A PCR analysis of the ubiquitin-like conjugation systems in macroautophagy

Abstract

A central part of the core macroauto-phagy (hereafter autophagy) machinery includes the two ubiquitin-like (Ubl) conjugation systems that involve the Ubl proteins Atg8 and Atg12.¹ Although the functions of these proteins have not been fully elucidated, they play critical roles in autophagosome formation. For example, Atg8 is involved in cargo recognition,^2,3 and the amount of Atg8 in part determines the size of the autophagosome,⁴ whereas Atg12 is part of a trimer that may function as an E3 ligase to facilitate Atg8 conjugation to phosphatidylethanolamine and determine, in part, the site of the conjugation reaction.⁵ Thus, fully functional autophagy requires both the Atg8 and Atg12 conjugation systems. Dysfunctional autophagy is associated with various human pathophysiologies including cancer, neurodegeneration, gastrointestinal disorders and heart disease. So, if you are wondering whether autophagy is operating properly in your own body, what can you do? The problem is that there are relatively few methods for analyzing autophagy in vivo.^6-11 Minimally, you might want to find out if the relevant genes are intact and have the correct sequence. Considering the rapid advances being made in DNA sequencing technology, it is likely only a matter of time before people can submit a DNA sample and obtain a rapid readout of particular genes, or their entire genome. Thus, anticipating the future, we decided to analyze a select set of autophagy-related (ATG) genes, with a focus on those encoding components of the Ubl conjugation systems, by a polymerase chain reaction (PCR)-based method that combines science with art.

Key words: :

• autophagy
• collaboration
• gel electrophoresis
• membrane
• primer

Introduction

The Atg8 and Atg12 conjugation systems have been relatively well characterized with regard to the chain of events that culminate in the final products (Fig. 1).^12-17 In brief, yeast Atg8 is synthesized with a C-terminal arginine that is removed by the Atg4 protease to expose the penultimate glycine residue. Atg8 is then activated by Atg7, an E1-like homolog, and subsequently conjugated to phosphatidylethanolamine through the action of Atg3, an E2-like enzyme. Atg12 is also activated by Atg7, and then conjugated to an internal lysine of Atg5 in a reaction dependent on Atg10, another E2-like component. Atg5 binds noncovalently to Atg16, which dimerizes to generate a trinary dimer referred to as Atg12–Atg5-Atg16 (the en dash between “Atg12” and “Atg5” denotes a covalent bond). A similar series of events describes the conjugation systems in higher eukaryotes.

Figure 1. Schematic outline of the Atg8 and Atg12 conjugation systems.

Atg8–PE initially lines both sides of the phagophore. Much of the Atg8 is released from PE by a second Atg4-dependent cleavage reaction, although some of the Atg8–PE on the concave side of the phagophore membrane remains trapped within the completed autophagosome; in the case of selective types of autophagy, the cargo may block access to Atg8–PE that interacts with various cargo receptors or tags. It is not known what regulates the cleavage of Atg8–PE, or whether this is temporally controlled to prevent premature deconjugation. Using a yeast multiple-knockout strain that lacks the known genes required for autophagosome formation,¹⁸ it was shown that Atg8–PE can be generated at multiple sites, not just the phagophore assembly site (PAS) as is seen in wild-type cells.¹⁹ When Atg4 and additional components are added back, Atg8–PE (detected as a GFP chimera) is detected at the PAS. Thus, there appears to be a posttranslational aspect to Atg8 regulation whereby the access of Atg4 to Atg8–PE is controlled at the PAS, whereas any conjugation that occurs at other locations is corrected by immediate Atg4-dependent deconjugation.

At present, there are 35 Atg proteins identified in fungi. It is striking that 8 of them, almost one-quarter of the total, are involved in the conjugation of Atg8 to PE and Atg12 to Atg5. Although we do not know the precise functions of all of the proteins involved in the conjugation systems, we clearly know much about the relevant protein-protein interactions. In addition, almost all of the structural information that is available for Atg proteins has focused on the Atg8 and Atg12 systems.^20-27 Accordingly, in planning our genomic analysis of autophagy we concentrated on the genes that correspond to the eight proteins involved in the generation of Atg8–PE and Atg12–Atg5-Atg16.

Results

The standard procedure at Yonder Biology is to obtain cheek cells from the interested individual(s), isolate the DNA, and amplify short tandem repeats that produce a visually pleasing pattern determined empirically. For the analysis of the autophagy conjugation systems, however, it was necessary to design specific oligonucleotide primers to each of the relevant genes (Table 1). Primer pairs were designed based on sequences in the NCBI UniSTS database (Table 2). Autophagy primer sets were then ordered from Eurofins MWG Operon.

