Accelerated aging diseases and their genetic causes Complex pattern of aging markers in primary human fibroblasts презентация

markers in primary human fibroblasts

doubling time increases, cell morphology alteres, larger rotund cells appear, which are regarded as older, in comparison with smaller oblong ones (Мikhelson, 1984; Lorenzini et al., 2005). Biochemical markers appear simultaneously with morphological alterations. They characterize aging cells in culture, defined as “aging markers”, which include changes in chromatine, nucleus and cytoplasmatic skeleton, high level of non-repaired DNA damages, etc. (Campisi, 2005); as well as in cell instability towards action of damaging agents, primarily hydrogen peroxide (Chen et al., 1998, Ryter et al., 2007).

associated with aging, its activity dramatically increasing in old cells (Dimri et al., 1995; Lee et al., 2006)

with premature ageing with Hutchinson-Gilford syndrome (children’s progeria), and the Werner one (adult progeria), normally serve as model for the detection of ageing markers
Fibroblasts taken from old donors, and from the progeria patients, are currently regarded as containing more ageing markers, than those taken from young healthy donors (Scafidi, Misteli, 2006, Sedelnikova et al., 2008).
Fibroblasts of skin of other mammals (mice) may also be applied for the detection of ageing markers (taking into account the Hyflick limit)

site
15 (21)
LMNA 1q21.2
FACE-1/ZMPSTE24 1q34
Skin atrophy.
Bird’s face
Loss of subcutaneous fat
Hair loss.
Arteriosclerosis
Increased metabolic rate
Hypogonadism
Grow hormone insensitivity?
Slow growth.
Reduced replicative life span of cultured cells
Shot telomeres
DNA repair defects?
Silent mutation in (1-4)-β-Galactosyl-transferase gene (594C>T)

of the accumulation of aberrant product of the LMNA gene, progerin, were demonstrated in the course of the study of cells of patients with Hutchinson-Gilford syndrome, as well as of cell cultures, acquired from aged donors (Scaffidi, Misteli, 2006). Results acquired by these authors, taken in complex with the present-day data concerning participation of components of nuclear lamina, including lamin А in the wide range of cell processes. Allow to regard structural alterations of nuclear lamina as the basic process for induction of markers of ageing, associated with various levels of cell regulation .

Aging. Science 312 : 1059 – 1063

Farnezyl-protein-transpherase

Ras-converting enzyme or Zmpste24

S-adenosyl- metionin;
isiprenyl-carboxymethyl-transpherase

Zmpste24

lamine А

«progerin»

Rusinol,
Sinensky, 2006

362 : 440–445.

Ramirez et al., 2006

et al., 2008)
Healthy donor (11 у.) – 5 %
Healthy donor (87 y.) - 20%
Atypical Werner’s syndrome – 40%
Hutchinson-Gilford’s syndrome – 80%

cells in culture, cells from old donors, and from Progeria patients (Sedelnikova et al., 2004,2008).
This phenomenon may be linked to both accumulation of either non-repaired DSBs or, modifications of chromatine, apprehended as DSBs by protein kinases АТМ and DNA-PK after DNA damage (Stiff et al., 2004), or else, ATR kinase during replication halt (Takahashi, Ohnishi, 2005); as well as by appearance of uncapping telomeres (Hao et al., 2004).
As a result, accumulation in cell population of cells with foci of γ-Н2АХ occurs, which forms an objective criterion of ageing on cell level.

aging. Science. 312:1059-1063

regulation with no changes in the DNA sequence itself are considered as epigenetic determinants. In recent years, the role of epigenetic mechanisms in the process of carcinogenesis (Jones, Baylin, 2002), as well as in cellular and organismal aging (Wilson, Jones, 1983; Issa, 2003; Fraga et al., 2005) was demonstrated.

aging. Science. 312:1059-1063

a RecQ family helicase.
Skin atrophy.
Hair graying/loss.
Arteriosclerosis
Osteoporosis
Muscle atrophy
Cataracts
Hyperlipidemia
Mild diabetes melitus, type 2
Hypogonadism
Cancer (sarcomas)
Slow growth.
Reduced replicative life span of cultured cells
Chromosome rearrangements
Sensitivity to 4-NQO and camptothecin (topoisomerase I poison)
Increased mutation rate, particulary DNA deletions
Rapid telomere shortening during cellular life span.
DNA repair defects
DNA replication defects (?)

