OUR RESEARCH PROJECTS
Project 1: Understanding
the role of sumoylation during spermatogenesis (supported by NIH)
Post-translational
modification by Small Ubiquitin-like Modifiers or SUMO proteins has been
identified as an important regulatory event that is implicated in several
cellular processes, seemingly based on the cell type. Recent findings from our
laboratory suggest diverse and potentially multiple roles of SUMO in testicular
function and spermatogenesis however, SUMO targets remain uncharacterized in
the testis due to the complex multicellular nature of testicular tissue, the
inability to maintain and manipulate spermatogenesis in vitro, and the technical challenges
involved in identifying low-abundance endogenous SUMO targets. In this study, we performed cell-specific
identification of sumoylated proteins using concentrated cell lysates prepared
with de-sumoylation inhibitors from freshly purified spermatocytes and
spermatids. Hundred and twenty proteins were uniquely identified in the
spermatocyte and/or spermatid fractions. The identified proteins are involved in the regulation of
transcription, stress response, microRNA biogenesis, regulation of major enzymatic
pathways, nuclear-cytoplasmic transport, cell cycle control, acrosome
biogenesis, and other processes. Several proteins with important roles during
spermatogenesis were chosen for further characterization by
co-immunoprecipitation, co-localization and in-vitro sumoylation studies. GPS-SUMO software was used to identify consensus and non-consensus
sumoylation sites within amino acid sequences of the proteins. The analyses
confirmed the sumoylation of several novel, previously uncharacterized, SUMO targets,
such as CDK1, RNAP II,
CDC5, MILI, DDX4, and Stk31.
Furthermore, several proteins that were previously identified as
SUMO targets in somatic cells (e.g., KAP1, MDC1) were identified as SUMO
targets in germ cells. Many of these proteins have a unique role in
spermatogenesis, particularly during meiotic progression. Notably, the
inhibition of sumoylation blocked the G2/M transition in spermatocytes in vitro. Co-localization studies
suggest a role of sumoylation in modifying proteins within heterochromatic and
intra-chromasomal sub-domains of germ cells. This research opens a novel avenue
for numerous further studies of SUMO at the level of individual targets.
Project 2: Understanding
the effect of tobacco smoke on cell signaling (FAMRI supported)
2.1) Sperm
project
Sperm are highly dependent on posttranslational
modifications of proteins. Massive phosphorylation on tyrosine residue is
required for sperm capacitation. Sumoylation has also been recently implicated
in spermatogenesis and sperm functions. Cigarette smoke is known to cause
oxidative stress in different tissues, and several studies suggest that it
causes oxidative stress in sperm. Whether tobacco affects posttranslational
modifications in human sperm is currently unknown. We have showed that a short exposure of human sperm to
physiological concentrations of cigarette smoke extract (CSE) causes the
partial de-sumoylation of many sperm proteins. Furthermore, the presence of a
low concentration of CSE in the human tubal fluid during an induction of in
vitro capacitation inhibits the capacitation-associated increase in protein
phosphorylation. Collectively, changes in posttranslational modifications may
be one of the mechanisms through which exposure to tobacco can negatively
affect sperm functions and cause fertility problems.
2.2) CDK14
project:
DNA arrays have been employed to monitor gene expression
patterns in testis of mice exposed to tobacco smoke for 24 weeks and compared
to control animals. The results of the analysis revealed significant changes in
expression of several genes that may have a role in spermatogenesis. Cdk14 was
chosen for further characterization because of a suggested role in the testis
and in regulation of Wnt signaling. RT-PCR analysis confirmed down regulation
of Cdk14 in mice exposed to cigarette smoke (CS). Cdk14 is expressed in all
testicular cells; spermatogonia- and Sertoli-derived cell lines treated with
cigarette smoke extract (CSE) in vitro showed down-regulation of CDK14 mRNA and
protein levels as well as down-regulation of β-catenin levels. CS-induced
down-regulation of CDK14 mRNA and protein levels was also observed in several
lung epithelium-derived cell lines including primary normal human bronchial
epithelial cells (NHBE), suggesting that the effect is not restricted to the
testis. Similar to testicular cells, CS-induced down-regulation of CDK14 in
lung cells correlated with decreased levels of β-catenin, a finding suggesting
impaired Wnt signaling. In the lungs, CDK14 was localized to the alveolar and
bronchial epithelium.
FUNDING
Funding sources
And The Mitrani foundation.
