The cell theory. Progress in studies on cell-cell communications

Current data confirm the fundamental statement of the cell theory concerning the cell reproduction in a series of generations (omnis cellula e cellula). Cell communities or ensembles integrated by the signaling systems established in prokaryotes and protists and functioning in multicellular organisms including mammals are considered as the structural and functional unit of a multicellular organism. The cell is an elementary unit of life and basis of organism development and functioning. At the same time, the adult organism is not just a totality of cells. Multinucleated cells in some tissues, syncytial structure, and structural-functional units of organs are adaptations for optimal functioning of the multicellular organism and manifestations of cell-cell communications in development and definitive functioning. The cell theory was supplemented and developed by studies on cell-cell communications; however, these studies do not question the main generalizations of the theory.     

NMR Conformational and Dynamic Consequences of a Gly to Ser Substitution in an Osteogenesis Imperfecta Collagen Model Peptide

Close packing of three chains in a standard collagen triple helix requires Gly as every third residue. Missense mutations replacing one Gly by a larger residue in the tripeptide repeating sequence in type I collagen are common molecular causes of osteogenesis imperfecta. The structural and dynamic consequences of such mutations are addressed here by NMR studies on a peptide with a Gly-to-Ser substitution within an α1(I) sequence. Distances derived from nuclear Overhauser effects indicate that the three Ser residues are still packed in the center of the triple helix and that the standard 1-residue stagger is maintained. NMR dynamics using H-exchange and temperature-dependent amide chemical shifts indicate a greater disruption of hydrogen bonding and/or increased conformational flexibility C-terminal to the Ser site when compared with N terminal. This is consistent with recent suggestions relating clinical severity with an asymmetric effect of residues N- versus C-terminal to a mutation site. Dynamic studies also indicate that the relative position between a Gly in one chain and the mutation site in a neighboring staggered chain influences the disruption of the standard hydrogen-bonding pattern. The structural and dynamic alterations reported here may play a role in the etiology of osteogenesis imperfecta by affecting collagen secretion or interactions with other matrix molecules.Mutations in collagen result in a variety of connective tissue diseases (1, 2), with the clinical phenotype depending on the location and function of the collagen type. For instance, mutations in type I collagen, the major collagen in bone, lead to a bone disorder, osteogenesis imperfecta (OI),³ whereas mutations in type III collagen, which is present in high amounts in blood vessels, lead to aortic rupture in Ehlers-Danlos syndrome type IV (1, 2). All collagens have a triple helix motif composed of three polyproline II-like chains that are staggered by 1 residue and supercoiled about a common axis. The smallest residue Gly is typically present as every 3rd residue in each chain because of the tight packing of the chains, which generates the characteristic (Gly-Xaa-Yaa)_n repeating sequence. The Gly residues are all buried in the center, and the structure is stabilized by interchain N–H (Gly) … CO (Xaa) hydrogen bonds (3–5). The most common type of mutation leading to collagen disorders is a missense mutation that replaces 1 Gly in the repeating sequence by a larger residue.The best characterized collagen disease is OI, or brittle bone disease, which is distinguished by fragile bones due to mutations in type I collagen (2, 6). More than 400 Gly substitution missense mutations in the α1(I) and α2(I) chains of type I collagen have been reported to lead to OI (7). The severity of the disease varies widely from mild cases with multiple fractures to perinatal lethal cases (2, 6, 7). A single base change in a Gly codon can lead to one of 8 residues (Ser, Ala, Cys, Val, Arg, Asp, Glu, Trp) or a missense mutation. The smallest residue Ala is underrepresented in OI, suggesting that it may not always lead to pathology, whereas Ser mutations are overrepresented, corresponding to the most common substitutions observed. The 152 mutations leading to a Gly to Ser substitution account for ∼39% of all missense mutations in the α1(I) of type I collagen (7), with 115 associated with mild phenotypes and 37 associated with lethal phenotypes.The identity of the residue replacing Gly may be a determinant in the clinical severity of OI. Model peptide studies indicate that the degree of triple helix destabilization depends on the residue replacing Gly, with a ranking of the least destabilizing to the most destabilizing Ala,Ser8). There is some correlation between clinical severity of OI cases and this destabilization scale, with the strongly destabilizing residues Val, Arg, Asp, and Glu associated largely with lethal phenotypes (8). However, as cited above, a Gly to Ser mutation can lead to a mild, a severe, or a lethal OI case, with no obvious molecular explanation. Other factors suggested to contribute to clinical phenotype include the rigidity of its immediate sequence environment; its location with respect to the C terminus; its proximity to salt bridges; and its presence at an interaction site, such as the binding site for proteoglycans on collagen fibrils (7, 9). A recent study of the stability of OI collagens supported the importance of the domain location of the mutation (10), whereas a network analysis of the mutations suggested the importance of a destabilizing tripeptide sequence C-terminal to the mutation site (11).The standard triple helix conformation must undergo some structural perturbation as a result of a Gly replacement that is likely to relate to the development of the disorder. Thus it is important to define the structural consequences of a Gly substitution. It has not proved possible to obtain molecular information for the long collagen molecules themselves, but model collagen peptides have proved amenable to x-ray crystallography and NMR techniques (12, 13). The structure of a peptide containing a Gly to Ala substitution near the center of the peptide (Pro-Hyp-Gly)₁₀ has been solved by x-ray crystallography (5). This structure shows an overall straight molecule with standard triple helical structures at both ends and a localized conformational deformation at the Ala replacement site. The direct N–H (Gly) … CO (Xaa) hydrogen bond is replaced by a water-mediated hydrogen bond N–H (Ala) … H₂O … CO (Xaa).Here, NMR spectroscopy is used to define the structural and dynamic effect of a Gly to Ser replacement through the application of recently developed NMR methodology on selectively ¹³C/¹⁵N doubly labeled collagen peptides (14). This strategy includes chain assignments, measurement of NOEs, and scalar J-couplings to define the conformation of the peptide. These results combined with NMR hydrogen exchange experiments and temperature-dependent chemical shift data demonstrate the disturbed dynamic features and hydrogen bonding around the Ser substitution site. The NMR data of the Gly to Ser peptide are compared with the NMR and x-ray high resolution structure of the peptide containing a Gly to Ala substitution (5).     

