Three mammalian nuclear lamin proteins, lamin B1, lamin B2 and the lamin A precursor, prelamin A, undergo canonical farnesylation and processing at CAAX motifs. In the case of prelamin A, there is an additional farnesylation-dependent endoproteolysis, which is defective in two congenital diseases: Hutchinson-Gilford progeria (HGPS) and restrictive dermopathy (RD). These two diseases arise respectively from defects in the prelamin A substrate and the enzyme (ZmpSte24) that processes it. Recent work has shed light on the roles of the lamin proteins and the enzymes involved in their farnesylation-dependent maturation. Other experimental work, including mouse model studies, have examined the possibility that farnesyl transferase inhibitors can represent effective treatment for HGPS. However, there are concerns about their use for this purpose given the potential for alternative prenylation pathways.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is caused by a point mutation in the LMNA gene, resulting in production of a truncated farnesylated-prelamin A protein (progerin).We previously reported that XPAmislocalized to the progerin-inducedDNAdouble-strand break (DSB) sites, blocking DSB repair, which led to DSB accumulation,DNA damage responses, and early replication arrest inHGPS. In this study, the XPA mislocalization to DSBs occurred at stalled or collapsed replication forks, concurrent with a significant loss of PCNA at the forks, whereas PCNA efficiently bound to progerin. This PCNA sequestration likely exposed ds-ssDNA junctions at replication forks for XPA binding. Depletion of XPA or progerin each significantly restored PCNAat replication forks.Our results suggest that although PCNAismuchmore competitive than XPAin binding replication forks, PCNA sequestration by progerin may shift the equilibrium to favor XPA binding. Furthermore, we demonstrated that progerin-induced apoptosis could be rescued by XPA, suggesting that XPAreplication fork binding may prevent apoptosis in HGPS cells. Our results propose a mechanism for progerininduced genome instability and accelerated replicative senescence in HGPS. - Hilton, B. A., Liu, J., Cartwright, B.M.,Liu,Y.,Breitman,M.,Wang,Y., Jones,R.,Tang, H.,Rusinol,A.,Musich,P.R.,Zou,Y.Progerin sequestrationof PCNApromotes replication fork collapse andmislocalization ofXPAin laminopathy-related progeroid syndromes.

Indiana University-Purdue University Indianapolis (IUPUI) / Atherosclerosis is a chronic disease that happens mostly in aged people, and recently studies have showed many similarities between Hutchinson Gilford Progeria Syndrome (HGPS) cells and aging cells, implicating dysfunctions of lamin A/C in aging process and atherosclerosis, as HGPS is caused by a mutated form of lamin A/C. Blood flow in arteries is generating shear stress that is mostly applied on endothelial cells that align along inner blood vessel wall. At the same time, endothelial cells are also under stretch by periodic arterial pulses. Considering the fact that atherosclerosis is prone to developing at arterial branches with disturbed shear and increased stretch, it is highly possible that laminar flow and proper stretch force are regulating endothelium to function appropriately. In this thesis, the investigation of what effects laminar flow or cyclic stretch can bring to endothelial cells was conducted, and examination of lamin A/C expression under mechanical forces were elaborated and incorporated with cell senescence. Results showed that laminar shear stress and stretch force can regulate lamin A/C expression in different patterns, which were impaired in senescent cells.

