The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body

The spliceosomal snRNAs U1, U2, U4, and U5 are synthesized in the nucleus, exported to the cytoplasm to assemble with Sm proteins, and reimported to the nucleus as ribonucleoprotein particles. Recently, two novel proteins involved in biogenesis of small nuclear ribonucleoproteins (snRNPs) were identified, the Spinal muscular atrophy disease gene product (SMN) and its associated protein SIP1. It was previously reported that in HeLa cells, SMN and SIP1 form discrete foci located next to Cajal (coiled) bodies, the so-called "gemini of coiled bodies" or "gems." An intriguing feature of gems is that they do not appear to contain snRNPs. Here we show that gems are present in a variable but small proportion of rapidly proliferating cells in culture. In the vast majority of cultured cells and in all primary neurons analyzed, SMN and SIP1 colocalize precisely with snRNPs in the Cajal body. The presence of SMN and SIP1 in Cajal bodies is confirmed by immunoelectron microscopy and by microinjection of antibodies that interfere with the integrity of the structure. The association of SMN with snRNPs and coilin persists during cell division, but at the end of mitosis there is a lag period between assembly of new Cajal bodies in the nucleus and detection of SMN in these structures, suggesting that SMN is targeted to preformed Cajal bodies. Finally, treatment of cells with leptomycin B (a drug that blocks export of U snRNAs to the cytoplasm and consequently import of new snRNPs into the nucleus) is shown to deplete snRNPs (but not SMN or SIP1) from the Cajal body. This suggests that snRNPs flow through the Cajal body during their biogenesis pathway.     

Virus study for continuous low pH viral inactivation inside a coiled flow inverter

Continuous processing for the production of monoclonal antibodies (mAb) gains more and more importance. Several solutions exist for all the necessary production steps, leading to the possibility to build fully continuous processes. Low pH viral inactivation is a part of the standard platform process for mAb production. Consequently, Klutz et al. introduced the coiled flow inverter (CFI) as a tool for continuous low pH viral inactivation. Besides theoretical calculations of viral reduction, no viral clearance study has been presented so far. In addition, the validation of continuous viral clearance is often neglected in the already existing studies for continuous processing. This study shows in detail the development and execution of a virus study for continuous low pH viral inactivation inside a CFI. The concept presented is also valid for adaptation to other continuous viral clearance steps. The development of this concept includes the technical rationale for an experimental setup, a valid spiking procedure, and finally a sampling method. The experimental results shown represent a viral study using xenotropic murine leukemia virus as a model virus. Two different protein A (ProtA) chromatography setups with varying pH levels were tested. In addition, one of these setups was tested against a batch experiment utilizing the same process material. The results show that sufficient low pH viral inactivation (decadic logarithm reduction value >4) was achieved in all experiments. Complete viral inactivation took place within the first 14.5 min for both continuous studies and the batch study, hence showing similar results. This study therefore represents a successful virus study concept and experiment for a continuous viral inactivation step. Moreover, it was shown that the transfer from batch results to the continuous process is possible. This is accomplished by the narrow residence time distribution of the CFI, showing how close the setup approaches the ideal plug flow and with that batch operation.     

Metallic electrodes and leads in simultaneous EEG-MRI: specific absorption rate (SAR) simulation studies

The purpose of this study was to investigate the changes in specific absorption rate (SAR) in human-head tissues while using nonmagnetic metallic electroencephalography (EEG) electrodes and leads during magnetic resonance imaging (MRI). A realistic, high resolution (1 mm(3)) head model from individual MRI data was adopted to describe accurately thin tissues, such as bone marrow and skin. The RF power dissipated in the human head was evaluated using the FDTD algorithm. Both surface and bird cage coils were used. The following numbers of EEG electrodes/leads were considered: 16, 31, 62, and 124. Simulations were performed at 128 and 300 MHz. The difference in SAR between the electrodes/leads and no-electrodes conditions was greater with the bird cage coil than with the surface coil. The peak 1 g averaged SAR values were highest at 124 electrodes, increasing to as much as two orders of magnitude (x172.3) at 300 MHz compared to the original value. At 300 MHz, there was a fourfold (x3.6) increase of SAR averaged over the bone marrow, and a sevenfold (x7.4) increase in the skin. At 128 MHz, there was a fivefold (x5.6) increase of whole head SAR. Head models were obtained from two different subjects, with an inter-subject whole head SAR variability of 3%. .     

Chemical cross-linking between lysine groups in vimentin oligomers is dependent on local peptide conformations

Previous studies have shown that cytoplasmic intermediate filaments, other than the keratins, are each constructed from a single type of polypeptide chain. Studies involving chemical crosslinking between lysine groups have shown that assembly of the filaments begins with the formation of dimers in which the peptide chains are parallel and in exact register, and that these dimers further associate in antiparallel patterns having specific degrees of overlap. In the present study, molecular modeling of the conformations of vimentin molecules indicated that lysine side chains in identical positions in regions of α-helix in parallel chains might be unable to be linked because they are on opposite sides of the coiled coil hydrophobic core. Examination of published data on chemical crosslinking of lysines in vimentin confirmed that there were no instances of linkage within dimers between the nine pairs of identical lysines that lie more than one position within α-helical regions in parallel chains. Even among linkages that apparently were between dimers, only one of the 11 linkage products identified involved lysines that were both within an α-helical region. In 10 of the 11 identified linkages between dimers, one or both of the linked lysines were in regions of random coil conformation. These results of molecular modeling indicate that relative motion between polypeptide chains in oligomers of intermediate filament proteins is not sufficient to overcome an orientation of lysine groups that is unfavorable for their chemical linkage. This finding supports the interpretations of keratin cross-linking data indicating that parallel homodimers are the basis for keratin intermediate filament assembly. © 1996 Wiley-Liss, Inc.     

