11 MZB I - Devuyst 2

In ER and cis-Golgi network:
• The N-glycosyl group is first modified to produce a high mannose-containing oligo.
• Phosphate addition to C6 is catalyzed by N-acetylglucosamine phosphotransferase.
• This enzyme binds to and recognizes a signal patch on the folded hydrolase.
• N-acetyl glucosamine is then removed to produce a mannose-6-phosphate

(The GlcNAc phosphotransferase enzyme that recognizes lysosomal hydrolases in the Golgi apparatus has separate catalytic and recognition sites. The catalytic site binds both high-mannose N-linked oligosaccharides and UDP-GlcNAc. The recognition site binds to a signal patch that is present only on the surface of lysosomal hydrolases. The GlcNAc is cleaved off by a second enzyme, leaving the M6P exposed (not shown).)

Bei einer Thesaurismose (früher Thesaurose) – von griech.: thesaurisma (Schatz, Vorrat) – oder Speicherkrankheit handelt es sich um eine Synthese oder Abbau betreffende Stoffwechselstörung. Jede Krankheit dieser Gruppe verursacht eine Anhäufung eines spezifischen Stoffwechselprodukts mit einem spezifischen Verteilungsmuster im Körper.

Defective lysosomal enzymes or defective targeting:
- Thesaurismoses – Lysosomal Storage Diseases 

• Secretory pathway: From ER → Golgi → endo/lysosomes or plasma membrane (pink) 
• Endocytic pathway: From extracellular space → lysosomes (green)

From Golgi – secretory - to lysosome (via endosome)
From plasma membrane - endocytosis - to endosomes

• Cargo receptors – captured by adaptins – bind to clathrin molecules (cytosol)
• Basket network – coated pit – dynamin assembly around the neck of vesicle
• Dynamin (GTPase) – pinch off the vesicle
• Removal of adaptin-coat – naked vesicle to fuse with target membrane

COP Proteins are a type of vesicle coat proteins.
-> COP-coated vesicles most commonly mediate transport between ER and Golgi

• Caused by mutations in OCRL1
  -> PtdIns(4,5)P2 5-Phosphatase
•X-linkedmultisystemic disorder
  1. Proximal tubular dysfunction (Fanconi Syndrome)
  2. Bilateral congenital cataracts
  3. Severe mental retardation (40 < IQ < 54) .

PtdIns(4,5)P2 5-Phosphatase 

• Distribution: Trans-Golgi network (major sorting site)

Role: Modulates the pool of membrane PtdIns(4,5)P2 :
1. Regulation of lysosomal enzymes delivery
2. Regulation of cell junctions and intracellular trafficking

• Specificity of vesicular transport: markers on vesicles – receptors on membrane
• Rab proteins: small GTPases on vesicles – Tethering proteins on membranes
  -> Rab Proteins Help Ensure the Specificity of Vesicle Docking
• Complement: vesicular (v-SNARE) – target membrane (t-SNARE)
  ->SNARE proteins seem to have a central role both in providing specificity and in catalyzing the fusion of vesicles with the target membrane

(SNARE proteins exist ascomplementary sets—vesicle membrane SNAREs, called v-SNAREs, and target membrane SNAREs, called t-SNAREs. v-SNARESs and t-SNAREs have characteristic helical domains. When a v-SNARE interacts with a t-SNARE, the helical domains of one wrap around the helical domains of the other to form stable trans-SNARE complexes, which lock the two membranes together.)

• Fusion: closer contact than docking - two bilayers within 1.5 nm
• Lipid intermix & water expulsion: high energy required, specific signals
• SNARE complex plays a central role + other proteins

• Identification : Rab proteins
• Fusion : SNARE complexes
• NSF (ATPase) & α‐SNAP: dissociation of SNARE complex

COPI-coated vesicles and COPII-coated vesicles transport material early in the secretory pathway: COPII-coated packages bud from the ER, and COPI-coated packages bud from pre-Golgi compartments and Golgi cisternae.

    GOLGI
COPII proteins ↑
COPI proteins ↓
       ER

-> consituitive Sceretion is not regulated

Beispiele für regulierten sekretorischen Weg:
Hepatozyt, exokrine Zellen, endokrine Zelle, Neurone

(The two pathways diverge in the trans Golgi network. The constitutive secretory pathway operates in all cells. Many soluble proteins are continually secreted from the cell by this pathway, which also supplies the plasma membrane with newly synthesized lipids and proteins. Specialized secretory cells also have a regulated secretory pathway, by which selected proteins in the trans Golgi network are diverted into secretory vesicles, where the proteins are concentrated and stored until an extracellular signal stimulates their secretion)

"Uptake of material into a cell by an invagination of the plasma membrane and its internalization in a membrane-bounded vesicle."

