Biología molecular

1. Developmental Consequences of
Defective ATG7-Mediated
Autophagy in Humans
By: Juan Diego Gómez & Sara María Gómez
Medicine- UPB
3rd semester
Molecular Biology

2. Introduction
AUTOPHAGY
Protection from cytotoxicity, pathogens and protein
aggregates. Helps metabolism recycling
Process:
ULK1 complex
receives signal
Formation of transient
double membrane bound
structure called phagophore.
Expansion: becomes an
autophagosome
LC3-II recruits the
cytoplasmic cargo so the
autophagosome can
take it
Autophagosome fuses with
endolysosomal system: Hydrolitic
enzymes degrade the cargo

3. ATG-7
It encodes as an enzyme which activates
ATG12.
Preautophagosomal phagophore is promoted by
this gen.
It facilitates lipidation of the protein
LC3-I.

4. General objective
Highlight the importance of the AGT7 gene in the autophagy
process by the study of five unrelated families with recessive ATG7
variants
cerebellar hipoplasia
Thin posterior corpus callosum
Ataxia
Musculoskeletal abnormalities
Development delay
Facial dysmorphism

5. Inmunohistochemistry
Identification of a
tissue.
It is made by a specific interaction (antigen-
antibody).
Antibody is previously
labeled.

6. Sequencing
methodology
With two pioneering methods, it
has some aspects in common and
it is classified into chemical and
enzymatic techniques.
CHEMICAL Chemical hydrolysis.
hydrolysis.
Original design aplicable
aplicable to DNA
DNA seqcuences (<250
It has three stages.
ENZYMATIC DNA is not degrated.
Interruption of the
synthesis of a
complementary strand.
It is described in three
sections.

7. Immunoblot
Is a laboratory technique used to identify a
specific protein in tissue. It is useful for the
diagnosis of multiple pathologies, it is
affordable and effective

8. INMUNOFLUORESCENCE
Is a type of immunohistochemistry technique that
utilizes fluorophores to visualize various cellular
antigens such as proteins. It can be used to
visualize the localization of various cellular
components within cells and tissues

9. results
Five families with ataxia, developmental
delay and ATG7 variants.
Circles: female
members
Squares: male
members
Shaded
symbols:
affected
members
Diamonds:
unknown
members

10. results
Biopsy indicated that ATG7 was
undetectable.
ATG7 levels were severely decreased in the
patient´s myoblasts.
P62 high levels were found in the patient´s
myoblasts.

11.  AGT7 protein was undetectable
in patient 1 and was decreased in
through6and10
 P62levelswereincreased
 B-actine was used as a loading
results
Analyze
d
proteins
Patientsand
control
+With
-
Without

12. results
control
Analyzed
proteins
Wild type
and
variants of
AGT7
Analyzed proteins
control
Densitometric analysis of the ratio of
LC3 to GAPDH, normalized to
wild-type values.
Introduction of
plasmids encoding
wild-type ATG7 into
immortalized fibroblasts
from Patient 1 for 24
hours

13. Our comprehensive clinical investigation of these
patients consolidates the critical importance of basal
autophagy in human neural and musculoskeletal
integrity. Many of the clinical features observed in these
patients are recapitulated in the conditional Atg7-
knockout mouse models, including brain abnormalities
Komatsu M, Wang Q
Komatsu M, Waguri S
It has also been shown that canonical autophagy can
still proceed in the absence of ATG proteins, albeit at a
reduced rate as a result of impaired inner
autophagosomal membrane degradation
Tsuboyama K, Koyama-Honda I, Sakamaki Y, Koike M,
Morishita H, Mizushima N.
These findings strengthen our understanding of
autophagy in human disease and expand the spectrum of
clinical phenotypes and genetic loci associated with
congenital autophagy-deficient syndromes. Given that
the perinatal lethality of Atg5-null mice can be avoided
through selective restoration of autophagy in the nervous
system.
Yoshii SR, Kuma A, Akashi T
DISCUSSION

14. Conclusions
Important cells processes and
mechanisms that are not available to
our eyes or through a microscope,
such as autophagy, can be studied by
molecular techniques The molecular techniques are fundamental for
the analysis and study of genetic diseases and
consequently it allows the improvement of the
diagnosis with methods such as immunoblotting
and exome sequencing
Molecular techniques also give us a light of options to
treat genetic diseases so damaging as the one of the
present study, for example by methods such as genetic
therapy

15. Done by: Sara Gómez

16. Done by: Juan Diego Gómez