The spectroscopic properties of the G_U state closely resemble those of the molten globule stabilized by the organic solvent from the native protein (called the G_N state), indicating that glycerol can stabilize the molten globule of cytochrome c either from the native or the acid-denatured protein. The G_U and the G_N states show spectroscopic (and, thus, structural) properties and stabilities comparable to those of molten globules stabilized by different effectors, despite the fact that the mechanisms involved in the molten globule formation may significantly differ. This implies in cytochrome c a hierarchy for the rupture (native-to-molten globule) or the formation (unfolded-to-molten globule) of intramolecular interactions leading to the stabilization of the molten globule state of the protein, independently from the effector responsible for the structural transition, in accord with the sequential model proposed by Englander and collaborators.     

Temperature-dependent splicing of beta-globin pre-mRNA

A T→G mutation at nucleotide 705 of human β-globin intron 2 creates an aberrant 5′ splice site and activates a cryptic 3′ splice site upstream. In consequence, the pre-mRNA is spliced via aberrant splice sites, despite the presence of the still functional correct sites. Surprisingly, when IVS2-705 HeLa or K562 cells were cultured at temperatures below 30°C, aberrant splicing was inhibited and correct splicing was restored. Similar temperature effects were seen for another β-globin pre-mRNA, IVS2-745, and in a construct in which a β-globin intron was inserted into a coding sequence of EGFP. Temperature-induced alternative splicing was affected by the nature of the internal aberrant splice sites flanking the correct sites and by exonic sequences. The results indicate that in the context of thalassemic splicing mutations and possibly in other alternatively spliced pre-mRNAs, temperature is one of the parameters that affect splice site selection.     

Effect of dimethyl sulfoxide on the structure and the functional properties of horseradish peroxidase as observed by spectroscopy and cyclic voltammetry

Electrochemical biosensors have found wide application in food and clinical areas, as well as in environmental field. A large number of articles focused on horseradish peroxidase (HRP)-based biosensors have been published in the last decade, due to the capability of HRP to quantitatively detect the presence of hydrogen peroxide produced in a reaction. At present a large body of multi-enzymatic amperometric biosensors are realized by entrapping HRP together with other enzymes into a polymeric matrix; these systems represent a promising example of simple, low-cost electrochemical tools for the analysis of bioanalytes in solution, such as glucose, choline and cholesterol. Due to the fact that polymers used for HRP entrapping are soluble in organic solvents and that many solvents are strong denaturants of aquo-soluble proteins, in this paper we investigate (in particular, by circular dichroism and electron paramagnetic spectroscopies) the effect of dimethyl sulfoxide, one of the organic solvents employed for polymer solubilization, on the structure and the functionality of HRP, in order to determine the effect induced by the solvent concentration on the structure and activity of the hemoprotein. This is relevant for basic and applied biochemistry, HRP being largely employed in bioinorganic chemistry and sensor area.     

The interplay between folding-facilitating mechanisms in Trypanosoma cruzi endoplasmic reticulum

Lectin (calreticulin [CRT])-N-glycan-mediated quality control of glycoprotein folding is operative in trypanosomatid protozoa but protein-linked monoglucosylated N-glycans are exclusively formed in these microorganisms by UDP-Glc:glycoprotein glucosyltransferase (GT)-dependent glucosylation. The gene coding for this enzyme in the human pathogen Trypanosoma cruzi was identified and sequenced. Even though several of this parasite glycoproteins have been identified as essential components of differentiation and mammalian cell invasion processes, disruption of both GT-encoding alleles did not affect cell growth rate of epimastigote form parasites and only partially affected differentiation and mammalian cell invasion. The cellular content of one of the already identified T. cruzi glycoprotein virulence factors (cruzipain, a lysosomal proteinase) only showed a partial (5-20%) decrease in GT null mutants in spite of the fact that >90% of all cruzipain molecules interacted with CRT during their folding process in wild-type cells. Although extremely mild cell lysis and immunoprecipitation procedures were used, no CRT-cruzipain interaction was detected in GT null mutants but secretion of the proteinase was nevertheless delayed because of a lengthened interaction with Grp78/BiP probably caused by the detected induction of this chaperone in GT null mutants. This result provides a rationale for the absence of a more drastic consequence of GT absence. It was concluded that T. cruzi endoplasmic reticulum folding machinery presents an exquisite plasticity that allows the parasite to surmount the absence of the glycoprotein-specific folding facilitation mechanism.