Two main types ofendocytosis are distinguished on the basis of the size of the endocytic vesicles formed. One type is called phagocytosis (“cellular eating”), which involves the ingestion of large particles, such as microorganisms or dead cells via large vesicles called phagosomes (generally >250 nm in diameter). The other type is pinocytosis (“cellular drinking”), which involves the ingestion of fluid and solutes via small pinocytic vesicles (about 100 nm in diameter). 

• Endocytosis: Continuous uptake of fluids, large & small molecules
  → Specialized cells: uptake of large particles and cells
• Size of endocytic vesicles:
  * Pinocytosis: small vesicles (< 150 nm)
  * Phagocytosis : large vesicles, phagosomes (>250 nm)

The endocytic part of the cycle often begins at clathrin-coated pits (Region of plasma membrane of animal cells that is coated with the protein clathrin on its cytosolic face. Such regions are continually forming and budding off by endocytosis to form intracellular clathrin-coated vesicles containing extracellular fluid and the materials dissolved in it.)

-> Selective form of pinocytosis that increases the efficiency of internalization of macromolecules

• >1000‐fold more effective than pinocytosis: concentrating mechanism
• Minor components of the ECF can be taken up in large amounts • Clearance of proteins and drugs
• Metabolization of vitamins, iron, hormones, choleterol

Epithelial cells lining the proximal tubule of the kidney

(Most cholesterol is transported in the blood as cholesteryl esters in the form of lipid-protein particles known as low-density lipoproteins (LDL). When a cell needs cholesterol for membrane synthesis, it makes transmembrane receptor proteins for LDL and inserts them into its plasma membrane. Once in the plasma membrane, the LDL receptors diffuse until they associate with clathrin-coated pits that are in the process of forming. Since coated pits constantly pinch off to form coated vesicles, any LDL particles bound to LDL receptors in the coated pits are rapidly internalized in coated vesicles. After shedding their clathrin coats, the vesicles deliver their contents to early endosomes, which are located near the cell periphery. Once the LDL and LDL receptors encounter the low pH in the endosomes, LDL is released from its receptor and is delivered via late endosomes to lysosomes. There the cholesteryl esters in the LDL particles are hydrolyzed to free cholesterol, which is now available to the cell for new membrane synthesis. If too much free cholesterol accumulates in a cell, the cell shuts off both its own cholesterol synthesis and the synthesis of LDL receptor proteins, so that it ceases either to make or to take up cholesterol.)

Mutations LDLR : Familial hypercholesterolemia
= the presence of high levels ofcholesterol in the blood

s.B.

Three pathways from the endosomal compartment in an epithelial cell are shown. Retrieved receptors are returned (1) to the same plasma membranedomain from which they came (recycling) or (2) to a different domain of the plasma membrane (transcytosis). (3) Receptors that are not specifically retrieved from endosomes follow the pathway from the endosomal compartment to lysosomes, where they are degraded (degradation).

Unter Transzytose versteht man einen rezeptorvermittelten Transport von Substanzen durch Zellen hindurch.

• Specialized enzymes of lysosomes : synthesized in ER → Golgi
• In ER an cis-Golgi: enzymes are tagged with mannose-6-phosphate sugar
• Trans-Golgi: M6P recognized by M6P receptor → sorted to lysosomes
• Binding of M6P in trans-Golgi and release in the lumen of lysosome: pH dependent
• M6P receptors are recycled to the trans-Golgi

=(The precursors of lysosomal hydrolases are covalently modified by the addition of mannose 6-phosphate (M6P) groups in the cis Golgi network. They then become segregated from all other types of proteins in the trans Golgi network because adaptins in the clathrin coat bind the M6P receptors, which, in turn, bind the modified lysosomal hydrolases. The clathrin-coated vesicles produced bud off from the trans Golgi network and fuse with late endosomes. At the low pH of the late endosome, the hydrolases dissociate from the M6P receptors, and the empty receptors are recycled to the Golgi apparatus for further rounds of transport.)