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<p dir="ltr">Premature aging disorders such as mandibuloacral dysplasia disorder (MAD) can be caused by improper maturation of nuclear scaffolding protein lamin A from its precursor prelamin A. ZMPSTE24 is responsible for both the earlier C-terminal CAAX cleavage and a subsequent N-terminal upstream cleavage during the posttranslational processing of prelamin A to lamin A. Although ZMPSTE24’s structure and function are well characterized, the role of the 4 apparent openings into its hollow inner chamber remains unknown. We hypothesize that one of these entrances, portal 1, is the initial substrate entry point based on its proximity to the zinc-coordinating active site. Unfortunately, ZMPSTE24 is difficult to express and purify. Fortunately, the yeast homolog, Ste24, not only shares many structural and functional similarities to ZMPSTE24 but is also much easier to express and purify in an active state. Therefore, we will use Ste24 and its substrate <b>a</b>-factor as a model system for ZMPSTE24 and its substrate prelamin A to deduce whether portal 1 acts as the primary substrate entry point. We examined portal 1’s function in primary substrate entry by observing how the incubation of portal 1 mutants engineered with cysteine residues around the portal with cysteine-reactive bismaleimide crosslinkers affects the activity of the C-terminal CAAX cleavage. If crosslinking of the cysteine residues occludes the portal, we hypothesize that activity will decrease because substrate cannot enter. The cysteine-less enzyme Ste24 (QA), which cannot react with the crosslinkers, was engineered by mutagenesis to contain 1 or 2 new cysteines at specific positions around this portal. We hypothesize that portal 1 occlusion with cysteine reactive bismaleimide crosslinkers will inactivate the enzyme by preventing substrate entry. We monitored changes in CAAX cleavage activity with a radioactive endoprotease-coupled CAAX assay.</p><p dir="ltr">In crude membranes derived from yeast expressing QA Ste24, activity was not inhibited in the presence of either BMH or BMOE crosslinker. For the single cysteine-containing mutants M210C, T267C, I307C, and V311C, each crosslinker similarly decreased activity over 50%. For the double cysteine-containing mutants M210C-I307C, T267C-I307C, and T267C-V311C, we found between 20-60% decreased activity in the presence of the crosslinker which has a length most similar to the distance between the two cysteines. These results closely reflect previous data and further suggest that CAAX activity of the enzyme may be decreased due to the occlusion of the primary entry site, portal 1.</p><p dir="ltr">With purified QA Ste24, changes in activity were less apparent. Activity for purified QA was not decreased in the presence of either crosslinker. Single cysteine-containing mutants did not show decreased activity in the presence of either crosslinker. Unlike what was observed in crude membrane preparations, the double-cysteine containing mutants exhibited minimal decrease in activity in the presence of the crosslinker that has a length most similar to the distance between the two cysteines.</p><p dir="ltr">In crude membrane preparations, the cysteine-containing QA Ste24 mutants have diminished activity in the presence of crosslinkers. This may be due to the occlusion of the primary entry point, portal 1. However, we recognize the possibility that the decrease in activity was the result of the occlusion of the exit portal site. It is imperative that further experiments confirm that exit portal occlusion is not occurring. For purified cysteine-containing QA Ste24 mutants, the negligible decrease in activity suggests either that the portal 1 is not the primary substrate entry point or that the conditions of the assay were not optimized to generate inhibition. For example, the concentration of crosslinker was not sufficient in the presence of excess lipid and the reconstitution mixture sequestered the crosslinker. Further optimization of the reaction conditions is warranted. The cysteine-containing QA Ste24 mutants must be assessed for free thiols to determine how successful the reaction with the crosslinkers is. A more developed understanding of how all four portals function in the Ste24 CAAX processing of <b>a</b>-factor will be very insightful towards the mechanism of ZMPSTE24 in lamin A CAAX processing and may catalyze new targets of study for ZMPSTE24-related diseases like MAD-B.</p>

Patients of Hutchinson-Gilford Progeria Syndrome (HGPS) display a rate of aging up to ten times that of normal human populations. It might be expected that HGPS cells would have a decreased ability to repair DNA damage through the cell cycle as compared to normal cells such as those of the BJ cell line since DNA damage accumulation is a hallmark phenotype of aging. On earth, we are exposed to far more ultraviolet-B (UV-B, 280-315 nm) and UV-A (315-400 nm) than UV-C (100-280 nm) radiation, since the latter is filtered-out by the atmospheric ozone layer. The relative sensitivity of prematurely aging HGPS cells to UV-B irradiation is unknown. It was hypothesized that the normal fibroblast cell line (BJ) would exhibit a higher rate of DNA repair and a higher level of cell viability after exposure to ultraviolet radiation than would be observed with the HGPS cells, and that these differences would be greater as the HGPS cells age in culture. A Cell-Titer Blue Viability Assay (Promega) was used to determine the effect of UV-B and UV-C on metabolic activity, an indicator for cell viability, in HGPS, BJ, and A549 (a human lung carcinoma) cells. A translesion DNA synthesis protein, pol-η, and several other DNA transcription and repair-related proteins also were hypothesized to be altered in the HGPS cell line, both before and after UV-induced DNA damage, as compared to the BJ cell line. Western blotting was used to monitor these proteins in BJ and HGPS cells following UV-C exposure. No differences in short-term viability were observed between BJ and HGPS cells, reflecting similarities in their repair abilities on the cellular level; however, there were significant differences in long-term viability. Enzyme Linked Immunosorbant Assays (ELISA) revealed a significant difference in DNA repair at the molecular level. Moreover, Western blotting revealed differences in the amounts of several repair-related proteins following UV exposure, including pol-η, an important trans-lesion synthesis protein. Although the difference in DNA repair did not appear at the cellular level, it is apparent that HGPS cells show a greater sensitivity to both UV-B and UV-C irradiation as compared to normal BJ fibroblasts and A549 carcinoma cells.

