Hotspots Of Instability Research Articles

CDR1 Composition Can Affect Nanobody Recombinant Expression Yields.

The isolation of nanobodies from pre-immune libraries by means of biopanning is a straightforward process. Nevertheless, the recovered candidates often require optimization to improve some of their biophysical characteristics. In principle, CDRs are not mutated because they are likely to be part of the antibody paratope, but in this work, we describe a mutagenesis strategy that specifically addresses CDR1. Its sequence was identified as an instability hot spot by the PROSS program, and the available structural information indicated that four CDR1 residues bound directly to the antigen. We therefore modified the loop flexibility with the addition of an extra glycine rather than by mutating single amino acids. This approach significantly increased the nanobody yields but traded-off with moderate affinity loss. Accurate modeling coupled with atomistic molecular dynamics simulations enabled the modifications induced by the glycine insertion and the rationale behind the engineering design to be described in detail.

Xq22 deletions and correlation with distinct neurological disease traits in females: Further evidence for a contiguous gene syndrome

Xq22 deletions that encompass PLP1 (Xq22-PLP1-DEL) are notable for variable expressivity of neurological disease traits in females ranging from a mild late-onset form of spastic paraplegia type 2 (MIM# 312920), sometimes associated with skewed X-inactivation, to an early-onset neurological disease trait (EONDT) of severe developmental delay, intellectual disability, and behavioral abnormalities. Size and gene content of Xq22-PLP1-DEL vary and were proposed as potential molecular etiologies underlying variable expressivity in carrier females where two smallest regions of overlap (SROs) were suggested to influence disease. We ascertained a cohort of eight unrelated patients harboring Xq22-PLP1-DEL and performed high-density array comparative genomic hybridizationand breakpoint-junction sequencing. Molecular characterization of Xq22-PLP1-DEL from 17 cases (eight herein and nine published) revealed an overrepresentation of breakpoints that reside within repeats (11/17, ~65%) and the clustering of ~47% of proximal breakpoints in a genomic instability hotspot with characteristic non-B DNA density. These findings implicate a potential role for genomic architecture in stimulating the formation of Xq22-PLP1-DEL. The correlation of Xq22-PLP1-DEL gene content with neurological disease trait in female cases enabled refinement of the associated SROs to a single genomic interval containing six genes. Our data support the hypothesis that genes contiguous to PLP1 contribute to EONDT.

LINE- and Alu-containing genomic instability hotspot at 16q24.1 associated with recurrent and nonrecurrent CNV deletions causative for ACDMPV.

Transposable elements modify human genome by inserting into new loci or by mediating homology-, microhomology-, or homeology-driven DNA recombination or repair, resulting in genomic structural variation. Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a rare lethal neonatal developmental lung disorder caused by point mutations or copy-number variant (CNV) deletions of FOXF1 or its distant tissue-specific enhancer. Eighty-five percent of 45 ACDMPV-causative CNV deletions, of which junctions have been sequenced, had at least one of their two breakpoints located in a retrotransposon, with more than half of them being Alu elements. We describe a novel ∼35kb-large genomic instability hotspot at 16q24.1, involving two evolutionarily young LINE-1 (L1) elements, L1PA2 and L1PA3, flanking AluY, two AluSx, AluSx1, and AluJr elements. The occurrence of L1s at this location coincided with the branching out of the Homo-Pan-Gorilla clade, and was preceded by the insertion of AluSx, AluSx1, and AluJr. Our data show that, in addition to mediating recurrent CNVs, L1 and Alu retrotransposons can predispose the human genome to formation of variably sized CNVs, both of clinical and evolutionary relevance. Nonetheless, epigenetic or other genomic features of this locus might also contribute to its increased instability.

