Autophagy - Recycling as a Survival Factor - FocusOn128

Introduction

Autophagy is a highly conserved cellular degradation process. In this degradation pathway in mammalian cells constituents of cytosol and organelles are sequestered into a double-membrane vesicle and delivered into the lysosome. This results in breakdown and eventual recycling of the resulting macromolecules.

In eukaryotic cells autophagy is divided into three major mechanistically different pathways: Macroautophagy is a catabolic process involving dynamic membrane rearrangement. Cytoplasmic components are sequestered into atuophagosomes. These are double-membrane vesicles which fuse with lysosomes in mammalian cells resulting in degradation. Microautophagy is a mechanism for the uptake of cytosolic components into the lysosome by invagination, protrusion and septation of the lysosome membrane. These components will be degraded by lysosomal enzymes. Chaperone-mediated autophagy (CMA) allows selective degradation of soluble proteins in lysosomes. In contrast to the other mammalian forms of autophagy, vesicle formation or major changes in the lysosomal membrane are not required. The target proteins are recognized at their KFERQ-like motif by a chaperone complex. They directly cross the lysosomal membrane to reach the lumen, where they are rapidly degraded.

Beclin-1

Beclin-1 is found to play an important role in autophagy initiation and autophagosome clearance. It is a coiled-coil Bcl-2-interacting protein which is homologous to the yeast autophagy gene APG6. This mammalian autophagy gene can inhibit tumorigenesis and is expressed at reduced levels in human breast carcinoma. This suggests that defects in autophagy proteins may contribute to the development or progression of tumors. Beclin-1 initiates autophagy by the formation of a complex with phosphoinositide-3-kinase, class 3 (PIK3C3). A protein called UVRAG (UV radiation resistance associated gene) activates the beclin-1 PIK3C3 complex. In addition, beclin-1 levels have been shown to play an imortant role on Alzheimer disease pathology shown with transgenic mice.

Fig.1: Immunocytochemistry staining of A431 cells using beclin-1 antibody Cat.-No. AP20057PU-N at 1 µg/ml
Fig.1: Immunocytochemistry staining of A431 cells using beclin-1 antibody Cat.-No. AP20057PU-N at 1 µg/ml

PIST

PIST or GOPC (Golgi-associated PDZ and coiled-coil motif-containing protein) is described as a protein binding partner to beclin-1, which can synergize to induce autophagy.

Fig. 2: Immunohistochemistry of PIST in rat colon tissue with PIST antibody Cat.-No. AP20060PU-N at 1 µg/ml
Fig. 2: Immunohistochemistry of PIST in rat colon tissue with PIST antibody Cat.-No. AP20060PU-N at 1 µg/ml
Fig. 3: Western blot analysis of PIST in PC-3 cell lysate with PIST antibody Cat.-No. AP20061PU-N at (A) 1, (B) 2 and (C) 4 µg/ml
Fig. 3: Western blot analysis of PIST in PC-3 cell lysate with PIST antibody Cat.-No. AP20061PU-N at (A) 1, (B) 2 and (C) 4 µg/ml
Fig. 4: Western blot analysis of extract from human HeLa cells using LC3A antibody Cat.-No. AP08079PU-N
Fig. 4: Western blot analysis of extract from human HeLa cells using LC3A antibody Cat.-No. AP08079PU-N
Fig. 5: Formalin-fixed, paraffin-embedded kidney tissue stained with ATG7 antibody Cat.-No. AP08525PU-N
Fig. 5: Formalin-fixed, paraffin-embedded kidney tissue stained with ATG7 antibody Cat.-No. AP08525PU-N
* = activated;  PE = Phosphatidylethanolamine
* = activated; PE = Phosphatidylethanolamine

LC3

Microtubule-associated protein 1 light chain 3 (LC3), a homolog of yeast APG8, is most abundant in autophagosomal membranes and well established as a marker for autophagosome and autophagic activity. The polyubiquitin-binding protein p62/SQSTM1 is an alternative marker to LC3 for autophagy which is described to bind directly to ATG8/LC3.

ATG/APG

Autophagy-related genes: The identification of the ATG genes in yeast and the finding of orthologs in other organisms, reveals the conservation of the machinery in all eukaryotes.

APG7 is essential for autophagy conjugation systems, autophagosome formation, starvation-induced bulk degradation of proteins and organelles. It has been suggested that it is essential for multisubstrates such as ATG8-like proteins and ATG12. ATG7, a member of the autophagy family of proteins, is the human gene product similar in homology to yeast APG7. In yeast the protein shows homology to the ATP-binding and catalytic sites of the E1 ubiquitin-activating enzymes.

