Study on the role of DYNLT1 in controlling human male fertility through in silico analysis and in vitro validation.

1. Meghna Salil Das (Reg. no.-3622029)
Internal Guide: Dr. Bala Shankar. K, Sathyabama
Institute of Science and Technology, Chennai.
External Guide: Dr. G. Pradeep Kumar, RGCB-TVM

2. ABSTRACT
DYNLT1 is a member of a gene family identified within the t-complex of the mouse, which has been linked
with male germ cell development and function in the mouse and the fly. This gene is associated with male
sterility in drosophila and mouse. Later it was found that the sterile human male’s samples also showed
absence or dysfunction of DYNLT1. But the exact pathway through which the gene effects the male sterility is
yet to be studied. This study aimed at predicting the interacting protein partners of DYNLT1 to find
association with any protein that directly control the male infertility. The in vitro validation of DYNLT1
showed association with TDP43 and HSP90 proteins whose roles in controlling male sterility have already
been studied.

3. DYNLT1 :
Dynein light chain protein (12.45kDa) is encoded by the DYNLT1 gene, in mouse it is
a t- complex gene – chromosome 17. In humans this gene is present in chromosome 6.
This protein has both dynein dependent and dynein independent functions.
DYNLT1 is a protein that plays important role in the male germ cell development in
mouse and drosophila. The dysfunction of this protein is directly associated to
infertility in these species.
Why is it studied?
• The aberrant expression of this protein is reported in human spermatozoa and
associated with male factor infertility.
• Even though the dysfunction or absence of DYNLT1 is linked with infertility in
human males, the actual pathway or its role in infertility is yet to be studied.
INTRODUCTION

4. …CONTINUED
Graph showing the expression levels of DYNLT1
transcript in different body tissues

5. OBJECTIVE
I. To predict the interacting partners of DYNLT1
II. To find the biological networks in which DYNLT1 is involved
III. To validate the predicted interacting proteins by IP, LS-MS/MS or MALDI
ToF and Western Blot Analysis

6. IN SILICO ANALYSIS
I. TOOLS AND DATABASES:
i. UNIPROT
ii. NCBI
iii. EMBOSS-NEEDLE & WATER
iv. STRING
v. BioGRID
vi. MINT
vii. DIP
viii. MENTHA.UNIROMA2
ix. HuRI
x. GeneCards
xi. KEGG
xii. REACTOME
xiii. EXPRESSION ATLAS
xiv. THE PROTEIN ATLAS
Reference Databases
Similarity search
tools
PPI databases &
prediction tools
Pathway
databases
Expression analysis
database

7. II. PROCEDURE:
Functional information of the
DYNLT1 gene and protein of mice
and humans was collected from
databases.
Pair-wise alignment was performed
with the protein sequence of human
and mouse to find the percentage
similarity.
The interacting proteins of DYNLT1
in both humans and mice were found
and compared. The common
interacting proteins were noted.
Based on the interacting proteins,
biological networks and pathways in
which DYNLT1 is involved were
predicted.

8. I. METHODS USED:
i. IP- Immunoprecipitation
ii. LC-MS/MS
iii. Western Blot Analysis
IN VITRO ANALYSIS
II. PROCEDURE:
Testes of a 3
month old
mouse was
taken as the
sample
The tissues
from the
sample were
extracted and
mixed with
NP-40 lysis
buffer
Then tissue
were
homoge-
nized and
centrifuged at
4°C.
Supernatant
containing
crude protein
extract was
used for IP
after
quantification
i. Sample Preparation:
Note : The DYNLT1 protein in mouse is 93.8% (106/113) similar that of humans.

9. ii. Immunoprecipitation
SDS PAGE was done to separate the protein of interest its interactome.
Proteins were eluted by boiling with RapiGest SF. Pellet was discarded.
The pellet was washed twice with PBS buffer
Centrifuged at 14000rpm for 30mins at 4°C
50µl of protein A agarose bead was added to the antigen-antibody complex and incubated at
4°C for 4 hrs.
1µg of primary antibody(anti-DYNLT1 polyclonal antibody) was added and incubated at 4°C
overnight.
100mg of protein lysate was diluted to 200µl with sterile PBS (Phosphate Buffered Saline).

