Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability

Naturally-occurring single α-helices (SAHs), are rich in Arg (R), Glu (E) and Lys (K) residues, and stabilized by multiple salt bridges. Understanding how salt bridges promote their stability is challenging as SAHs are long and their sequences highly variable. Thus, we designed and tested simple de novo 98-residue polypeptides containing 7-residue repeats (AEEEXXX, where X is K or R) expected to promote salt-bridge formation between Glu and Lys/Arg. Lys-rich sequences (EK3 (AEEEKKK) and EK2R1 (AEEEKRK)) both form SAHs, of which EK2R1 is more helical and thermo-stable suggesting Arg increases stability. Substituting Lys with Arg (or vice versa) in the naturally-occurring myosin-6 SAH similarly increased (or decreased) its stability. However, Arg-rich de novo sequences (ER3 (AEEERRR) and EK1R2 (AEEEKRR)) aggregated. Combining a PDB analysis with molecular modelling provides a rational explanation, demonstrating that Glu and Arg form salt bridges more commonly, utilize a wider range of rotamer conformations, and are more dynamic than Glu-Lys. This promiscuous nature of Arg helps explain the increased propensity of de novo Arg-rich SAHs to aggregate. Importantly, the specific K:R ratio is likely to be important in determining helical stability in de novo and naturally-occurring polypeptides, giving new insight into how single α-helices are stabilized

Mutations in the SPG7 gene cause chronic progressive external ophthalmoplegia through disordered mitochondrial DNA maintenance.

Despite being a canonical presenting feature of mitochondrial disease, the genetic basis of progressive external ophthalmoplegia remains unknown in a large proportion of patients. Here we show that mutations in SPG7 are a novel cause of progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions. After excluding known causes, whole exome sequencing, targeted Sanger sequencing and multiplex ligation-dependent probe amplification analysis were used to study 68 adult patients with progressive external ophthalmoplegia either with or without multiple mitochondrial DNA deletions in skeletal muscle. Nine patients (eight probands) were found to carry compound heterozygous SPG7 mutations, including three novel mutations: two missense mutations c.2221G>A; p.(Glu741Lys), c.2224G>A; p.(Asp742Asn), a truncating mutation c.861dupT; p.Asn288*, and seven previously reported mutations. We identified a further six patients with single heterozygous mutations in SPG7, including two further novel mutations: c.184-3C>T (predicted to remove a splice site before exon 2) and c.1067C>T; p.(Thr356Met). The clinical phenotype typically developed in mid-adult life with either progressive external ophthalmoplegia/ptosis and spastic ataxia, or a progressive ataxic disorder. Dysphagia and proximal myopathy were common, but urinary symptoms were rare, despite the spasticity. Functional studies included transcript analysis, proteomics, mitochondrial network analysis, single fibre mitochondrial DNA analysis and deep re-sequencing of mitochondrial DNA. SPG7 mutations caused increased mitochondrial biogenesis in patient muscle, and mitochondrial fusion in patient fibroblasts associated with the clonal expansion of mitochondrial DNA mutations. In conclusion, the SPG7 gene should be screened in patients in whom a disorder of mitochondrial DNA maintenance is suspected when spastic ataxia is prominent. The complex neurological phenotype is likely a result of the clonal expansion of secondary mitochondrial DNA mutations modulating the phenotype, driven by compensatory mitochondrial biogenesis

Pro Libris: Lubuskie Pismo Literacko-Kulturalne, nr 4 (2008)

172 s. : il

Identification of functional differences between recombinant human α and β cardiac myosin motors

The myosin isoform composition of the heart is dynamic in health and disease and has been shown to affect contractile velocity and force generation. While different mammalian species express different proportions of α and β myosin heavy chain, healthy human heart ventricles express these isoforms in a ratio of about 1:9 (α:β) while failing human ventricles express no detectable α-myosin. We report here fast-kinetic analysis of recombinant human α and β myosin heavy chain motor domains. This represents the first such analysis of any human muscle myosin motor and the first of α-myosin from any species. Our findings reveal substantial isoform differences in individual kinetic parameters, overall contractile character, and predicted cycle times. For these parameters, α-subfragment 1 (S1) is far more similar to adult fast skeletal muscle myosin isoforms than to the slow β isoform despite 91% sequence identity between the motor domains of α- and β-myosin. Among the features that differentiate α- from β-S1: the ATP hydrolysis step of α-S1 is ~ten-fold faster than β-S1, α-S1 exhibits ~five-fold weaker actin affinity than β-S1, and actin·α-S1 exhibits rapid ADP release, which is >ten-fold faster than ADP release for β-S1. Overall, the cycle times are ten-fold faster for α-S1 but the portion of time each myosin spends tightly bound to actin (the duty ratio) is similar. Sequence analysis points to regions that might underlie the basis for this finding

Impact of GnRH analogues on oocyte/embryo quality and embryo development in in vitro fertilization/intracytoplasmic sperm injection cycles: a case control study

<p>Abstract</p> <p>Background</p> <p>Despite the clinical outcomes of ovarian stimulation with either GnRH-agonist or GnRH-antagonist analogues for in vitro fertilization (IVF) being well analysed, the effect of analogues on oocyte/embryo quality and embryo development is still not known in detail. The aim of this case-control study was to compare the efficacy of a multiple-dose GnRH antagonist protocol with that of the GnRH agonist long protocol with a view to oocyte and embryo quality, embryo development and IVF treatment outcome.</p> <p>Methods</p> <p>Between October 2001 and December 2008, 100 patients were stimulated with human menopausal gonadotrophin (HMG) and GnRH antagonist in their first treatment cycle for IVF or intracytoplasmic sperm injection (ICSI). One hundred combined GnRH agonist + HMG (long protocol) cycles were matched to the GnRH antagonist + HMG cycles by age, BMI, baseline FSH levels and by cause of infertility. We determined the number and quality of retrieved oocytes, the rate of early-cleavage embryos, the morphology and development of embryos, as well as clinical pregnancy rates. Statistical analysis was performed using Wilcoxon's matched pairs rank sum test and McNemar's chi-square test. P < 0.05 was considered statistically significant.</p> <p>Results</p> <p>The rate of cytoplasmic abnormalities in retrieved oocytes was significantly higher with the use of GnRH antagonist than in GnRH agonist cycles (62.1% vs. 49.9%; P < 0.01). We observed lower rate of zygotes showing normal pronuclear morphology (49.3% vs. 58.0%; P < 0.01), and higher cell-number of preembryos on day 2 after fertilization (4.28 vs. 4.03; P < 0.01) with the use of GnRH antagonist analogues. The rate of mature oocytes, rate of presence of multinucleated blastomers, amount of fragmentation in embryos and rate of early-cleaved embryos was similar in the two groups. Clinical pregnancy rate per embryo transfer was lower in the antagonist group than in the agonist group (30.8% vs. 40.4%) although this difference did not reach statistical significance (P = 0.17).</p> <p>Conclusion</p> <p>Antagonist seemed to influence favourably some parameters of early embryo development dynamics, while other morphological parameters seemed not to be altered according to GnRH analogue used for ovarian stimulation in IVF cycles.</p

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5\ub4-splice-site selection causing tissue-specific defects

\ua9 The Author(s) 2024.The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A &gt; C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5’-splice site (5’SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5’SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches