The conformational space sampled by the two-domain protein calmodulin has been explored by an approach based on four sets of NMR observables obtained on Tb 3؉ -and Tm 3؉ -substituted proteins. The observables are the pseudocontact shifts and residual dipolar couplings of the C-terminal domain when lanthanide substitution is at the N-terminal domain. Each set of observables provides independent information on the conformations experienced by the molecule. It is found that not all sterically allowed conformations are equally populated. Taking the N-terminal domain as the reference, the C-terminal domain preferentially resides in a region of space inscribed in a wide elliptical cone. The axis of the cone is tilted by Ϸ30°with respect to the direction of the N-terminal part of the interdomain helix, which is known to have a flexible central part in solution. The C-terminal domain also undergoes rotation about the axis defined by the C-terminal part of the interdomain helix. Neither the extended helix conformation initially observed in the solid state for free calcium calmodulin nor the closed conformation(s) adopted by calcium calmodulin either alone or in its adduct(s) with target peptide(s) is among the most preferred ones. These findings are unique, both in terms of structural information obtained on a biomolecule that samples multiple conformations and in terms of the approach developed to achieve the results. The same approach is in principle applicable to other multidomain proteins, as well as to multiple interaction modes between two macromolecular partners. C almodulin (CaM) is a paradigm case in structural biology. The following brief survey of the history of the structural and dynamic studies on this protein serves the double purpose of putting the present findings in proper perspective and of acknowledging those pieces of previous information that were essential to allow the present approach to be developed and to yield novel structural information.CaMs are two-domain proteins belonging to the large family of EF-hand proteins (1-3). They contain Ϸ150 amino acid residues, organized into two domains of Ϸ70 aa each and connected by a short linker. Each domain is made up of two special helix-loop-helix motifs (EF-hand motifs) that can bind a calcium ion in the loop. The two loops are held close to one another by two short antiparallel ␤-strands forming a threehydrogen bond stretch of ␤-sheet. The function of CaM in cell cytoplasm is that of responding to sudden rises of calcium concentration by binding up to four calcium ions in the four EF-hand loops, by changing conformation because of metal binding, and by thus becoming able to recognize, bind to, and activate, a number of proteins and enzymes (1,(4)(5)(6)(7)(8). Early x-ray data (9) showed the four-calcium (Ca 2 ) N (Ca 2 ) C CaM form (subscripts N and C refer to the calcium atoms bound by the N-and C-terminal domains, respectively) to have a dumbbell shape, with helix 4, the last helix of the N-terminal domain, and helix 5, the first helix of the C-termi...

Approximately 75% of all breast cancers express the oestrogen and/or progesterone receptors. Endocrine therapy is usually effective in these hormone-receptor-positive tumours, but primary and acquired resistance limits its long-term benefit 1,2 . Here we show that in mouse models of hormone-receptor-positive breast cancer, periodic fasting or a fasting-mimicking diet 3-5 enhances the activity of the endocrine therapeutics tamoxifen and fulvestrant by lowering circulating IGF1, insulin and leptin and by inhibiting AKT-mTOR signalling via upregulation of EGR1 and PTEN. When fulvestrant is combined with palbociclib (a cyclin-dependent kinase 4/6 inhibitor), adding periodic cycles of a fasting-mimicking diet promotes long-lasting tumour regression and reverts acquired resistance to drug treatment. Moreover, both fasting and a fasting-mimicking diet prevent tamoxifen-induced endometrial hyperplasia. In patients with hormone-receptor-positive breast cancer receiving oestrogen therapy, cycles of a fasting-mimicking diet cause metabolic changes analogous to those observed in mice, including reduced levels of insulin, leptin and IGF1, with the last two remaining low for extended periods. In mice, these long-lasting effects are associated with long-term anti-cancer activity. These results support further clinical studies of a fasting-mimicking diet as an adjuvant to oestrogen therapy in hormone-receptor-positive breast cancer.Growth factor signalling through the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAP kinase) axes enhances oestrogen receptor activity and frequently underlies endocrine resistance in breast tumours 1,2,6 . Water-only fasting or plant-based diets that are simultaneously low in calories, sugar and protein and proportionally high in fat (fasting-mimicking diets (FMDs)) reduce circulating growth factors such as insulin and IGF1 2,6,7 . Therefore, we hypothesized that these dietary interventions could be used to enhance the activity of oestrogen therapy (ET) and delay endocrine resistance.Low-serum, low-glucose cell culture conditions designed to mimic the effects of fasting or FMD (referred to as short-term starvation, STS) increased the anti-tumour activities of tamoxifen and fulvestrant in HR + /HER2breast cancer (BC) cell lines, and similar results were obtained in mouse xenografts of the same cell lines subjected to weekly cycles of fasting or FMD (Fig. 1a, Extended Data Figs. 1, 2a, b). STS also increased the anti-tumour activity of tamoxifen in tumour organoids from patients with HR + BC 8 , and weekly FMD cycles prevented acquired resistance to tamoxifen in mice (Extended Data Fig. 2c, d). Enhancement of ET activity through STS was dependent on the reduction in serum, but not glucose, as adding back glucose to the growth medium did not affect the observed potentiation (Extended Data Fig. 3a).In mice, besides increasing β-hydroxybutyrate levels (Extended Data Fig. 3b) and lowering blood glucose (from 6.3 ± 0.6 mmol l −1 to 4.1 ± 0....

Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates called “drusen”. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates SQSTM1/p62 mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD.