Вакуолярная система
Сравнительный объем, занимаемый основными элементами цитоплазмы в гепатоците. компартмент % от общего объема клетки Примерное число на клетку Цитозоль 54 Митохондрии 22 1700 Гр. ЭПР 9 Гл. ЭПР и аппарат Гольджи 6 Ядро 6 1 Пероксисомы 1 400 Лизосомы 1 300 Эндосомы 1 200
% от всех мембран Тип мембраны гепатоцит 5000 мкм 3 Экзокринная клетка поджелудочной железы 1000 мкм 3 Общий объем мембран Плазматическая мембрана Гр. ЭПР Гл. ЭПР Аппарат Гольджи Митохондрии Наружная мембрана Внутренняя мембрана ЯО Секреторные везикулы Лизосомы Пероксисомы Эндосомы 110,000 мкм 2 2 35 16 7 7 0.2 not determined 0.4 0.4 0.4 13,000 мкм 2 5 60 <1 10 4 3 3 not determined not determined not determined Соотношение различных типов мембран
Субдомены ЭПР субдомены функции белки Гр. ЭПР Транслокация Складывание и олигомеризация Гликозилирование Деградация Sec61 complex, TRAP, TRAM, BiP PDI, Calnexin, Calreticulin, BiP Oligosaccharide transferase EDEM, Derlin1 Гл. ЭПР Детоксикация Метаболизм липидов Метаболизм гема Метаболизм Са Метаболизм гликогена Метаболизм стероидов Семейство цитохром Р450 HMG-CoA reductase Cytochrome b(5) IP3 receptors Ядерная оболочка Транспорт: ядерные поры Заякоревание хроматина POM121, GP210 Lamin B receptor Экспортный домен Экспорт белков и липидов в секреторный путь Sar1p, Sec12p, Sec16p Контактные зоны Транспорт липидов LTPs
белки животные дрожжи растения Образование тубул Reticulons DP1 Lunapark family Lnp1p Yop1p Reticulons DP1 Слияние тубул Atlastin Sey1 RHD3 Пласты Climp63, p180, kinectin
Model for interactions among the HSP proteins in the tubular ER. In this schematic diagram, atlastin-1 and M1 spastin interact directly with the ER-shaping proteins, including REEP1, as well as with one another (not shown). These interactions very likely occur through the hydrophobic hairpins of each of these proteins inserted into the membrane, though the first hydrophobic domain may exist in the ER membrane instead as a more traditional transmembrane segment (see Figure Figure1J).1 J). It is unclear whether the larger atlastin-1 hydrophobic segments completely span the membrane. REEP1 makes direct contact with the microtubule cytoskeleton through its C-terminal cytoplasmic domain. The M1 isoform of the spastin ATPase also binds to microtubules, through the MIT domain or a region adjacent to it, and is involved in microtubule severing, coupling changes in ER morphology with microtubule dynamics. GTP, GTP-binding domain; MTB, microtubule-binding domain.
Атластин
Гранулярный ЭПР
Extra length peptide Normal length peptide Протеазы
Extra length peptide Normal length peptide Протеазы
Сигнальная последовательность
Сигнальная последовательность белок последовательность Preproalbumin Met-Lys- Trp -Val- Thr - Phe - Leu - Leu - Leu - Leu - Phe -Ile-Ser- Gly -Ser-Ala- Phe -Ser ↓ Arg... Pre-IgG light chain Met-Asp-Met-Arg-Ala-Pro-Ala-Gln- Ile-Phe-Gly-Phe-Leu-Leu-Leu-Leu-Phe -Pro-Gly- Thr-Arg-Cys ↓ Asp... Prelysozyme Met- Arg -Ser- Leu - Leu -Ile- Leu -Val- Leu - Cys - Phe - Leu -Pro- Leu -Ala-Ala- Leu - Gly ↓ Lys...
