The effects of overexpression of lamin a and two mutants associated with premature aging on stem cell differentiation and proliferation.

The nuclear lamina is composed of A and B-type lamins and performs a number of functions within the nucleus. Mutations within lamins give rise to a group of diseases called laminopathies including autosomal dominant Emery-Dreifuss muscular dystrophy and lipodystrophy. Hutchinson Gilford Progeria Syndrome (HOPS), or so-called premature aging, is a rare and devastating laminopathy disorder caused by mutations in LMNA that leads to the production of a truncated mutant form of prelamin A, called progerin or LAA50, that cannot undergo proteolytic processing to yield mature lamin A. In an atypical form of HOPS, a point mutation at amino acid 644 alters the Zmpste24 endoproteolytic cleavage site, causing the production of mutant prelamin A. Why and how these prelamin A mutants cause such devastating phenotypes is not folly understood. It has been suggested that the mutant prelamin A may affect the ability of adult stem cells to self-renewal and differentiate, which are essential processes in order to replace damage of old tissues, critical for organismal longevity. Herein, expression plasmids containing two mutations found in classical and atypical HOPS were transiently overexpressed in clonal rat dermal papilla (DP) 9 cells, which, in vivo, reside at the base of the hair follicle and play an important role in hair follicle cycling. DP9 cells showed an increased number of nuclear abnormalities compared to control cells, which have previously been shown to be characteristic of cells from HGPS patients. DP9 cell lines stably overexpressing either FLAG-prelamin A， FLAG-prelamin ΑΔ50 or FLAG-prelamm A(R644C) were created. To assess the ability of the stem cells to self-renew, stable cell lines were routinely passaged and counted. Results indicate that stable overexpression of FLAG-prelamin ΑΔ50 and FLAG-prelamin A(R644C) inhibits the ability of stem cells to self-renew in vitro. Previous reports have shown that DP9 cells may be directed towards both adipogenic and osteogenic lineages. Therefore, in this study, stable DP9 cell lines expressing FLAG- prelamin A， FLAG-prelamin ΑΔ50 or FLAG-prelamin A(R644C) were exposed to an adipogenic medium for six days. Results showed that overexpression of FLAG-prelamin A, FLAG-prelamm ΑΔ50 and FLAG-prelamm A(R644C) inhibited the accumulation of intracellular lipids, reflecting a decreased ability to differentiate in vitro. Taken together, these results suggest the lipodystrophy and alopecia associated with HGPS may be due to the failure of adult stem cell populations within each tissue to both self-renew and differentiate, and may underpin the disease pathogenesis. Furthermore, these results have implications for other laminopathies that produce mutant prelamin A

The multiple roles of A-type lamins in cellular aging, cell cycle progression and the DNA damage response.

A-type lamins are a group of type V intermediate filaments whose main members are lamin A and C. Lamins A/C are components of the nuclear lamina and are encoded by the LMNA gene. Lamins A/C have a variety of cellular functions, including maintaining the structural integrity of the nucleus and the regulation of signal transduction pathways, transcription factors and DNA replication. Mutations in LMNA give rise to a diverse spectrum of diseases, termed laminopathies, which include premature aging syndromes. In Chapter 3, I sought to understand the role of wild type lamin A in normal cellular aging. Lamin A C-terminal cysteine residues were irreversibly oxidized during the in vitro aging of human dermal fibroblasts (HDFs), which impaired the ability of lamin A to form disulfide bonds, causing loss of function. Furthermore, loss of these cysteine residues induced premature senescence, suggesting that these cysteine residues are important for lamin A function during cellular aging. In Chapter 4, I extended previous findings implicating A-type lamins in the control of cell cycle progression. Loss of A-type lamins or its nucleoplasmic binding partner, LAP2α, caused delayed G1/S-phase progression, reduced cellular proliferation and cell cycle exit. Proliferative defects could not be rescued via treatment with anti-oxidants. In Chapters 5 and 6, I addressed the role of wild type mature lamin A/C in the DNA damage response (DDR). A-type lamins interact with the DDR mediator protein 53BP1 via its Tudor domain. Loss of LMNA caused endogenous DNA damage and loss of 53BP1 protein levels. Furthermore, loss of LMNA resulted in defective DNA repair that ultimately led to increased sensitivity to DNA damage. Together, the data presented here extends previous findings implicating A-type lamins in cell cycle progression and provides novel insights into the cellular roles of A-type lamins in cellular aging and the DNA damage response