Table 1. Autophagy-related genes used in this analysis

Lane	Gene	Marker accession #	Size bp	Home sapiens
1	ATG3	G20743	147	chromosome 3
2	ATG3	RH120928	303	chromosome 3
3	ATG4B	RH47370	133	chromosome 2
4	ATG4B	D2S2042	125	chromosome 2
5	ATG5	RH93335	85	chromosome 6
6	ATG5	RH78069	149	chromosome 6
7	ATG7	D3S3454	160	chromosome 3
8	MAP1LC3B	D16S2663	>231	chromosome 16; 9
9	MAP1LC3B	D16F298S2	154	chromosome 16
10	ATG10	D5S806	197	chromosome 5
11	ATG12	RH79593	236	chromosome 5
12	ATG12	WI-14939	125	chromosome 5
13	ATG16L1	WI-12735	150	chromosome 2
14	ATG16L1	G16449	98	chromosome 2
Table 2. Autophagy primer sequences
Gene primer set
ATG 3GTC CCT TAT TAG AGA AAC TGT AT
AGA ATA TGA CTA CAC AAG ACA CT
ATG 3GCC AGT TGC ACT TCC AAT AAA AC
CCC AGA TGT TAA ACC TGT CCA TC
ATG 4BAG GTG CTA ACA CGG GGC T
CGG GAG GCC TTA CTG CTT
ATG 4BCT CTG ATC TCA TCT CAG GTC TGG
TCC ACA GGT CTT GCA TTC TG
ATG 5GGC AGC AAG AAA TTC TAT GAC A
ATT TTG ACT GGT CGT CAT TGG
TG 5GGC ATT GTA GGC TTG ACT TAC C
GCC TTT GCA GTA GGC TTG AG
TG 7GTG ACT GTG CTT TCA GGT
GGA TGT GAC TGC TTG TTC
MAP 1LC3BGG ATA TAG GTC ACC CCT CAG
GTT AAA GGA GTT CCT GTC ACC
MAP 1LC3BGT CAG GAC CTT CAG CAG TTT ACA G
CTC AGC TAG AGA GAT CTC CCT AAG
ATG 10CAC CTG AAC TGC TGA ATG TG
TCC AAT GGT TAA TTG CAT CC
ATG 12TTC CAA CTT CTT GGT CTG GG
TGG AGG AAA AAT GAG ATC TTC G
ATG 12TTT TTT TGA GAT GGG GTC TCA
AAA AAA ATT ATT AGG CTG CAG AGG
ATG 16L1GTC AGG CCT TAT CAC ACT CTC C
CAG AAC CTC TCT CCC TGT CC
CAG AAC CTC TCT CCC TGT CC
AAA GGA ACA GTG ACT GTA GCA GC

Cheek cells were collected from the inside of the cheeks of two subjects using a sterile cotton swab (Fig. 2). Two sets of cells were collected and referred to as DK1 and DK2. DNA was isolated from cheek cells using a modified Qiagen DNeasy protocol. Autophagy DNA markers were amplified from the DNA corresponding to DK1 and DK2 using the PCR method with the autophagy-specific primer sets. The primer sets were tested using a standard PCR protocol as described in Materials and Methods (Fig. 3). Depending on the banding pattern, two primer sets were used for certain genes (Table 2).

Figure 2. Procedure for collecting cheek cells.

Figure 3. An initial test gel was run to qualify the primer sets and visualize the banding size/pattern.

Amplified DNA markers were run on a 4% agarose gel to separate the bands and visualize the DNA. The gel was stained with ethidium bromide, and illuminated under UV light. Several images were taken with a high-quality camera, and the images were transferred to a computer. The image was cropped using Photoshop, colorized and sized for printing (Fig. 4).

Figure 4. The final gel-loading scenario was determined and a new gel was run to visualize the autophagy markers in a manner that was consistent with the known metabolic pathway cascade as depicted in Figure 1. All DNA bands are from DK1 unless otherwise specified. Lanes correspond to the following: (1) 100 base DNA ladder; (2) Atg8 (MAP1LC3) with Atg4 (RH47370, D2S2042); (3) Atg8 (MAP1LC3B/D16S2663) with Atg7 (D3S3454); (4) Atg8 (MAP1LC3B/D16S2663 and D16F298S2) with Atg3 (RH120928); (5) Atg12 (RH79593) with Atg7 (D3S3454); (6) Atg12 (RH70503; DK2 DNA) with Atg5 (RH93335) and Atg10 (D5S806; DK2 DNA); (7) Atg12 (RH79593) with Atg5 (RH93335) and ATG16L1 (ATG16L1, WI-12735 added digitally); (8) 100 base DNA ladder. The sequence for yeast ATG8 was added to the background; this sequence was quadrupled on the final image for aesthetic reasons. The final gels are shown in the (A) zebra pattern, (B) an experimental customized pattern and (C) igloo pattern (without the background sequence).

Discussion

All primer sets worked on the first attempt at amplification on both individuals, suggesting that the assay was robust and the loci highly conserved. Four markers required optimization due to multiple banding: ATG4B (RH47370), ATG12 (W1-14939), ATG12 (W1-12735) and ATG16L1 (G16449). Increasing PCR stringency by increasing the annealing temperature failed to fully resolve the multiple banding issue.