Protein Signal Sequence Length Charge BiP MKLSLVAAMLLLLSAARA 18 +1 Apo-A1 MKAAVLTLAVLFLTGSQA 18 +1 TGF- β 2 MHYCVLSAFLILHLVTVAL 19 0 Interferon γ MKYTSYILAFQLCIVLG 17 +1 Glucagon MKSIYFVAGLFVMLVQG 19 +1 Choriogonadotropin MEMFQGLLLLLLLSMGGTWA 20 -1 EGF-receptor MRPSGTAGAALLALLAALCPRA 24 +1 Growth hormone MATGSRTSLLLAFGLLCLPWLQEGSA 26 +1 Котрансляционный перенос
translocating chain-associating membrane protein translocon-associated protein β α
Сигнальная пептидаза
15 to 20
C-tail-anchored proteins (tail-anchored proteins ) Target SNAREs (syntaxin), Vesicle SNAREs (synaptobrevin), Giantin ( Golgi tethering protein ), Sec61γ, Sec61β, Cytochrome b(5), Heme oxygenase I and II, UBC 6 ( ER degradation ), Bcl-2, Bax, Tom5, Tom6
Сигнальная пептидаза BiP олигосахаридтрансфераза Гликозидазы 1, 2 Калнексин, калретикулин PDI -протеиндисульфидизомераза Ero1p
(a) Exposed hydrophobic regions (yellow) that will eventually be buried upon folding or oligomerization are often recognized by the Hsp70 chaperone BiP (red). (b) The lectins calreticulin (blue) and calnexin bind to sugar moieties possessing one terminal glucose residue (gray hexagon), which can be found on incompletely folded glycoproteins. (c) PDIs (green) form mixed disulfide bonds with free thiol groups to catalyze disulfide bond formation, reduction, or isomerization. Hsp70 BiP калретикулин / калнексин PDIs
PDI -протеиндисульфидизомераза
Ero1 Prx4
Glc 3 Man 9 GlcNAc 2 Asn-X-Ser/Thr Первичное гликозилирование 12-14акт
Калнексин Калретикулин
calreticulin
Glc, glucose; Man, mannose; UDP, uridine diphosphate; UMP, uridine monophosphate.) Протеиндисульфидизомераза – ERp57 ERAD – ЭПР-ассоциированная деградация белков sec61
Sep15 UGT1 ERp57 калретикулин BiP PDI
EDEM – ER degradation-enchancing alfa-mannosidase-like protein BiP
Убиквитин лигазы Doa10 Hrd1
BiP ATF6 UPR unfolded protein response ATF6 (activating transcription factor 6) IRE1 (inositol requiring 1), PERK/PEK (PKR-like endoplasmic reticulum kinase/pancreatic eIF2a kinase), bZIP (basic leucine zipper) domain-containing transcription factors IRE1 PERK BiP BiP ЭПР
BiP ATF6 UPR unfolded protein response ATF6 (activating transcription factor 6) IRE1 (inositol requiring 1), PERK/PEK (PKR-like endoplasmic reticulum kinase/pancreatic eIF2a kinase), bZIP (basic leucine zipper) domain-containing transcription factors IRE1 PERK BiP BiP ЭПР
UPR unfolded protein response ATF6 (activating transcription factor 6) IRE1 (inositol requiring 1), PERK/PEK (PKR-like endoplasmic reticulum kinase/pancreatic eIF2a kinase), bZIP (basic leucine zipper) domain-containing transcription factors
СИНТЕЗ ЛИПИДОВ В ЭПР
phosphatidylcholine sphingomyelin phosphatidylserine phosphatidylethanolamine S=S SH N C
Синтез фосфатидилхолина. fatty acyl-coenzyme A (fatty acyl CoA), glycerol 3-phosphate, and cytidine-bisphosphocholine (CDP-choline).
22 реакции
steroid regulator element-binding proteins (SREBP), SREBP cleavage-activating protein (SCAP ) SP1 and SP2 протеазы
SREB2 - активация генов, вовлеченных в синтез холестерола SREB 1а и SREB1 с– активация генов, вовлеченных в синтез жирных кислот Insig DHCR7 дигидрохолестерол редуктаза Холестерол ацилтрансфераза - ACAT нейтральная холестерол эфир гидролаза - nCEH Много холестерола – нарушение Са помп –нарушение калнексина и калретикулина – стресс ЭПР HMG CoA редуктаза
Microsomal triglyceride transfer protein (MTP), Ацетил СоА холестерол трансфераза (АСАТ)
lipid-transfer proteins (LTPs)- ceramide transport protein (CERT)
Невезикулярный транспорт стерола Везикулярный и невезикулярный– механизмы сортинга +мембраны должны различаться способностью связывать холестерол Напр. Сфинголипиды имееют высокую аффинность
Компартменты ЭПР
Гладкий ЭПР Семейство Функции Состав CYP1 метаболизм лекарств и стероидов (особенно эстрогена ) 3 подсемейства, 3 гена, 1 псевдоген CYP2 метаболизм лекарств и стероидов 13 подсемейств, 16 генов, 16 псевдогенов CYP3 метаболизм лекарств и стероидов (включая тестостерон ) 1 подсемейство, 4 гена, 2 псевдогена CYP4 метаболизм арахидоновой кислоты 6 подсемейств, 12 генов, 10 псевдогенов CYP5 синтез тромбоксана A 2 1 подсемейство, 1 ген CYP7 биосинтез желчных кислот, участие в метаболизме стероидов 2 подсемейства, 2 гена CYP8 различные 2 подсемейства, 2 гена CYP11 биосинтез стероидов 2 подсемейства, 3 гена CYP17 биосинтез стероидов, 17-альфа гидроксилаза 1 подсемейство, 1 ген CYP19 биосинтез стероидов (ароматаза, синтезирующая эстроген ) 1 подсемейство, 1 ген CYP20 не установлены 1 подсемейство, 1 ген CYP21 биосинтез стероидов 2 подсемейства, 1 ген, 1 псевдоген CYP24 биодеградация витамина D 1 подсемейство, 1 ген CYP26 гидроксилирование ретиноловой кислоты 3 подсемейства, 3 гена CYP27 различные 3 подсемейства, 3 гена CYP39 7-альфа-гидроксилирование 24-гидроксихолестерола 1 подсемейство, 1 ген CYP46 холестерол 24-гидроксилаза 1 подсемейство, 1 ген CYP51 биосинтез холестерола 1 подсемейство, 1 ген, 3 псевдогена
Компартменты ЭПР A single stage-9 Drosophila oocyte surrounded by follicle cells. Within the oocyte, Gurken protein (red) is restricted to the dorsal/anterior corner near the nucleus (N) in dots representing the tER–Golgi units. The ER in all cells is green. (Courtesy ofBram Herpers, Department of Cell Biology, UMC Utrecht, The Netherlands.)