Tools for decoding ubiquitin signaling in DNA repair

The maintenance of genome stability requires dedicated DNA repair processes and pathways that are essential for the faithful duplication and propagation of chromosomes. These DNA repair mechanisms counteract the potentially deleterious impact of the frequent genotoxic challenges faced by cells from both exogenous and endogenous agents. Intrinsic to these mechanisms, cells have an arsenal of protein factors that can be utilised to promote repair processes in response to DNA lesions. Orchestration of the protein factors within the various cellular DNA repair pathways is performed, in part, by post-translational modifications, such as phosphorylation, ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs). In this review, we firstly explore recent advances in the tools for identifying factors involved in both DNA repair and ubiquitin signaling pathways. We then expand on this by evaluating the growing repertoire of proteomic, biochemical and structural techniques available to further understand the mechanistic basis by which these complex modifications regulate DNA repair. Together, we provide a snapshot of the range of methods now available to investigate and decode how ubiquitin signaling can promote DNA repair and maintain genome stability in mammalian cells

Baryon Number Violation and String Topologies

In supersymmetric scenarios with broken R-parity, baryon number violating sparticle decays become possible. In order to search for such decays, a good understanding of expected event properties is essential. We here develop a complete framework that allows detailed studies. Special attention is given to the hadronization phase, wherein the baryon number violating vertex is associated with the appearance of a junction in the colour confinement field. This allows us to tell where to look for the extra (anti)baryon directly associated with the baryon number violating decay.Comment: 1+55 pages, 30 figure

MRNIP/C5orf45 Interacts with the MRN Complex and Contributes to the DNA Damage Response.

Through an RNAi-based screen for previously uncharacterized regulators of genome stability, we have identified the human protein C5orf45 as an important factor in preventing the accumulation of DNA damage in human cells. Here, we functionally characterize C5orf45 as a binding partner of the MRE11-RAD50-NBS1 (MRN) damage-sensing complex. Hence, we rename C5orf45 as MRNIP for MRN-interacting protein (MRNIP). We find that MRNIP is rapidly recruited to sites of DNA damage. Cells depleted of MRNIP display impaired chromatin loading of the MRN complex, resulting in reduced DNA end resection and defective ATM-mediated DNA damage signaling, a reduced ability to repair DNA breaks, and radiation sensitivity. Finally, we show that MRNIP phosphorylation on serine 115 leads to its nuclear localization, and this modification is required for MRNIP's role in promoting genome stability. Collectively, these data reveal that MRNIP is an important component of the human DNA damage response

Author Correction:C16orf72/HAPSTR1/TAPR1 functions with BRCA1/Senataxin to modulate replication-associated R-loops and confer resistance to PARP disruption

Correction to: Nature Communications https://doi.org/10.1038/s41467-023-40779-9, published online 17 August 2023

QCD Event Generators

This report is a survey on QCD Event Generator issues of relevance for LEP 2. It contains four main sections: a summary of experience from LEP 1, extrapolations to LEP 2 energies, Monte Carlo descriptions and standardization issues.Comment: 84 pages, LaTeX2e, eps figures included in file using filecontents environments, gzipped, uuencoded, to appear in the proceedings of the LEP 2 Worksho

Loss of ubiquitin E2 Ube2w rescues hypersensitivity of Rnf4 mutant cells to DNA damage

SUMO and ubiquitin play important roles in the response of cells to DNA damage. These pathways are linked by the SUMO Targeted ubiquitin Ligase Rnf4 that catalyses transfer of ubiquitin from a ubiquitin loaded E2 conjugating enzyme to a polySUMO modified substrate. Rnf4 can functionally interact with multiple E2s, including Ube2w, in vitro. Chicken cells lacking Rnf4 are hypersensitive to hyroxyurea, DNA alkylating drugs and DNA crosslinking agents, but this sensitivity is suppressed by simultaneous depletion of Ube2w. Cells depleted of Ube2w alone are not hypersensitive to the same DNA damaging agents. Similar results were also obtained in human cells. These data indicate that Ube2w does not have an essential role in the DNA damage response, but is deleterious in the absence of Rnf4. Thus, although Rnf4 and Ube2w functionally interact in vitro, our genetic experiments indicate that in response to DNA damage Ube2w and Rnf4 function in distinct pathways