Saccharomyces cerivisiae as a model system for kidney disease: what can yeast tell us about renal function?

Ion channels, solute transporters, aquaporins, and factors required for signal transduction are vital for kidney function. Because mutations in these proteins or in associated regulatory factors can lead to disease, an investigation into their biogenesis, activities, and interplay with other proteins is essential. To this end, the yeast, Saccharomyces cerevisiae, represents a powerful experimental system. Proteins expressed in yeast include the following: 1) ion channels, including the epithelial sodium channel, members of the inward rectifying potassium channel family, and cystic fibrosis transmembrane conductance regulator; 2) plasma membrane transporters, such as the Na(+)-K(+)-ATPase, the Na(+)-phosphate cotransporter, and the Na(+)-H(+) ATPase; 3) aquaporins 1-4; and 4) proteins such as serum/glucocorticoid-induced kinase 1, phosphoinositide-dependent kinase 1, Rh glycoprotein kidney, and trehalase. The variety of proteins expressed and studied emphasizes the versatility of yeast, and, because of the many available tools in this organism, results can be obtained rapidly and economically. In most cases, data gathered using yeast have been substantiated in higher cell types. These attributes validate yeast as a model system to explore renal physiology and suggest that research initiated using this system may lead to novel therapeutics.     

Dissecting a bacterial collagen domain from Streptococcus pyogenes: sequence and length-dependent variations in triple helix stability and folding

To better investigate the relationship between sequence, stability, and folding, the Streptococcus pyogenes collagenous domain CL (Gly-Xaa-Yaa)(79) was divided to create three recombinant triple helix subdomains A, B, and C of almost equal size with distinctive amino acid features: an A domain high in polar residues, a B domain containing the highest concentration of Pro residues, and a very highly charged C domain. Each segment was expressed as a monomer, a linear dimer, and a linear trimer fused with the trimerization domain (V domain) in Escherichia coli. All recombinant proteins studied formed stable triple helical structures, but the stability varied depending on the amino acid sequence in the A, B, and C segments and increased as the triple helix got longer. V-AAA was found to melt at a much lower temperature (31.0 °C) than V-ABC (V-CL), whereas V-BBB melted at almost the same temperature (～36-37 °C). When heat-denatured, the V domain enhanced refolding for all of the constructs; however, the folding rate was affected by their amino acid sequences and became reduced for longer constructs. The folding rates of all the other constructs were lower than that of the natural V-ABC protein. Amino acid substitution mutations at all Pro residues in the C fragment dramatically decreased stability but increased the folding rate. These results indicate that the thermostability of the bacterial collagen is dominated by the most stable domain in the same manner as found with eukaryotic collagens.     

CRISPRseek: A Bioconductor Package to Identify Target-Specific Guide RNAs for CRISPR-Cas9 Genome-Editing Systems