Взаимодействие с ПМ Взаимодействие с МТХ
Chromatin LAP2 a otefin LAP2 b LBR Lamin B Lamins A/C Emerin H2A, H2B, MAR/SAR? HP1, DNA? ? baf Inner nuclear membrane
Thus, the key events of ERAD are: (i) substrate recognition, (ii) substrate delivery to the cytosolic site of ubiquitin ligase action, (iii) substrate extraction from the ER, and (iv) delivery to the proteasome for degradation. Indeed, chaperones such as PDI, GRP94, and BiP can associate with ERAD components such as Derlins, OS-9, XTP3-B, and signal peptide peptidase [
phosphatidylcholine phosphatidylethanolamine glycerolipides phosphatidylserine sphingomyelin sphingolipides phosphatidylinosit phospholipides
Current Research: Lipid Biosynthesis; Cellular Lipid Trafficking and Mobilization; Lipoprotein Assembly and Secretion; Targeted Drug Delivery Apolipoprotein B (apoB) is responsible for the packaging of dietary and endogenous lipids into lipoprotein particles. These particles are distributed to peripheral tissues, such as muscle and adipose, where the lipids are either utilized for energy or stored. However, intravascular metabolism of apoB-containing lipoproteins, particular those originating from liver, give rise to low density lipoproteins (LDL), which in elevated concentrations in the blood cause atherosclerosis and heart disease. Furthermore, imbalances among hepatic and intestinal lipid biosynthesis, storage, utilization and secretion are important in the pathobiology of obesity, type 2 diabetes, and nonalcoholic fatty liver disease. Our laboratory is studying the process by which apoB, in combination with the microsomal triglyceride transfer protein (MTP) and other cofactors, controls lipid transport by the liver and intestine. Our most recent studies have focused on the unexpected evolutionary origins of lipid transport proteins. It is now clear that MTP is the ancestral member of this gene family and exists in divergent vertebrate and invertebrate species, whose last common ancestor diverged over 550 million years ago. During its long evolutionary history, MTP has acquired distinct functions enabling it to participate in a disparate array of lipid mobilization and transport pathways, ranging from primitive lipoprotein assembly in nematodes and arthropods, to bulk lipid transport and antigenic lipid presentation in humans. Our phylogenic dissection of MTP and apoB function coupled with ongoing structural, biochemical, and biophysical analyses, are providing new insights into mechanisms of lipid mobilization and secretion and strategies to beneficially control disturbances in lipid metabolism associated with chronic disease. As an outgrowth of our studies on the structure and function of apoB, we are also exploring the use of apoB’s lipid binding domains to achieve the packaging of lipophilic drugs. By fusing apoB to single chain antibodies unique to transformed cells, we hope to achieve the selective delivery of drug-containing complexes to cancer cells without affecting healthy cells and tissues. Figure Legend: Lipid Mobilization and Secretion by Lipoprotein Producing Cells. Neutral lipids such as triglycerides and cholesterol esters are synthesized by enzymes associated with the endoplasmic reticulum (ER) membrane (1). These lipids are either stored in the cytosol or consumed for energy production (Utilization). Lipid secretion requires the mobilization of cytosolic lipid, a step that involves cytosolic and membrane-associated factors that are not well defined (2). Microsomal triglyceride transfer protein (MTP), which resides in the lumen of the ER, possesses a neutral lipid transfer activity that is required for the generation of lumenal lipid droplets. MTP also functions directly on apolipoprotein B (apoB) to form precursor lipoprotein particles (5). These precursor particles fuse with lipid droplets to form mature intestinal chylomicrons of hepatic VLDL (6), which are then secreted via the classical secretory pathway (7).
Невезикулярный транспорт SCP2 -неспецифический транспорт жирных кислот и холестерола Кавеолин1+шапероны SCP2
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