ATG12 forms a conjugate with ATG5. This conjugate has a ubiquitin-protein ligase (E3)-like activity for protein lipidation in autophagy. ATG12 has also been shown to interact with ATG10 in human embryonic kidney cells in the presence of ATG7. At least two isoforms of ATG12 are known to exist.

LAMP1/2

Lysosomal-associated membrane proteins 1 and 2 (Lamp1/2) are two of the most abundant lysosomal components which have found to be involved in autophagy.

References

Jaeger AJ et al: Regulation of amyloid precursor protein processing by the Beclin 1 complex.
PLoS, One Jun 2010, 15; 5(6): e11102. [PMID 20559548 ]
Kametaka S et al: Apg14p and Apg6/Vps30p form a protein complex essential for autophagy in the yeast, Saccharomyces cerevisiae.
J Biol Chem, Aug 1998, 28; 273(35): 22284-91. [PMID 9712845 ]
Liang XH et al: Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein.
J Virol, Nov 1998; 72(11): 8586-96. [PMID 9765397 ]
Liang, XH et al: Induction of autophagy and inhibition of tumorigenesis by beclin 1.
Nature, Dec 1999; 402(6762): 672-6. [PMID 10604474 ]
Massey, AC et al: Chaperone-mediated autophagy in aging and disease.
Curr Top Dev Biol., 2006; 73: 205-35. [PMID 16782460 ]
Pankiv, S et al: p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy.
J Biol Chem, Aug 2007, 17; 282(33): 24131-45. [PMID 17580304 ]
Pickford, F et al: The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice.
J Clin Invest, Jun 2008; 118(6): 2190-9. [PMID 18497889 ]
Yue Z et al: A novel protein complex linking the delta 2 glutamate receptor and autophagy: implications for neurodegeneration in lurcher mice.
Neuron. Aug 2002, 29; 35(5): 921-33. [PMID 12372286 ]
Yang, Z et al: An overview of the molecular mechanism of autophagy.
Curr Top Microbiol Immunol, 2009; 335: 1-32. [PMID 19802558 ]

Further readings

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Primary Antibodies

Catalog No. Host Iso. Clone Pres. React. Applications  

APG5L / ATG5 (N-term) antibody

Kidney: Formalin-Fixed Paraffin-Embedded (FFPE) Rabbit IgG Aff - Purified Bov, Hu, Ms, Por, Pufferfish, Rt, Ze E, P, WB
50 µg / €470.00
  Acris Antibodies GmbH

APG7L / ATG7 (N-term) antibody

Kidney: Formalin-Fixed Paraffin-Embedded (FFPE) Rabbit IgG Aff - Purified Hu, Ms ICC/IF, P, WB
50 µg / €470.00
  Acris Antibodies GmbH

APG7L / ATG7 Control Peptide

APG7L / ATG7
50 µg / €160.00
  Acris Antibodies GmbH

APG7L / ATG7 Control Peptide

APG7L / ATG7
50 µg / €160.00
  Acris Antibodies GmbH

APG7L / ATG7 antibody

APG7L / ATG7 Rabbit Purified Hu E, WB
0.1 mg / €440.00
  Acris Antibodies GmbH

APG7L / ATG7 Control Peptide

APG7L / ATG7 Purified
50 µg / €190.00
  Acris Antibodies GmbH

APG8 / ATG8 antibody

Figure 1. Immunoblot of APG8 fusion protein. Anti-APG8 antibody generated by immunization with recombinant yeast APG8 was tested by immunoblot with other anti-UBL antibodies against E.coli lysates expressing the APG8-GFP fusion protein. All UBLs possess limited homology to Ubiquitin and to each other, therefore it is important to know the degree of reactivity of each antibody against each UBL. Panel A shows total protein staining using ponceau. Panel B shows specific reaction with APG8 using a 1:4,000 and 1:8,000 dilution of IgG fraction of Rabbit-anti-APG8 (Yeast) followed by reaction with a 1:15,000 dilution of HRP Goat-a-Rabbit IgG MX. All primary antibodies were diluted in TTBS buffer supplemented with 5% non-fat milk and incubated with the membranes overnight at 4° C. E.coli lysate proteins were separated by SDS-PAGE using a 15% gel. Similar experiments (data not shown), where other UBL fusion proteins were separated and probed with this antibody showed no reactivity of anti-APG8 with other UBLs. This data indicates that anti-APG8 is highly specific and does not cross react with other UBLs. A chemiluminescence system was used for signal detection (Roche). Other detection systems will yield similar results. Data contributed by M. Malakhov, www.lifesensors.com, personal communication. Rabbit Purified Ye E, WB
0.5 mg / €440.00
  Acris Antibodies GmbH

