N-terminal truncations of human bHLH transcription factor Twist1 leads to the formation of aggresomes

Abstract

In the cell, misfolded proteins are processed by molecular chaperone-mediated refolding or through ubiquitin-mediated proteosome system. Dysregulation of these mechanisms facilitates the aggregation of misfolded proteins and forms aggresomes in the juxta nuclear position of the cell which are removed by lysosome-mediated autophagy pathway in the subsequent cell division. Accumulation of misfolded proteins in the cell is hallmark of several neurological disorders and other diseases including cancer. However, the exact mechanism of aggresome formation and clearance is not thoroughly understood. Reports have shown that several proteins including p300, p53, TAU, α-synuclein, SOD, etc. contain intrinsically disordered region (IDR) which has the tendency to form aggresome. To study the nature of aggresome formation and stability of the aggresome, we have chosen Twist1 as a model protein since it has IDR regions. Twist1 is a bHLH transcription factor which plays a major role in epithelial mesenchymal transition (EMT) and shown to interact with HAT domain of p300 and p53. In the present study, we generated several deletion mutants of human Twist1 with different fluorescent tags and delineated the regions responsible for aggresome formation. The Twist1 protein contains two NLS motifs at the N-terminal region. We showed that the deletions of regions spanning the amino acids 30-46 (Twist1Δ30-46) which lacks the first NLS motif form larger and intense aggregates while the deletion of residues from 47 to 100 (Twist1Δ47-100) which lacks the second NLS motif generates smaller and less intense aggregates in the juxta nuclear position. This suggests that both the NLS motifs are needed for the proper nuclear localization of Twist1. The aggresome formation of the Twist1 deletion mutants was confirmed by counterstaining with known aggresome markers: Vimentin, HDAC6, and gamma tubulin and further validated by MG-132 treatment. In addition, it was found that the aggresomes generated by the Twist1Δ30-46 construct are more stable than the aggresome produced by the Twist1Δ47-100 construct as well as the wild-type Twist1 protein. Taken together, our data provide an important understanding on the role of IDR regions on the formation and stability of aggresomes.

USF1 defect drives p53 degradation during Helicobacter pylori infection and accelerates gastric carcinogenesis

“…This could represent an Hp strategy to prevent USF1 transcriptional function, impairing its tumour suppressive activity and DNA repair functions. Alternatively, USF1 foci could correspond to protein aggregation due to infection-induced misfolding, as recently reported the formation of aggresomes by Twist1, another b-HLH-LZ transcription factor 48…”

“…Alternatively, USF1 foci could correspond to protein aggregation due to infection-induced misfolding, as recently reported the formation of aggresomes by Twist1, another b-HLH-LZ transcription factor. 48 …”

A novel insight into neurological disorders through HDAC6 protein–protein interactions

“…Furthermore, HDAC6’s catalytic domains are equally important due to their interaction with microtubule tracks 36 . Through interaction with microtubules, HDAC6 facilitates the autophagic degradation and clearance of several protein aggregates including HTT 36 , LC3 36 , ATXN3 34 , CFTR 8 , KALRN (kalirin-7) 38 , SNCAIP 38 , MAPT 29 , the aggresome marker VIM 39 , and E3- ligase proteins STUB1 (CHIP) 40 , and TRIM50 41 . Moreover, HDAC6 also tightly forms a complex with PRKN (Parkin), a PD-related protein, and under proteasome dysfunction, mediates the bidirectional transport of this complex on microtubules towards aggresomes 42 .…”