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Dr. Anne Bertolotti is a cell and molecular biologist with an established lab at the Medical Research Council Laboratory of Molecular Biology in England. She has devoted her career to the study of protein function and mechanisms that contribute to protein misfolding. This is an important area of study because misfolded proteins are implicated in a variety of diseases and disorders such as Parkinson’s and Alzheimer's, which are highly prevalent among the aged population. Her lab has focused particularly on understanding the conditions that lead to a breakdown in the protein quality control system as the body ages. These contributions can lead to revolutionary therapeutic advances for many of the pervasive neurodegenerative diseases that are prevalent amongst the human population.

Education & Career
Dr. Anne Bertolotti has a vast array of educational experiences from diverse schools and environments. She has successfully completed her Ph.D in 1998 from Strasbourg University where she worked with Dr. Laszlo Tora and Pr. Pierre Chambon. She immediately went on to complete her post-doctoral research from 1998 to 2000 with Pr. David Ron at The Skirball Institute of Biomolecular Medicine. During her post-doc, she obtained a position at the French National Institute of Health and Medical Research (INSERM) in 1999. In 2004, she was elected as a Young Investigator at the European Molecular Biology Organization (EMBO). Her research has been acknowledged and values in Europe and around the world, indicated by her becoming an EMBO member in 2013. Her extensive research has led to her being awarded the Hooke Medal in 2014 by the British Society for Cell Biology. The Hooke Medal is awarded to emerging leaders in the field of cell biology. Since 2006, she has been a group leader at the Medical Research Council Laboratory of Molecular Biology (LMB) in Cambridge, UK for the division of neurobiology.

ATPase Dedicated Chaperone of 17 kDA (Adc-17)
Protein folding is a very complex physical process of folding a polypeptide into a three-dimensional structure with help from numerous molecules and enzymes. The blueprint for folding is encoded in every type of protein structure, starting from the primary amino acid sequence up to the association of multiple folded proteins into a quaternary structure. Due to this complexity, there are many steps during this process which can go awry leading to misfolding of proteins due to extracellular, intracellular, or genetic factors. Protein misfolding frequency and severity varies from cell to cell, however there are mechanisms in place to re-fold proteins into their correct structure. This regulation helps to maintain proteostasis, which is the homeostasis of protein levels in the cell. An important complex involved in this is the proteasome, which degrades misfolded or damaged proteins to prevent cell stress caused by accumulation of those proteins. Much of Dr. Bertolotti's work revolves around further understanding how the proteasome is regulated under disease states.

The proteasome is necessary for cell survival, and proteasome levels increase in response to cell stress, such as protein misfolding stress. In yeast, the up-regulation of proteasomes is caused by Rpn-4, which is a transcription factor which regulates proteasome subunit levels. However, generating more proteasome subunits is not sufficient to up-regulate proteasome activity since assembling subunits into a functional proteasome is quite complex. The assembly process requires a regulatory particle (RP) which has 6 ATPases (Rpt1-Rpt-6). Dr. Bertolotti and her team identified a stress produced protein, Adc-17, which facilitates early steps in RP assembly and is crucial to adapting proteasome assembly to increased demands. In the absence of Adc-17, proteasome defects became more pronounced. Thus, Adc-17 seems to function by increasing its levels to adjust proteasome assembly in times of proteasome stress. Additionally, in Adc-17 knockout mutants, cell fitness was decreased along with levels of Rpt-6. Rpt-6 is known to be necessary for proteasome form and function, which explains a decrease in fitness with decreasing Rpt-6. It was found that adequate amounts of adc17 were necessary to maintain normal levels of Rpt6, and to form the Rpt-3, Rpt-6 dimer, which occurs early in RP formation. It was also found that Adc17 was capable of suppressing the severe growth defects caused by multiple distinct mutants. Dr. Bertolotti's research looked at both wildtype and cim3-1 mutant cells and found that the central particle regulatory particles were increases in cim3-1 mutants, indicating that these mutants had deficient proteasomes. This research shows how cells do have internal mechanisms for rescuing cell functioning by adjusting proteasome assembly to meet increasing proteasome demands in times of stress, and here it was shown that Adc-17 is one such inducible chaperone that adjusts proteasome assembly.

Target of Rapamycin Complex 1 (TORC1)
This experiment was done with the intent of gaining understanding of the pathway that allows cells to maintain proteasome homeostasis. The authors found a pathway at which the central molecule is [[CRTC1|TORC1 ]]. Inhibition of TORC1 by the mitogen activated protein kinase leads to an increase of proteasome subunits under stress conditions. The authors found that when the stress and nutrient sensitive regulator sfp1 is removed, TORC1 becomes hyperactive which prevents Adc17 induction when the cell is put under stress by tunicamycin. Therefore by selectively inhibiting TORC1 it was found Adc17 activity could be induced and an increase in proteasome abundance was observed in yeast cells. It was found that Mpk-1 is important in tunicamycin induced stress events in yeast cells. It acts to induce Adc17 by inhibiting the TORC1 signal. Due to the necessary work by Adc17 in the early proteasome assembly it was hypthesised that mpk1 is a master regulator of the proteasome. It was found that when mpk1 was deleted cells showed no increase in proteasome levels when tunicamycin, and rapamycin caused stress in the cell. This type of defect was known from other studies to be due to RP assembly deficiencies. In comparing mpk1 deficient cells to Adc17 deficient cells it was found the effects were more severe in mpk1 deficient cells which suggested that mpk1 has multiple functions in assembly, not just Adc17 activation. It was then found that mpk1 acts to increase proteasome levels in times of stress. Due to the conserved structure of the pathway the tests in yeast cells may be applicable in mammalian cells.