APG16L / ATG16L1 Control Peptide

APG16L / ATG16L1
50 µg / €160.00
  Acris Antibodies GmbH

ATG12 / APG12 antibody

Figure 1. Immunoblot of APG12 fusion protein. Anti-APG12 antibody generated by immunization with recombinant yeast APG12
was tested by immunoblot against yeast lysates expressing the APG12-GFP fusion protein and other UBL fusion proteins. All UBLs
possess limited homology to Ubiquitin and to each other, therefore it is important to know the degree of reactivity of each antibody
against each UBL. Panel A shows total protein staining using ponceau. Panel B shows positions of free GFP or GFP containing
recombinant proteins present in each lysate preparation after reaction with a 1:1,000 dilution of anti-GFP followed by reaction with a 1:15,000 dilution of HRP Donkey-a-Goat IgG MX. Panel C shows specific
reaction with APG12 using a 1:2,000 dilution of IgG fraction of Rabbit-anti-APG12 (Yeast) followed by reaction with a
1:15,000 dilution of HRP Goat-a-Rabbit IgG MX. All primary antibodies were diluted in TTBS buffer supplemented
with 5% non-fat milk and incubated with the membranes overnight at 4° C. Yeast lysate proteins were separated by SDS-PAGE using a
15% gel. This data indicates that anti-APG12 is highly specific and does not cross react with other UBLs. A chemiluminescence
system was used for signal detection (Roche). Other detection systems will yield similar results. Data contributed by M. Malakhov,
Lifesensors Inc., personal communication. Rabbit Purified S. cerevisiae, Ye E, WB
0.5 mg / €440.00
  Acris Antibodies GmbH

Beclin-1 Control Peptide

Beclin-1
50 µg / €160.00
  Acris Antibodies GmbH

Beclin-1 Control Peptide

Beclin-1
50 µg / €160.00
  Acris Antibodies GmbH

CD107a / LAMP1 antibody

CD107a / LAMP1 Rat IgG2a 1D4B APC Ms F
0.1 mg / €290.00
  Acris Antibodies GmbH

CD107a / LAMP1 antibody

CD107a / LAMP1 Rat IgG2a 1D4B FITC Ms F
0.5 mg / €380.00
  Acris Antibodies GmbH

CD107a / LAMP1 antibody

CD107a / LAMP1 Rat IgG2a 1D4B Purified Ms C, EM, F, IP, WB
0.5 mg / €330.00
  Acris Antibodies GmbH

CD107a / LAMP1 Control Peptide

CD107a / LAMP1
50 µg / €160.00
  Acris Antibodies GmbH

CD107b / LAMP2 antibody

AP07767PU-N LAMP2 antibody staining of Formalin-Fixed Paraffin-Embedded Human Placenta at 5 µg/ml followed by biotinylated goat anti-rabbit IgG secondary antibody, alkaline phosphatase-streptavidin and chromogen. Rabbit Aff - Purified Hu, Ms ICC/IF, P, WB
50 µg / €470.00
  Acris Antibodies GmbH

CD107b / LAMP2 Control Peptide

CD107b / LAMP2
50 µg / €160.00
  Acris Antibodies GmbH

CD107b / LAMP2 antibody

Staining of permeabilised mouse peritoneal macrophages with Rat Anti Mouse CD107b -FITC conjugated (SM2211F/FT). Rat IgG1 M3/84 FITC Ms F
0.1 mg / €280.00
  Acris Antibodies GmbH

CD107b / LAMP2 antibody

Staining of mouse peritoneal macrophages with RAT ANTI MOUSE CD107b (SM2211P)following
permeabilsation with Leucoperm Rat IgG1 M3/84 Purified Ms C, F, IP, P, WB
0.25 mg / €390.00
  Acris Antibodies GmbH

GOPC / PIST antibody

Brain, cerebellum: Formalin-Fixed Paraffin-Embedded (FFPE) Rabbit Aff - Purified Hu, Ms, Rt P, WB
50 µg / €470.00
  Acris Antibodies GmbH

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