Fachsprachlich wird als mitochondriale DNA, kurz mtDNA, die zumeist zirkuläre, doppelsträngige DNA im Inneren (Matrix) der Mitochondrien bezeichnet.
-> in humans, the over 16000 base pairs of mitochondrial DNA encode for only 37 genes: 13 for Proteins, 22 for Transfer RNA

Mitochondrial Encephalopathy, Lactic Acidosis and Stroke-like episodes (MELAS)

Als MELAS-Syndrom bezeichnet man eine mitochondriale Enzephalomyopathie

Production of cellular components

• Interconnected spaces enclosed by a membrane (connected to nuclear envelope) membrane
• Synthesis of cell membrane components and export material
• Ribosomes: protein synthesis

Synthesis, packaging, routing of proteins

• Stacks of membrane-enclosed sacs
• Reception, modification and routing of proteins made in ER
• Surrounded by small vesicles (in – out traffic)

Intracellular digestion, garbage

• Lysosomes break down cellular waste products and macromolecules into simple compounds, which are then transferred back into the cytoplasm as new materials.
• To accomplish the digestion lysosomes utilize 40 different types of hydrolytic enzymes, which are manufactured in endoplasmic reticulum and modified in the Golgi.

Degradation of hydrogen peroxide

• Peroxisomes contain enzymes that convert (cytotoxic) hydrogen peroxide to water (oxidative
lysosomes reactions).
• Were first discovered by the Belgian scientist Christian de Duve, who also discovered y

The fate of proteins synthesized in cytosol depends on the AA sequence → sorting signals to direct proteins into nucleus/organelles/ER

KDEL is a target peptide sequence in the amino acid structure of a protein which prevents the protein from being secreted from the endoplasmic reticulum (ER). A protein with a functional KDEL motif will be retrieved from the Golgi apparatus by retrograde transport to the ER lumen.

• K—Lysine
• D—Aspartic acid
• E—Glutamic acid
• L—Leucine
  Lys-Asp-Glu-Leu.

#1 First cross-road: ± ER-specific Signal Sequence

#2 No adress for organelle: → Cytosolic proteins

#3 Mitochondria Signal Sequence

#5 Signal Sequence for Peroxisomes

#6 Signal Sequence for Nucleus

s.B.

"Fatty acid acylation is the covalent attachment of fatty acids to proteins."

• Protein acylation is a post-translational modification of proteins.
• Prominent type: fatty acylation, the addition of fatty acids to particular amino acids (e.g. myristoylation or palmitoylation)
• Protein acylation is a mechanism of biological signaling
mechanism signaling

-> Die Kopplung von Fettsäuren wie Myristinsäure oder Palmitinsäure führt zur Bindung der acylierten Proteine an die Zellmembran. Dieser Prozess kann für die Funktion des gesamten Proteins von großer Bedeutung sein.

Prenylation (also known as isoprenylation or lipidation) is the addition of hydrophobic molecules to a protein or chemical compound. It is usually assumed that prenyl groups (3-methyl-but-2-en-1-yl) facilitate attachment to cell membranes, similar to lipid anchors like the GPI anchor, though direct evidence is missing.

• Addition of hydrophobic molecules to a protein
• Facilitate attachment to cell membranes

-> Proteins are Imported in Mitochondria in an Unfolded State

Chaperone (engl. Anstandsdamen) sind Proteine, die neu synthetisierten Proteinen „helfen“, sich korrekt zu falten. 

s.B:

Mutations in PEXgenes coding for peroxin (PEX) proteins: Zellweger Syndrome

Cerebro-hepato-renal syndrome: accumulation of fatty acids that
are normally degraded in peroxisomes → multi-organoga dysfunction

Traffic in both directions through the pores:
Newly made proteins for the nucleus enter from the cytosol 
RNA molecules and ribosomal subunits are exported

Proteins bound for the nucleus are actively transported through nuclear pores

-> GTP hydrolysis provides energy driving nuclear transport

The secretory pathway is a series of steps a cell uses to move proteins out of the cell, a process known as secretion. The path of a protein destined for secretion has its origins in the rough endoplasmic reticulum, a membrane-bound compartment in the cell. The protein then proceeds through the many compartments of the Golgi apparatus and finally ends up in a vesicle that transiently fuses at the cell plasma membrane via permanent plasma membrane structures called porosomes, depositing the proteins on the outside of the cell.

Directed by ER signal sequence (hydrophobic AAs): Translocation
(1) water-soluble proteins: complete translocation – ER lumen
(2) Transmembrane proteins: partial translocation

→ ER sorting signal: Extended bloc of hydrophobic residues

Attached ribosomes (ER, nucleus): make proteins that are translocated into ER

Free ribosomes: proteins encoded by nuclear DNA in cytosol
 

A common pool of ribosomes is used to synthesize the proteins that stay in the cytosol and those that are transported into the ER. The ER signal sequence on a newly formed polypeptide chain directs the engaged ribosome to the ER membrane. The mRNA molecule remains permanently bound to the ER as part of a polyribosome, while the ribosomes that move along it are recycled; at the end of each round of protein synthesis, the ribosomalsubunits are released and rejoin the common pool in the cytosol.