10. (iii) Western blot analysis
The IP samples and the
adult testicular lysate
(input) were loaded and
resolved in SDS PAGE.
The separated proteins in
gel were transferred onto a
polyvinylidene difluoride
(PVDF) membrane
through electrophoresis.
After transfer the
membrane was cut
according to the size of the
protein of interest
The free area of membrane
was blocked using 3%
gelatin in 0.01% PBST for
1hr.
The blocker was
discarded and the
membrane was washed
using PBST solution for
3 times , 10mins each.
Primary antibody was diluted
in PBST, 1:1000 ratio and was
added to the membrane.
Then the membrane was
incubation for 2hrs at
constant rocking.
The primary antibody was
discarded and the
membrane was washed
thrice with PBST for
10mins each.
Secondary antibody (anti isotypic
ab conjugated with HRP enzyme)
was diluted in (1:2000 ratio) PBST
was added to the membrane and
incubated for 1hr @constant
rocking.
The secondary antibody
was discarded and the
membrane was washed
with PBST thrice for
10mins each.
The developer solution
was prepared (5ml
PBST + 0.1%NiCl2 +
0.05% DAB + 10µl
H2O2)

11. Western Blot Process
Gel preparation SDS PAGE setup
Membrane Transfer Rocker Developing the blot

12. SILVER STAINING
SIGMA Silver Staining Protocol:

13. RESULTS OF IN SILICO ANALYSIS
• CORUM • DIP
• STRING
• MINT
• HuRI

14. • MENTHA.UNIROMA2

15. • BIOGRID

16. • INTACT

17. • PATHWAYS

18. • A total 251 interacting proteins of DYNLT1 were identified
using the above mentioned tools.
• Out of which 21 proteins were found in more than one tool.
• The proteins which showed highest no. of interactions with
DYNLT1 or the proteins with the highest interaction score
varied from tool to tool.

19. Result for PANTHER Analysis
Fig: different protein classes

20. IN VITRO ANALYSIS RESULTS:
Dual color
Protein
ladder used
AIRE (55/61kDa)
TDP43 (43kDa)
HSP90 (90kDa)
L IP1 IP2 IP3 BC Input
DYNLT1B (14kDa)
15kDa
10kDa
75kDa
55kDa
50kDa
37kDa
100kDa
75kDa
L – Ladder,
IP1-3 – Immuno pull down samples,
BC – protein A agarose bead control,
Input – Adult testicular lysate.
Silver Staining showing the interacting
proteins .
L IP1 IP2 IP3 BC Input

21. RESULTS
• A targeted Western blot analysis was done with the IP sample of DYNLT1 to check its
association with autoimmune regulator (AIRE). AIRE shows localization to the cytoskeletal
network and since DYNLT1 is a cytoskeletal motor complex protein, it was assumed to have
an association with AIRE. But the blot showed no specific association between the two.
• Western blot analysis was done for the same IP sample of DYNLT1 to check its association
with TAR DNA binding protein 43 (TDP43). TDP43 is a DNA binding protein. TDP was
found to gave a role in the cause of male fertility. The blot between DYNLT1 and TDP
showed some association.
• Western blot analysis was done for the IP samples with heat shock protein 90 (HSP90). The
interaction of this protein with DYNLT1 was predicted in the in silico analysis part. This
Western blot was done to validate the prediction. The blot showed association with the two.
• Silver staining was done to detect less concentration of protein from the IP sample and also
the adult mice testicular lysate. In this, it was detected that IP pulled down the proteins and
the silver staining showed a lot of interacting proteins.

22. CONCLUSION
The need for human infertility should be evaluated by using a multidisciplinary approach
involving genetics, epigenetics and at molecular levels to develop appropriate screens for
abnormal phenotypes and to discover more effective solutions for an infertile couple’s problems
is increasing. As more genes involved are discovered and the causes of infertility disorders
become better understood, the more infertility management and treatment will be improved.
DYNLT1 is one such gene whose protein dysfunction was shown in the analysis of infertile man.
The in silico analysis showed a wide range of interacting proteins. A few of which had direct
involvement in controlling human male infertility. This prediction suggest that the interaction or
these interacting proteins as an option for drug target. The in vitro validation showed the
association of DYNLT1 with TDP43 and HSP90. Both of these proteins have a role in causing male
infertility. The DYNLT1 protein can be studied further to know its function in male infertility.