Huntingin (Htt)
Huntington’s disease and a key protein involved in the pathogenesis of Huntington’s—huntingin (Htt)— are currently studied in order to clarify the function and mechanisms that Htt employs in pathogenesis of the disease. The proteolysis of mutant Htt is currently thought to be an early event in the progression of Huntington’s disease, hence this paper sought to further elucidate whether there was another process by which mutant Htt may trigger Huntington’s disease. Htt was found to co-localize with proteasomal chaperones, Rpt4 and Rpt6, which are members of the 19S ATPase family. Interestingly, Rpt4 and Rpt6 have been thought to have other functions, outside of their proteolytic role, such as the formation of protein aggregates. It was also found that misfolding of a fragment of huntingin, Htt73, is facilitated by Rpt4 and Rpt6. Rpt4 and Rpt6 were also found to enhance misfolding of Htt73 when it was conjugated to a poly(q) expansion which is known to cause impairment of proteolytic function and lead to protein aggregation. Also, the addition of the entire 19S proteasome also lead to Htt aggregation, in vitro, indicating that the entire proteasomal machinery can be implicated in facilitating misfolding of proteins with a poly(Q) expansion, and eventual aggregation. So given this data, Rpt4, Rpt6 and the entire 19S proteasome machinery may be implicated in remodelling proteins with a poly(Q) expansion and initiating aggregation of such proteins in a process that is independent of their degradative function. As such, this may provide a new direction for understanding a different process by which Htt progresses Huntington's disease.

Therapeutic Developments
Many of breakthroughs in research conducted in Dr. Bertolotti's lab has led to the development of drugs and therapies targeted at a variety of different neurodegenerative diseases.

Dr. Bertolotti and her lab focus therapeutic developments to restore proteostasis and promote cell survival in times of protein misfolding stress by targeting parts of cellular mechanisms that disrupt protein homeostasis. The three branches of the unfolded protein response (UPR) help regulate proteostasis by increasing translation of chaperone proteins to help with re-folding via the IRE1 and ATF6 pathways and/or downregulate global protein synthesis via the PERK pathway. The PERK pathway, when activated, leads to phosphorylation of the alpha subunit of eIF2a, which is a eukaryotic translation initiation factor that when phosphorylated ends up decreasing global translation and induces G1 cell cycle arrest preventing cell proliferation. Additionally, the phosphorylation of this initiation factor leads to an increase in transcription and translation of ATF4 (activating transcription factor) which in turn activates CHOP production. CHOP has 2 effects downstream, one being apoptosis and the other being activation of PPr1r15a, which recruits and forms a complex with ppc1 (a phosphatase) that dephosphorylates eIF2a to resume protein synthesis. Discovering further details regarding PERK pathway activation creates the opportunity to test therapeutic developments at various points within the pathway. Drugs known to alleviate protein misfolding and having anti-prion activity can be tested in relation to the PERK pathway in order to determine their mechanism of action, one such drug known for its anti-prion activity is Guanabenz. Dr. Bertolotti explored the activity of Guanabenz to determine if it was involved in protecting against accumulation of misfolded proteins. Upon adding Guanabenz to cells treated with tunicamycin (molecule that induces ER stress), they found that cell survival increased compared to tunicamycin treated cells without Guanabenz. This suggests that Guanabenz increased the cell viability and reversed cell death that was caused by increased ER stress. The addition of guanabenz to tunicamycin treated cells also led to sustained eIF2a levels and decreased CHOP expression, while the observed levels of phosphatases were significantly reduced in cells with both tunicamycin and guanabenz. This showed that there are molecules which can promote cell survival under times of stress by inhibiting a phosphatase to keep eIF2a phosphorylated and thus global protein translation reduced until proteostasis is restored. Many of the mechanisms involved with reducing the accumulation of misfolded proteins in the ER revolve around the phosphorylation of eIF2α to decrease protein synthesis and alleviate cell stress. Dr. Bertolotti’s lab focused on upstream negative regulators of eIF2 phosphorylation, including the PPP1R15A-PP1c complex. This was done to isolate inhibitors of PPP1R15A-PP1c that ultimately lead to sustained eIF2α, and therefore an enhanced stress response to the accumulation of misfolded proteins. Although Guanabenz showed promises of cell viability in vitro, it was not able to selectively inhibit different PP1c complexes in vivo. As a result, a derivative of GBZ, Sephin1, was instead found to selectively inhibit PPC1 in vivo. Importantly, Sephin1-treated mice were devoid of any adverse effects in weight, memory, and rotarod performance which were present in the guanabenz treated group. Sephin1 was also able to rescue the effects of a broad range of protein misfolding diseases, including Charcot-Marie-Tooth 1B (CMT1B) and amyotrophic lateral sclerosis (ALS). In conclusion, Sephin1 may have broad therapeutic effects that can be used to decrease ER stress in a variety of disease states.