A common feature of progeria syndromes is a premature aging phenotype and an enhanced accumulation of DNA damage arising from a compromised repair system. HGPS (Hutchinson-Gilford progeria syndrome) is a severe form of progeria in which patients accumulate progerin, a mutant lamin A protein derived from a splicing variant of the lamin A/C gene (LMNA). Progerin causes chromatin perturbations which result in the formation of DSBs (double-strand breaks) and abnormal DDR (DNA-damage response). In the present article, we review recent findings which resolve some mechanistic details of how progerin may disrupt DDR pathways in HGPS cells. We propose that progerin accumulation results in disruption of functions of some replication and repair factors, causing the mislocalization of XPA (xeroderma pigmentosum group A) protein to the replication forks, replication fork stalling and, subsequently, DNA DSBs. The binding of XPA to the stalled forks excludes normal binding by repair proteins, leading to DSB accumulation, which activates ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) checkpoints, and arresting cell-cycle progression.

Indiana University-Purdue University Indianapolis (IUPUI) / Endothelial cells form the lining of the walls of blood vessels and are continuously subjected to mechanical stimuli from the blood flow. Microtubule-organizing center (MTOC), also known as centrosome is a structure found in eukaryotic cells close to the nucleus. MTOC relocates relative to the nucleus when endothelial cells are exposed to shear stress which determines their polarization, thus it plays a critical role in cell migration and wound healing. The nuclear lamina, a mesh-like network that lies underneath the nuclear membrane, is composed of lamins, type V intermediate filament proteins. Mutations in LMNA gene that encodes A-type lamins cause the production of a mutant form of lamin A called progerin and leads to a rare premature aging disease known as Hutchinson-Gilford Progeria Syndrome (HGPS). The goal of this study is to investigate how fluid flow affects the cytoskeleton of endothelial cells. This thesis consists of two main sections; computational mechanical modeling and laboratory experimental work. The mechanical model was implemented using Ansys Workbench software as a tensegrity-based cellular model in order to simulate the state of an endothelial cell under the effects of induced shear stress from the blood fluid flow. This tensegrity-based cellular model - composed of a plasma membrane, cytoplasm, nucleus, microtubules, and actin filaments - aims to understand the effects of the fluid flow on the mechanics of the cytoskeleton. In addition, the laboratory experiments conducted in this study examined the MTOC-nuclear orientation of endothelial cells under shear stress with the presence of wound healing. Wild-type lamin A and progerin-expressing BAECs were studied under static and sheared conditions. Moreover, a custom MATLAB code was utilized to measure the MTOC-nuclear orientation angle and classification. Results demonstrate that shear stress leads to different responses of the MTOC orientation between the wild-type and progerin-expressing cells around the vertical wound edge. Future directions for this study involve additional experimental work together with the improved simulation results to confirm the MTOC orientation relative to the nucleus under shear stress.

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.

Hutchinson-Gilford Progeria Syndrome is described as a series of mutations within the lamin A gene leading to the accumulation of progerin in the nucleus, contributing to premature aging and affecting the epigenetic control. Epigenetic control, such as DNA methylation, relies on DNA methyltransferase enzymes. In human cells, heat shock (HS) leads to the formation of nuclear stress bodies (nSBs); ribonucleoprotein aggregates of Sat III RNA and RNA-binding proteins. The objectives of this study were to determine if epigenetic status induces varying responses to HS and assess the variability of nuclear proteins in similar conditions. Results show epigenetic modifications do not prevent a stress response; however the extent may be affected. In addition the functions of most nuclear antigens were not affected. It is most likely the sum of interactions at the inner nuclear membrane and nuclear lamina interface that result in nuclear strength pertaining to lamin A.