the face, forming the red “butterfly”

Low DNA synthesis (2 times) with the normal rate of DNA-polymerases α, β, γ
Chromosomes rearrangements
Icreased level of recombination

Bloom syndrome

besides the identification of all markers of aging described by Scaffidi P., Misteli T (2006), we observed a significant discrepancy between these cells and cells from old donors. The level of stable chromosomal aberrations in the investigated cells was not elevated and, by this the marker, normally showing the "real" biological age, our patient was consistent with her 9 years.

frequency analysed by FISH in human blood lymphocytes

Age dynamics

The patients with Werner’s syndrome demonstrate increased SCA level, corresponding to premature aging of organisms.
The lymphocytes from patient with Hutchinson-Gilford syndrome have no time for SCA formation

patient

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(leukemias and lymphomas)
Occulocutaneous telangiectasias
Progeroid skin and hear changes
Hypogonadism
Defectif cell cycle chekpoint arrest
Reduced replicative life span
Chromosomal rearrangements
Sensitivity to ionizing radiation
Inoppropriate apoptosis

Shot telomeres
Delayed/absence P53 induction and accumulation after DNA damage
Defects in repair of DNA double-strand breaks
Defects in V(D)J recombination

Ataxia-telangiectasia (Luis-Bar syndrome)

хромосомных аномалий, увеличенная чувствительность к ионизирующим излучениям и радиомиметикам, к УФ-свету и агентам сходного действия (таким, как 4-нит-рохинолиноксид)
Появляется у одного из 40 тыс. новорожденных, основные поражения отмечены в нервной и иммунной системах (мозжечковая атаксия, приводящая к нарушениям координации мышц, шатающейся походке и прогрессирующей умственной отсталости, кожным нарушениям, предрасположенности к раковым заболеваниям и др.)

donor 51 y. and АТ8SP (11 y.). For purposes of detection of the number of nuclei, the same field of vision is presented, after DAPI coloring.

1.08±1.07

4.06±1.28*

12.32±2.09**

of serine (S1219) in 53BP1 (Lee et al., 2008). This protein also plays an important role in the checkpoint response of the cell to damage (Iwabuchi et al., 2008, Eliezer et al., 2008). The appearance of foci 53ВР1, as well as foci of γ-Н2AХ can be regarded as one of the reliable markers of aging cells.
53ВР1 protein forms a traditional marker of repair processes (Wilson, Stern, 2008). Appearance and elimination of 53ВР1 foci after DNA damage tends to coincide with γ-Н2АХ dynamics, as they often colocalize in the zone of DSB

to occur in coloring by 53ВР1 antibodies in intact cells, while clear-cut foci form after DNA damage. Such foci tend to form in AT patients’ cells in many intact cells, i.e. 16% and 18% respectedly, while there are only 6 % of such cells in control.

donor 51 y. и АТ8SP (11 y.). DAPI

170,71 ± 1,91

134,45 ± 4,95*

93,01 ± 3,55*

Fluorescence intensity

donor 52 y, AT6SP (23 y.) and АТ8SP (11 y.)

level of trimethyl derivatives of histone H3 (K9 and K27), in contrast to the cells from old donors and other progeria, is not falling, but rather rising, as it is shown in tumor cells.
In the case of AT6SP, the level of 3meK9H3 in contrast to the cells from AT8SP is falling as in the cells of old donors, but 3meK27H3 rather rising, as in AT8SP.
The quantity of 3meK9H3 may serve as a prognostic marker of gravity of the disease

JA. 2003. A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome. Nat Genet.,33:497-501

донора VH-10 (вверху) и больной с синдромом Секкеля Sc1SP (внизу)

response during genome replication and is mutated in primordial dwarfism.
Nat Genet. 
48(1):36-43
The TRAF [tumor necrosis factor (TNF) receptor-associated factors] -interacting protein (TRAIP) functions as (RING)-type E3 ubiquitin ligase, but its physiological substrates are not yet known. 

