SINP Triennial Report - Saha Institute of Nuclear Physics

Biophysical Sciences Including Chemistry 23
effects by expressing N-terminal domain of Huntingtin (Htt) in specific cell lines, we expressed exon
1 of htt that codes for 40 glutamines (40Q) and 16Q in Neuro2A and HeLa cells. (2) Aggregates and
various apoptotic markers were detected at various time points after transfection. In addition, we checked
the alterations of expressions of few apoptotic genes by RT-PCR. (3) Cells expressing exon 1 of htt
coding 40Q at a stretch exhibited nuclear and cytoplasmic aggregates, increased caspase-1, caspase-2,
caspase-8, caspase-9/6, and calpain activations, release of cytochrome c and AIF from mitochondria in
a time-dependent manner. Truncation of Bid was increased, while the activity of mitochondrial complex
II was decreased in such cells. These changes were significantly higher in cells expressing N-terminal
Htt with 40Q than that obtained in cells expressing N-terminal Htt with 16Q. Expressions of caspase-
1, caspase-2, caspase-3, caspase-7, and caspase-8 were increased while expression of Bcl-2 was decreased
in cells expressing mutated Htt-exon 1. (4) Results presented in this communication showed that
expression of mutated Htt-exon 1 could mimic the cellular phenotypes observed in Huntington’s disease
and this cell model can be used for screening the agents that would interfere with the apoptotic pathway
and aggregate formation.
Majumder, Pritha; Raychaudhuri, Swasti; Chattopadhyay, Biswanath † ; Bhattacharyya, Nitai P
HYPK, a Huntingtin interacting protein, reduces aggregates and apoptosis induced by N-terminal
Huntingtin with 40 glutamines in Neuro2a cells and exhibits chaperone-like activity
Expansion of polymorphic glutamine (Q) numbers present at the protein Huntingtin (Htt) beyond 36Q
results in its misfolding and aggregation, and the aggregates recruit several other proteins. Here we
show that HYPK, initially identified as an Htt-interacting partner by yeast two-hybrid assay, physically
interacts with N-terminal Htt in Neuro2A cells and alters the numbers and distribution of aggregates
formed by N-terminal Htt with 40Q. HYPK also alters the kinetics of mutated N-terminal Htt-mediated
aggregate formation. Fluorescence recovery after photobleaching studies reveal that over-expression of
HYPK results in the appearance of Htt poly Q aggregates, which upon bleaching recovers similar to
80% of initial fluorescence intensity within 6 min. Fluorescence loss in photobleaching studies indicate
loss off fluorescence intensity of the aggregates with time in presence of HYPK. Over-expression of
this protein reduces poly Q-mediated caspase-2, caspase-3 and caspase-8 activations, whereas gamma
ray-induced activations of these enzymes are not affected. In vitro and in vivo studies demonstrate that
HYPK possesses a novel chaperone-like activity. We conclude that HYPK, without having any sequence
similarity with known chaperones, plays an effective role in protecting neuronal cells against apoptosis
induced by mutated N-terminal Htt by modulating the aggregate formation.
Raychaudhuri, Swasti; Sinha, Mithun; Mukhopadhyay, Debashis; Bhattacharyya, Nitai P
Resistance to induction of micronuclei, chromosomal aberrations and apoptosis by Co-60 gammaray
in a cell strain M5, derived from Chinese hamster V79 cells
The paper aims to investigate cytogenetic and apoptotic responses of gamma-irradiation in a radioresistant
cell strain designated as M5. Induced micronuclei, chromosomal aberrations, nuclear
fragmentation and nucleosomal ladders by gamma-irradiation were less at equal doses in M5 cells in
comparison with that obtained in the parental Chinese hamster V79 cells. However, at equal survival,