The organization of genome duplication is a critical determinant of the landscape of genome maintenance

Genome duplication is essential for cell proliferation, and the mechanisms regulating its execution are highly conserved. These processes give rise to a spatiotemporal organization of replication initiation across the genome, referred to as the replication program. Despite the identification of such programs in diverse eukaryotic organisms, their biological importance for cellular physiology remains largely unexplored. We address this fundamental question in the context of genome maintenance, taking advantage of the inappropriate origin firing that occurs when fission yeast cells lacking the Rad3/ATR checkpoint kinase are subjected to replication stress. Using this model, we demonstrate that the replication program quantitatively dictates the extent of origin de-regulation and the clustered localization of these events. Furthermore, our results uncover an accumulation of abnormal levels of single-stranded DNA (ssDNA) and the Rad52 repair protein at de-regulated origins. We show that these loci constitute a defining source of the overall ssDNA and Rad52 hotspots in the genome, generating a signature pattern of instability along the chromosomes. We then induce a genome-wide reprogramming of origin usage and evaluate its consequences in our experimental system. This leads to a complete redistribution of the sites of both inappropriate initiation and associated Rad52 recruitment. We therefore conclude that the organization of genome duplication governs the checkpoint control of origin-associated hotspots of instability and plays an integral role in shaping the landscape of genome maintenance.

How does Thermosensor Desk Measure Membrane Thickness? Tryptophan Fluorescence and Mutagenesis Analysis

The Bacillus subtilis histidine kinase DesK is a 5 transmembrane stretches- thermosensor suited to remodel membrane fluidity when temperature drops below 30 oC. We have recently designed a hybrid nanosensor with one transmembrane domain which is 5 times smaller than the parental protein. This chimerical protein fully retains, in vivo and in vitro, the sensing properties of the parental system and was called Minimal Sensor-DesK (MS-DesK). A recent paper (1) provides evidence that this perfectly simplified system could serve as a model to study a complex biological phenomena. Thus the MS-DesK is used here to study the conversion of a physical stimulus into a biological response that involves a change in the signaling transduction state.The N-terminus of TMS1 contains three hydrophilic aminoacids near the lipid-water interface creating an instability hot spot. We show that this boundary-sensitive motif controls the sensing and transmission activity by mutagenesis and in vitro reconstitution experiments. Accordingly, we hypothesize that membrane thickness is the temperature agent that determines the signaling state of the cold sensor by dictating the hydration level of the meta-stable hydrophilic spot. Tryptophan-labeled peptides corresponding to the sensor (functional and non-functional) transmembrane domain were synthesized and incorporated into membrane vesicles. Fluorescence spectroscopy and Circular Dichroism data of the peptides incorporated in liposomes of varying fatty acid chain length and different melting temperatures is presented.[1] Cybulski LE, Martin M, Mansilla MC, Fernandez A, de Mendoza D. Membrane thickness cue for cold sensing in a bacterium. Curr Biol. 20: 1539-1544, 2010

Thermosensor Desk Measures Membrane Thickness

The bacterium Bacillus subtilis adjusts the composition of membrane lipids to cope with temperature variations. The histidine kinase DesK is a five-pass transmembrane thermosensor suited to remodel membrane fluidity in B. subtilis according to temperature. To understand the mechanism of sensing, individual transmembrane segments (TMS) were fused to the cytoplasmic catalytic domain of DesK (DesKC) and the ability to respond to temperature analyzed. Surprisingly, a hybrid TMS composed of 17 aminoacids of the first TMS and the C-terminal 14-residue portion of TM5 fused to DesKC, fully retains in vivo and in vitro the sensing properties of full-length DesK [1]. Besides, when chimerical fusions of DesKC to either TMS1 or TMS5 are expressed in vivo individually in a desK- strain, thermosensing is lost. Nevertheless, when they are co-expressed thermosensing is recovered; suggesting that interactions between TMS1 and TMS5 are needed for signaling.The N-terminus of TMS1 contains three hydrophilic aminoacids near the lipid-water interface creating an instability hot spot. We showed that this boundary-sensitive motif controls the sensing and transmission activity. Accordingly, we hypothesize that membrane thickness is the temperature agent that determines the signaling state of the cold sensor by dictating the hydration level of the meta-stable hydrophilic spot. This hypothesis is supported through biochemical studies including in vitro reconstitution of the MS in liposomes with different chain length.[1] Cybulski LE, Martin M, Mansilla MC, Fernandez A, de Mendoza D. Membrane thickness cue for cold sensing in a bacterium. Curr Biol. 20: 1539-1544, 2010