DNA damage sites induced by UV laser microirradiation both in the absence and presence of pre-treatment with BrdU as a damage sensitizer. Representative images, before and after UV laser microirradiation. Scale bar, 5 μm. (b) GFP-TRAIP colocalizes with γH2AX and with RFP-PCNA at sites of UV laser-induced damage. Representative images of UV laser-irradiated GFP-TRAIP expressing cells immunostained for γH2AX (pre-sensitized with BrdU) or co-expressing RFP-PCNA (no BrdU pre-treatment) as indicated. Scale bar, 5 μm. (c, d) GFP-TRAIP is detected by a Proximity Ligation Assay (PLA) in close proximity to PCNA, an association enhanced after UV-induced damage. (c) Representative images of PLA signals/nucleus in doxycycline-inducible GFP-TRAIP HeLa cells before and after damage with 25 J/m2 UV-C. Scale bar, 5 μm. (d) Quantification of PLA signals/nucleus. Box plots, center line denote mean values, box 25/75 %, whiskers 5/95 %, data pooled from n=2 independent experiments, n>65 data points per condition per experiment; Mann Whitney rank sum test: *** p<0.001. (e) TRAIP accumulates at sites of localized UV damage, colocalising with RPA and γH2AX. Representative immunofluorescence images of MRC5 cells transfected with GFP-TRAIP or GFP alone after UV-C irradiation through 3 μm microfilters. Scale bar, 5 μm.

выявлен дефект NER
Дефекты эксцизионной репарации нуклеотидов (нарушение вырезания, застройки брешей и др.). Разнообразные дефекты репарационных процессов.
При XP-вариант наблюдается изменение параметров репликации ДНК
Сверхчувствительность к УФ-свету,
ведущая к появлению красных пятен на коже, переходящих в незаживающие коросты и нередко в рак кожи; неврологические расстройства); поражения век, бровей и глаз. Распространение: 1 случай на 250000 человек в Европе и США; 1 :40000 человек в Японии
 

skin cancer and internal neoplasms: autopsy report of four xeroderma pigmentosum patients (XP-A, XP-C and XP-D). Acta Neuropathol Commun.  1:4. doi: 10.1186/2051-5960-1-4.

XP patients studied. A and B: Case 1 XP-A patient (A) at age 17y with numerous freckle-like pigmented lesions on sun exposed skin and (B) at 37y with marked cachexia and thinning of subcutaneous tissues of face and chest. She had more that 100 surgical procedures on her face for removal of skin lesions. C: Case 2 XP-D patient at age 40y. She had been well protected from sun exposure since early childhood and had only few pigmented lesions and skin cancers. D: Case 3 XP-C patient, at age 29y with multiple freckle-like pigmented lesion on sun exposed skin and cheilitis. The patient underwent many surgical procedures for removal of skin cancers on her face. E and F: Case 4 XP-C patient, at age 28y (E) with multiple pigmented lesions, telangiectasia, cheilitis and corneal clouding. Multiple surgical procedures were performed on her face for removal of skin cancers and at age 48y (F) following exenteration of both orbits for treatment of ocular squamous cell carcinomas.

family),
XPB and XPD (helicases in TFII-H),
XPG (yeast Rad2 homologue, endonuclease)
20 (40?)
Bird’s face
Loss of subcutaneous fat
Skin photosensitivity
Neurodegeneration
Hypogonadism

UV-sensitivity
Impaired transcription-coupled nucleotide excision repair
Decreased recovery of transcription after irradiation
General RNA-polymerase II transcription defects

Cockayne syndrome (CS)

репарации повреждений от химических мутагенов и канцерогенов (по не УФ-свста), обусловленные дефектами эндонуклеаз, дефектами распознавания кросс-сшивок ДНК;
пониженная способность к апоптозу после ионизирующего облучения; двукратное удлинение G2-фазы клеточного цикла
Сверхчувствительность к химическим мутагенам и канцерогенам, уменьшение количества всех клеточных элементов крови, различные аномалии врожденных способностей, деформация пальцев и другие виды скелетных нарушений, урогенитальные нарушения, микроцефалия, микрофтальмия, дефекты уха и потеря слуха, сердечные и гастроинтестинальные нарушения

anemia genes.
Dong HDong H, Nebert DWDong H, Nebert DW, Bruford EADong H, Nebert DW, Bruford EA, Thompson DCDong H, Nebert DW, Bruford EA, Thompson DC, Joenje HDong H, Nebert DW, Bruford EA, Thompson DC, Joenje H, Vasiliou V.