Caspase-2 is localized at the Golgi complex and cleaves golgin-160 during apoptosis

Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors.     

Organization of designed nanofibrils assembled from alpha-helical peptides as determined by electron microscopy.

Self-assembling peptides present attractive platforms for engineering materials with controlled nanostructures. Recently, an alpha-helical fibril forming peptide (alphaFFP) was designed that self-assembles into nanofibrils at acid pH. Circular dichroism spectroscopy, electron-microscopy and x-ray fibre diffraction data showed that the most likely structure of alphaFFP fibrils is a five-stranded coiled coil rope. In the present study, scanning transmission electron microscopy (STEM) was used to improve our understanding of the alphaFFP fibril structure. The measurements of fibril mass per length suggest that there are ten alpha-helices in transverse sections of the fibrils. Based on the known data, it is proposed that a predominant fibrillar structure of alphaFFP is a dimer of alpha-helical five stranded protofilaments wrapped around a common axis. It is shown that these structures have an axial dimension of 58 +/- 16 nm and a width of 4 +/- 1 nm. A small number of thin fibrils is also observed in the negative stained preparation and STEM images. The thin fibrils may correspond to the single protofilament.     

Computational assessment of folding energy landscapes in heterodimeric coiled coils

The coiled coil structural motif consists of alpha helices supercoiling around each other to form staggered knobs‐into‐holes packing. Such structures are deceptively simple, especially as they often can be described with parametric equations, but are known to exist in various conformations. Even the simplest systems, consisting of 2 monomers, can assemble into a wide range of states. They can form canonical as well as noncanonical coiled coils, be parallel or antiparallel, where helices associate with different degrees of shift, tilt, and rotation. Here, we investigate the energy landscape of heterodimeric coiled coils by carrying out de novo folding simulations starting from amino acid sequence. We folded a diverse set of 22 heterodimers and demonstrate that the approach is capable of identifying the atomic details in the experimental structure in the majority of cases. Our methodology also enables exploration of alternative states that can be accessible in solution beyond the experimentally determined structure. For many systems, we observe folding energy landscapes with multiple energy minima and several isoenergetic states. By comparing coiled coils from single domains and those extracted from larger proteins, we find that standalone coiled coils have deeper energy wells at the experimentally determined conformation. By folding the competing homodimeric states in addition to the heterodimers, we observe that the structural specificity towards the heteromeric state is often small. Taken together, our results demonstrate that de novo folding simulations can be a powerful tool to characterize structural specificity of coiled coils when coupled to assessment of energy landscapes.     

Charged single alpha-helix: a versatile protein structural motif

A few highly charged natural peptide sequences were recently suggested to form stable alpha-helical structures in water. In this article we show that these sequences represent a novel structural motif called charged single alpha-helix (CSAH). To obtain reliable candidate CSAH motifs, we developed two conceptually different computational methods capable of scanning large databases: SCAN4CSAH is based on sequence features characteristic for salt bridge stabilized single alpha-helices, whereas FT_CHARGE applies Fourier transformation to charges along sequences. Using the consensus of the two approaches, a remarkable number of proteins were found to contain putative CSAH domains. Recombinant fragments (50-60 residues) corresponding to selected hits obtained by both methods (myosin 6, Golgi resident protein GCP60, and M4K4 protein kinase) were produced and shown by circular dichroism spectroscopy to adopt largely alpha-helical structure in water. CSAH segments differ substantially both from coiled-coil and intrinsically disordered proteins, despite the fact that current prediction methods recognize them as either or both. Analysis of the proteins containing CSAH motif revealed possible functional roles of the corresponding segments. The suggested main functional features include the formation of relatively rigid spacer/connector segments between functional domains as in caldesmon, extension of the lever arm in myosin motors and mediation of transient interactions by promoting dimerization in a range of proteins.     

Comparison of the structures and stabilities of coiled‐coil proteins containing hexafluoroleucine and t‐butylalanine provides insight into the stabilizing effects of highly fluorinated amino acid side‐chains

Highly fluorinated analogs of hydrophobic amino acids are well known to increase the stability of proteins toward thermal unfolding and chemical denaturation, but there is very little data on the structural consequences of fluorination. We have determined the structures and folding energies of three variants of a de novo designed 4‐helix bundle protein whose hydrophobic cores contain either hexafluoroleucine (hFLeu) or t‐butylalanine (tBAla). Although the buried hydrophobic surface area is the same for all three proteins, the incorporation of tBAla causes a rearrangement of the core packing, resulting in the formation of a destabilizing hydrophobic cavity at the center of the protein. In contrast, incorporation of hFLeu, causes no changes in core packing with respect to the structure of the nonfluorinated parent protein which contains only leucine in the core. These results support the idea that fluorinated residues are especially effective at stabilizing proteins because they closely mimic the shape of the natural residues they replace while increasing buried hydrophobic surface area.