Some of the autophagy DNA marker primer sets worked on yeast and dog DNA, suggesting that these human autophagy genes are highly conserved. This work was not confirmed, however, and was only attempted for use as a control for another project that Yonder Biology was working on in the lab.

Determining the autophagic response in vivo following treatments aimed at modulating autophagy is a major goal of researchers and clinicians who are attempting to evaluate the effect of anticancer drugs, or regimens designed to ameliorate neurodegenerative or other diseases. Although the method of analysis described here does not provide a practical method for analyzing macroautophagy in vivo, it highlights the need for additional research on this topic.

Materials and Methods

Table 2. Autophagy primer sequences

Gene primer set
ATG 3GTC CCT TAT TAG AGA AAC TGT AT
AGA ATA TGA CTA CAC AAG ACA CT
ATG 3GCC AGT TGC ACT TCC AAT AAA AC
CCC AGA TGT TAA ACC TGT CCA TC
ATG 4BAG GTG CTA ACA CGG GGC T
CGG GAG GCC TTA CTG CTT
ATG 4BCT CTG ATC TCA TCT CAG GTC TGG
TCC ACA GGT CTT GCA TTC TG
ATG 5GGC AGC AAG AAA TTC TAT GAC A
ATT TTG ACT GGT CGT CAT TGG
TG 5GGC ATT GTA GGC TTG ACT TAC C
GCC TTT GCA GTA GGC TTG AG
TG 7GTG ACT GTG CTT TCA GGT
GGA TGT GAC TGC TTG TTC
MAP 1LC3BGG ATA TAG GTC ACC CCT CAG
GTT AAA GGA GTT CCT GTC ACC
MAP 1LC3BGT CAG GAC CTT CAG CAG TTT ACA G
CTC AGC TAG AGA GAT CTC CCT AAG
ATG 10CAC CTG AAC TGC TGA ATG TG
TCC AAT GGT TAA TTG CAT CC
ATG 12TTC CAA CTT CTT GGT CTG GG
TGG AGG AAA AAT GAG ATC TTC G
ATG 12TTT TTT TGA GAT GGG GTC TCA
AAA AAA ATT ATT AGG CTG CAG AGG
ATG 16L1GTC AGG CCT TAT CAC ACT CTC C
CAG AAC CTC TCT CCC TGT CC
CAG AAC CTC TCT CCC TGT CC
AAA GGA ACA GTG ACT GTA GCA GC

Collecting cells

Cheek cells were collected from the inside of the cheek using a sterile cotton swab (DNA Genotek, ORAcollect OC-100).

Isolating the DNA

(1) Opening the cells: The cotton end of the collection swab was dispensed into a microcentrifuge tube and reagents to lyse the cells were added. Lysis was performed with the QIAamp DNA mini Kit (Qiagen, 51304). (2) Capturing the DNA: The DNA-cell lysis solution was then passed through a column using vacuum pressure; the DNA binds to the silica membrane in the column and the rest of the cell matter passes though the column. (3) Washing and eluting the DNA: To wash contaminants from the DNA, ethanol in varying concentrations (57% and 70%) was passed through the column using vacuum pressure. Water or Tris-EDTA (10 mM Tris, pH 8.0, 1 mM EDTA) was used to elute the DNA from the column.

Yeast DNA was isolated from California Ale Yeast (White Labs; WLP001).

Amplifying specific DNA regions by PCR

The DNA was transferred into a microtiter plate along with DNA Taq Polymerase, 1 μl 40 mM DNTPs (Monserate Biotech, 2001 and 8001) and 1 mM MgCl₂ in 20 μl reactions. The PCR was performed using an ABI GeneAmp PCR System 9700. One cycle of PCR consisted of raising the temperature to 90°C, 30 min to denature the DNA, followed by an annealing temperature of 54–64°C, 30 min and a 72°C extension reaction, 45 min. This cycle was repeated 25 to 30 times.

Separating the amplified segments of DNA by gel electrophoresis.

Nine microliters of the amplified DNA segments were removed from the PCR microtiter plate. A gel loading dye (GenScript, M00120) was added to each amplified DNA solution, and this solution was loaded into the wells of a 4% UltraPure agarose gel (Invitrogen, 16500500). The gel was run at 120 V for 90 min.

Imaging the DNA pattern.

The gel was submerged in an ethidium bromide solution for 20 min, rinsed (destained) and imaged on a Bio-Rad Gel Doc^TM XR⁺ System and the image was transferred to a computer.

Style and colorize DNA image.

Computer-aided design was used to crop, adjust, colorize and size the DNA portrait.

Acknowledgements

The authors thank Lisa Klionsky for “volunteering” to provide cheek cells.

Disclosure of Potential Conflicts of Interest

Andy Bass and Dean Sauer are the co-founders of Yonder Biology.