Fanconi anemia (FA) is a recessively inherited disease manifesting developmental abnormalities, bone marrow failure, and increased risk of malignancies. Whereas FA has been studied for nearly 90 years, only in the last 20 years have increasing numbers of genes been implicated in the pathogenesis associated with this genetic disease. To date, 19 genes have been identified that encode Fanconi anemia complementation group proteins, all of which are named or aliased, using the root symbol "FANC." Fanconi anemia subtype (FANC) proteins function in a common DNA repair pathway called "the FA pathway," which is essential for maintaining genomic integrity. The various FANC mutant proteins contribute to distinct steps associated with FA pathogenesis. Herein, we provide a review update of the 19 human FANC and their mouse orthologs, an evolutionary perspective on the FANC genes, and the functional significance of the FA DNA repair pathway in association with clinical disorders. This is an example of a set of genes--known to exist in vertebrates, invertebrates, plants, and yeast--that are grouped together on the basis of shared biochemical and physiological functions, rather than evolutionary phylogeny, and have been named on this basis by the HUGO Gene Nomenclature Committee (HGNC).

by the FA core complex via monoubiquitination (orange circles) regulates downstream genes involved in recombination repair of DNA crosslinks.

HSCT

Forty-three of 83 female FA patients (51.8%) and 17 of 46 male FA patients (37.0%) developed SCC (average age at SCC diagnosis for 48 FA patients without HSCT 30.0 years, for 12 FA patients after HSCT 25.8 years).

the most widely studied at present time in the framework of study of primary mechanisms of the organism ageing, potential factors, conditioning life span. Considerable interest directed at these specialized complexes, is conditioned by their unique functions in the securing of the cell genome (Blackburn, 2001). Apart from preventing the chromosome fusion, telomeres are responsible for their fixation at cell membrane (Podgornaya et al., 2000; Hediger et al., 2002; Rose et al., 2004), for the mitotic and meiotic segregation of chromosomes (Conrad et al.,1997; Kirk et al., 1997; Dynek, Smith, 2004), and for their meiotic coupling (Rockmill, Roeder, 1998), for the stabilization of broken chromosomes (Jager, Philippsen, 1989; Pennaneach et al., 2006), and for their defence against reparation systems (Shay, Wright, 2007; Mirsi et al., 2008), as well as being responsible for gene expression (Baur et al., 2001; Pedham et al., 2006).

senior age respondents

Regular correlation between telomere length with respondent age appears after having attained certain age, when adequate strategy of active longevity has been attained, and individual differences may be traced back.

by the FA core complex via monoubiquitination (orange circles) regulates downstream genes involved in recombination repair of DNA crosslinks.

a replication fork with an ICL

Анемия Фанкони (A, B, C, D1, D2, E, F, G, I, K, L, M, N, O, P, and Q). Эти белки делятся на 3 группы: (1) белки корового комплекса; (2) FANCI и FANCD2 белки, составляющие комплекс ID2 и (3) эффекторные белки
(A) FA активируется во время фазы S при обнаружении ICLs или сходных повреждений ДНК. Коровый комплекс FA привлекается в зону повреждения MHF1-MHF2-FANCM комплексом.
(B) Комплекс ID2 моноубиквитинируется и связывается с зоной повреждения ДНК. Комплекс B-L-100 способствует этому убиквитинированию совместно с еще двумя субкомплексами (A-G-20 and C-E-F)
(C) Нуклеазы XPF/FANCQ-ERCC1 совместно с SLX4/FANCP, incise the Dпроводят ДНК-инцизию
(D) Моноубиквитинированный комплекс ID2 привлекает белки репарации, включая BRCA1, BRCA2/FANCD1, FANCJ, PALB2/FANCN, and RAD51C/FANCO.
(E) После успешной репарации комплекс, ID2 деубиквитинизируется USP1-UAF1 , что способствует его высвобождению из хроматина.