Membrane Thickness Cue for Cold Sensing in a Bacterium

Thermosensors are ubiquitous integral membrane proteins found in all kinds of life. They are involved in many physiological roles, including membrane remodeling, chemotaxis, touch, and pain [1-3], but, the mechanism by which their transmembrane (TM) domains transmit temperature signals is largely unknown. The histidine kinase DesK from Bacillus subtilis is the paradigmatic example of a membrane-bound thermosensor suited to remodel membrane fluidity when the temperature drops below approximately 30°C [1, 4] providing, thus, a tractable system for investigating the mechanism of TM-mediated input-output control of thermal adaptation. Here we show that the multimembrane-spanning domain from DesK can be simplified into a chimerical single-membrane-spanning minimal sensor (MS) that fully retains, in vivo and in vitro, the sensing properties of the parental system. The MS N terminus contains three hydrophilic amino acids near the lipid-water interface creating an instability hot spot. Mutational analysis of this boundary-sensitive beacon revealed that membrane thickness controls the signaling state of the sensor by dictating the hydration level of the metastable hydrophilic spot. Guided by these results we biochemically demonstrated that the MS signal transmission activity is sensitive to bilayer thickness. Membrane thickness could be a general cue for sensing temperature in many organisms.

Engineering a Thermosensor To Dissect a Transmembrane Signaling System

The DesK-DesR two-component system regulates the order of membrane lipids in the bacterium Bacillus subtilis by controlling the expression of the des gene coding for the delta 5-acyl-lipid desaturase. In this work, we address the process by which DesK transmembrane segments (TMS) transmit temperature signals across the membrane by engineering the 5 TMS domain of the DesK into a single-TMS chimeric sensor. This so-called Minimal Sensor (MS) fully retains in vivo and in vitro the sensing input and transmission output of the parental system. Progressive deletions of TM segments revealed that only the first TM segment (TM1) is essential to regulate the kinase activity. Therefore, our engineered MS combines the N-terminal 17-residue portion of TM1 with the C-terminal 14-residue portion of TM5 which is naturally fused to the cytosolic catalytic domain. The MS N-terminus contains three hydrophilic aminoacids near the lipid-water interface creating an instability hot spot. This boundary-sensitive motif controls the sensing and transmission activity. Accordingly, we hypothesize that membrane thickness is the temperature agent that determines the signaling state of the cold sensor by dictating the hydration level of the meta-stable hydrophilic spot. This hypothesis is supported through the study of the signaling behavior of MS variants purposely constructed.

The Resilience of Israeli–Turkish Relations

Turkey was the first – and for decades the only – Islamic country to recognize the Jewish state, opening diplomatic relations in 1949. While Turkey became a member of NATO in 1952, and Israel served during the Cold War as a Western ally to counter Soviet alliances in the Arab world, relations between the two states were low-key through the decades of wars fought between Israel and the Arabs. Yet Turkey never severed the relationship despite Arab pressure to do so. With the end of the Cold War, Israel and Turkey emerged as the most democratic and economically dynamic states in the region. Their foreign pro-Western orientation and their self-perception as bastions of democratic and free market values in an unruly neighbourhood placed them, as was the case during the Cold War years, in the same strategic boat. In the early 1990s, with the end of the Cold War, during a period when the Arab-Israeli peace process gained momentum, Israeli –Turkish relations also moved into high gear. Diplomatic ties were upgraded to embassy-level status, joint military exercises began and intelligence cooperation was expanded. In addition, economic relations boomed. Annual trade between the two nations grew to US$2 billion in 2004, up from US$200 million in 1993, and since the mid-1990s Turkey has been the number one tourist destination for Israelis. The remarkable upgrading of relations with Israel was the result of the emergence of a evolving international constellation, following the breakdown of the Soviet Union, and the subsequent adoption of a new Turkish approach to a newly defined Greater Middle East. Turkey benefited only partially from the ‘peace dividends’ at the end of the Cold War, because it still found itself in a volatile strategic environment. While free to adopt a more assertive foreign policy than during the Cold War, 1 it also viewed itself as encircled by dangerous neighbours and surrounded by hotspots of instability. Thus, the main context for Turkey’s rapprochement with Israel was the reorientation of its foreign policy.