CRISPR-Cas systems are a diverse family of RNA-protein complexes in bacteria that target foreign DNA sequences for cleavage. Derivatives of these complexes have been engineered to cleave specific target sequences depending on the sequence of a CRISPR-derived guide RNA (gRNA) and the source of the Cas9 protein. Important considerations for the design of gRNAs are to maximize aimed activity at the desired target site while minimizing off-target cleavage. Because of the rapid advances in the understanding of existing CRISPR-Cas9-derived RNA-guided nucleases and the development of novel RNA-guided nuclease systems, it is critical to have computational tools that can accommodate a wide range of different parameters for the design of target-specific RNA-guided nuclease systems. We have developed CRISPRseek, a highly flexible, open source software package to identify gRNAs that target a given input sequence while minimizing off-target cleavage at other sites within any selected genome. CRISPRseek will identify potential gRNAs that target a sequence of interest for CRISPR-Cas9 systems from different bacterial species and generate a cleavage score for potential off-target sequences utilizing published or user-supplied weight matrices with position-specific mismatch penalty scores. Identified gRNAs may be further filtered to only include those that occur in paired orientations for increased specificity and/or those that overlap restriction enzyme sites. For applications where gRNAs are desired to discriminate between two related sequences, CRISPRseek can rank gRNAs based on the difference between predicted cleavage scores in each input sequence. CRISPRseek is implemented as a Bioconductor package within the R statistical programming environment, allowing it to be incorporated into computational pipelines to automate the design of gRNAs for target sequences identified in a wide variety of genome-wide analyses. CRISPRseek is available under the GNU General Public Licence v3.0 at http://www.bioconductor.org.     

The HSP70 Modulator MAL3-101 Inhibits Merkel Cell Carcinoma

Merkel Cell Carcinoma (MCC) is a rare and highly aggressive neuroendocrine skin cancer for which no effective treatment is available. MCC represents a human cancer with the best experimental evidence for a causal role of a polyoma virus. Large T antigens (LTA) encoded by polyoma viruses are oncoproteins, which are thought to require support of cellular heat shock protein 70 (HSP70) to exert their transforming activity. Here we evaluated the capability of MAL3-101, a synthetic HSP70 inhibitor, to limit proliferation and survival of various MCC cell lines. Remarkably, MAL3-101 treatment resulted in considerable apoptosis in 5 out of 7 MCC cell lines. While this effect was not associated with the viral status of the MCC cells, quantitative mRNA expression analysis of the known HSP70 isoforms revealed a significant correlation between MAL3-101 sensitivity and HSC70 expression, the most prominent isoform in all cell lines. Moreover, MAL3-101 also exhibited in vivo antitumor activity in an MCC xenograft model suggesting that this substance or related compounds are potential therapeutics for the treatment of MCC in the future.     

The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA,an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia

Early-onset torsion dystonia (EOTD) is a neurological disorder characterized by involuntary and sustained muscle contractions that can lead to paralysis and abnormal posture. EOTD is associated with the deletion of a glutamate (ΔE) in torsinA, an endoplasmic reticulum (ER) resident AAA⁺ ATPase. To date, the effect of ΔE on torsinA and the reason that this mutation results in EOTD are unclear. Moreover, there are no specific therapeutic options to treat EOTD. To define the underlying biochemical defects associated with torsinAΔE and to uncover factors that might be targeted to offset defects associated with torsinAΔE, we developed a yeast torsinA expression system and tested the roles of ER chaperones in mediating the folding and stability of torsinA and torsinAΔE. We discovered that the ER lumenal Hsp70, BiP, an associated Hsp40, Scj1, and a nucleotide exchange factor, Lhs1, stabilize torsinA and torsinAΔE. BiP also maintained torsinA and torsinAΔE solubility. Mutations predicted to compromise specific torsinA functional motifs showed a synthetic interaction with the ΔE mutation and destabilized torsinAΔE, suggesting that the ΔE mutation predisposes torsinA to defects in the presence of secondary insults. In this case, BiP was required for torsinAΔE degradation, consistent with data that specific chaperones exhibit either pro-degradative or pro-folding activities. Finally, using two independent approaches, we established that BiP stabilizes torsinA and torsinAΔE in mammalian cells. Together, these data define BiP as the first identified torsinA chaperone, and treatments that modulate BiP might improve symptoms associated with EOTD.     

A G protein–coupled receptor and the intracellular synthase of its agonist functionally cooperate

Export of newly synthesized G protein–coupled receptors (GPCRs) remains poorly characterized. We show in this paper that lipocalin-type prostaglandin D₂ (PGD₂) synthase (L-PGDS) interacts intracellularly with the GPCR DP1 in an agonist-independent manner. L-PGDS promotes cell surface expression of DP1, but not of other GPCRs, in HEK293 and HeLa cells, independent of L-PGDS enzyme activity. In addition, formation of a DP1–Hsp90 complex necessary for DP1 export to the cell surface is dependent on the interaction between L-PGDS and the C-terminal MEEVD residues of Hsp90. Surprisingly, PGD₂ synthesis by L-PGDS is promoted by coexpression of DP1, suggesting a possible intracrine/autocrine signaling mechanism. In this regard, L-PGDS increases the formation of a DP1–ERK1/2 complex and increases DP1-mediated ERK1/2 signaling. Our findings define a novel cooperative mechanism in which a GPCR (DP1) promotes the activity of the enzyme (L-PGDS) that produces its agonist (PGD₂) and in which this enzyme in turn acts as a cofactor (of Hsp90) to promote export and agonist-dependent activity of the receptor.