ответ на сигнал о повреждении ДНК (фосфорилирование ATR/ATM),через FA/BRCA патвэй [1], формируется коровый комплекс FA – состоящий из FANCA (A), FANCB (B), FANCC (C), FANCE (E), FANCF (F), FANCG (G), FANCM (M), и FANCL (L) белков плюс FAAP20, FAAP24, и FAAP100 (FAAP). Этот комплекс связывается UBE2T(T) через FANCL, моноубиквитинируя и активируя димер FANCD2/I. FANCD2/I (D2/I) переносится в зону повреждения и привлекает туда эе белки-эффекторы, включая BRCA1 (S), BRCA2 (D1), RAD51 (R), BRIP1 (J), PALB2 (N), RAD51C (O), SLX4 (P), and ERCC4 (Q), и ждругие репарационные факторы (FAN1). Через FANCM/BS патвэй [2], FA коровый комплекс связывается с комплексом BS путем взаимодействия FANCM-RMI1и TopoIIIα с BS, также привлекая его в район повреждения. Через FANCD2/ATM патвэй [3],, FANCD2 фосфорилируется ATM и колоколизуется с комплексом NMR , что вызывает S-арест. complex

mammals (Homo sapiens, Mus musculus, and Gallus gallus), amphibian (African clawed toad, Xenopus laevis), fish (zebrafish, Danio rerio), sea squirt (Ciona intestinalis), insect (Drosophila melanogaster), worm (Caenorhabditis elegans), yeast (Saccharomyces cerevisiae), and plant (Arabidopsis thaliana). FANC genes are grouped into three classes. Group I includes nine genes that encode proteins that form the FA core complex; group II encodes FANCD2 and FANCI that form the D2/I complex; group III comprises eight genes that encode FA effector proteins that function downstream of D2/I complex. Lower eukaryotes tend to be missing orthologues of the FA core complex genes. A = FANCA, B = FANCB, C = FANCC, D2 = FANCD2, E = FANCE, F = FANCF, G = FANCG, I = FANCI, L = FANCL, M = FANCM, D1 = BRCA2/FANCD1, J = BRIP1/FANCJ, N = PALB2/FANCN, O = RAD51C/FANCO, P = SLX4/FANCP, Q = ERCC4/FANCQ/XPF, R = RAD51/FANCR, S = BRCA1/FANCS, T = UBE2T/FANCT. If we extend this gene family update to include prokaryotes, it might be noted that, whereas no orthologs of any of the 19 eukaryotic FANC genes exist in prokaryote genomes, RAD51 (as a nineteenth FANC member in living organisms) qualifies as a homologue of bacterial RecA

the FANCD2 protein indicating the amino-terminal NLS (nuclear localization signal) domain (green), CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation) domain (maroon), PIP-box (PCNA-interacting protein motif) (orange), and the C-terminal EDGE motif (purple). Functionally-characterized phosphorylation sites (teal) and K561 monoubiquitination site (yellow) are indicated by small circles. (B) Schematic of the FANCI protein indicating the Leu (leucine zipper) domain (light blue), ARM (armadillo repeat) domain (pink), and C-terminal EDGE motif (purple) and NLS domain (green). The S/TQ motif (teal) and K523 site of monoubiquitination (yellow) are indicated by small circles. (C) Mouse Fanci-Fancd2 heterodimer crystal structure represented as both surface and ribbons with domains indicated. This structure was solved by the Pavletich group in 2011(PDB ID: 3S4W).

FA core complex

. (A) The Skp1/Cullin/F-box protein (SCF) complex includes the Cullin protein, which acts as a scaffold to bridge the catalytic E3 ubiquitin ligase RBX1 to the adaptor protein Skp1, and the F-box protein.  The F-box protein recognizes and recruits the target protein for ubiquitination by the E2 ubiquitin-conjugating enzyme, UBC. (B) We propose that the FANCA protein is structurally analogous to Cullin, and may link the E3 ubiquitin ligase FANCL with the putative adaptor protein FANCC. FANCC has been shown to interact with both FANCA and FANCE, indicating that it may function analogously to Skp1.  FANCE may be analogous to the F-box protein. FANCE is known to interact directly with FANCD2 and may facilitate its monoubiquitination of FANCL and UBE2T.

upon monoubiquitination of FANCD2 and FANCI, which would preclude further ubiquitination. (A) The ID2 heterodimer inactivation model. Following monoubiquitination, ID2 heterodimerization occurs and is stabilized through a noncovalent interaction between monoubiquitin covalently linked to FANCI K523 and the FANCD2 CUE domain. There is also possibly a reciprocal interaction between monoubiquitinated FANCD2 K561 and an UBD in the carboxy-terminus of FANCI, shielding FANCD2 from further ubiquitination. (B) The FANCD2 self-inactivation model. Monoubiquitination could promote an intramolecular association between ubiquitin covalently attached to K561 and the amino-terminal CUE domain, resulting in a closed conformation. (C) The E3 ubiquitin ligase dissociation model. Once FANCL is autoubiquitinated, the ubiquitin moiety may interact noncovalently with the CUE domain on FANCD2 enabling monoubiquitination of FANCD2 on K561. This interaction is predicted to be weak and short-lived, leading to rapid dissociation of FANCL and FANCD2, precluding further ubiquitination.

humans, are less contradictory, and provide a picture which is much more stable. It is necessary to assess more accurately the character of dispersion of all markers studied by decades, and to form a reliable scale.

It is just this observation which demonstrates that progeria is a deeply pathological aging and its analogy with the natural aging is limited.

в свою очередь фосфорелирует серин в 215 положении у CDC25C, что приводит к блокированию ее функций. Фосфорелированная форма CDC25C связывается с белком 14-3-3σ, что поддерживает еe каталитическую неактивность и способствует переходу в цитоплазму и секвестрированию.

Вторая ветвь G2-чекпойнта опосредуется через ATR/CHK1 активацию. При этом пути одновременно фосфорелируется-выключается белок CDC25А, а также фосфорелируется серин-549 белка Wee1 (пртеинкиназа), что облегчает его связывание с тем же белком 14-3-3σ и приводит к усилению ингибиторной активности киназ по отношению к CDC2(CDK1). Это придает второй ветви большую гибкость в контроле и консолидации G2-ареста.

ее повышенной нестабильности, причем этот эффект определяет протяженность G2-ареста.
Циклин В во время G1 и S фаз имеет цитоплазматическую локализацию и перемещается в ядро только к началу митоза. Белок 14-3-3σ приводит к секвестрированию циклина В в цитоплазме в ответ на повреждение ДНК.

участие в митозе, включая вход и выход из него. PLK1 является позитивным регулятором CDC25C-активности в необлученных клетках и, специфически фосфорелируя ее, способствует вхождению в митоз. PLK3, напротив, активируется АТМ в ответ на повреждение ДНК, взаимодействует с CDC25C, фосфорелируя ее по серину-216, что приводит к ингибированию ее активности.

взаимодействие PCNA c Р21, CDC25C, и CDC2(Cdk1)-циклин В, но не одновременное, а последовательное. Связывание Р21 и CDC25C с комплексом PCNA-CDC2-циклин В является совершенно особым и не позволяет CDC25C дефосфорелировать CDК1 для активации митоза. Р21 может блокировать САК, которая активирует CDК1 путем фосфорелирования триптофана в 161 положении.

активирует транскрипцию трех вовлеченных в него белков: GADD45, P21 и 14-3-3σ и подавляет транскрипцию CDC2(CDK1) и циклина В.

Есть данные о вовлеченности в G2-арест BRCA1, опосредованно через ATM/ATR или напрямую, через активацию CHK1, но механизм этого остается неясным.

G2-реакция клетки на облучение зависит от фазы цикла, в которую это произошло.

же после облучения, захватывает клетки, облученные непосредственно в G2-фазе и является АТМ-зависимым, проходящим и независимым от дозы. Он приводит к резкому снижению митотического индекса.
Второй, который развивается позже, в клетках, облученных на более ранних стадиях клеточного цикла, является АТМ-независимым, зато зависимым от дозы и приводит к накоплению клеток в фазе G2.

заболеваний), нарушение вырезания димеров тbмина или недостаточная скорость застройки брешей после вырезания нуклеотидов, возможны нарушения транскрипции
Нехватка серы в белках волос и их луковиц, ведущая к ломкости волос, "тигровость" волос (чередование светлых и темных полос по длине волоса, выявляемое под микроскопом); ихтиоз; часто умственная и физическая отсталость; дефекты полового развития; аномалии кожи и зубов; нередки раковые заболевания
 

системам репарации - NER и HR