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The hydrophobic effect is a major driving force in protein folding. A complete understanding of this effect requires the description of the conformational states of water and protein molecules at different temperatures. Towards this goal, we characterise the cold and hot denatured states of a protein by modelling NMR chemical shifts using restrained molecular dynamics simulations. A detailed analysis of the resulting structures reveals that water molecules in the bulk and at the protein interface form on average the same number of hydrogen bonds. Thus, even if proteins are ‘large’ particles (in terms of the hydrophobic effect, i.e. larger than 1 nm), because of the presence of complex surface patterns of polar and non-polar residues their behaviour can be compared to that of ‘small’ particles (i.e. smaller than 1 nm). We thus find that the hot denatured state is more compact and richer in secondary structure than the cold denatured state, since water at lower temperatures can form more hydrogen bonds than at high temperatures. Then, using Φ-value analysis we show that the structural differences between the hot and cold denatured states result in two alternative folding mechanisms. These findings thus illustrate how the analysis of water-protein hydrogen bonds can reveal the molecular origins of protein behaviours associated with the hydrophobic effect.

Folding and function may impose different requirements on the amino acid sequences of proteins, thus potentially giving rise to conflict. Such a conflict, or frustration, can result in the formation of partially misfolded intermediates that can compromise folding and promote aggregation. We investigate this phenomenon by studying frataxin, a protein whose normal function is to facilitate the formation of iron-sulfur clusters but whose mutations are associated with Friedreich's ataxia. To characterize the folding pathway of this protein we carry out a Φ-value analysis and use the resulting structural information to determine the structure of the folding transition state, which we then validate by a second round of rationally designed mutagenesis. The analysis of the transition-state structure reveals that the regions involved in the folding process are highly aggregation-prone. By contrast, the regions that are functionally important are partially misfolded in the transition state but highly resistant to aggregation. Taken together, these results indicate that in frataxin the competition between folding and function creates the possibility of misfolding, and that to prevent aggregation the amino acid sequence of this protein is optimized to be highly resistant to aggregation in the regions involved in misfolding.

SH3 domains are very abundant protein-protein interactions modules, involved in the regulation of several cellular processes. Whilst they have been associated to allosteric communication pathways between contiguous domains in multi-domain proteins, there is lack of information regarding the intra-domain allosteric cross-talk within the SH3 moiety. Here we scrutinize the presence of an allosteric network in the C-terminal SH3 domain of Grb2 protein, upon binding the Grb2-associated binding 2 protein. To explore allostery, we performed double mutant cycle analysis, a powerful quantitative approach based on mutagenesis in conjunction with kinetic experiments. Data reveal the presence of an unexpected allosteric sparse network that modulates the affinity between the SH3 domain and its physiological partner.

Nuclear activated β -catenin plays a causative role in colorectal cancers (CRC) but remains an elusive therapeutic target. Using human CRC cells harboring different Wnt/ β -catenin pathway mutations in APC/KRAS or β-catenin/KRAS genes, and both genetic and pharmacological knockdown approaches, we show that oncogenic β -catenin signaling negatively regulates the expression of NHERF1 (Na + /H + exchanger 3 regulating factor 1), a PDZ-adaptor protein that is usually lost or downregulated in early dysplastic adenomas to exacerbate nuclear β -catenin activity. Chromatin immunoprecipitation (ChIP) assays demonstrated that β -catenin represses NHERF1 via TCF4 directly, while the association between TCF1 and the Nherf1 promoter increased upon β -catenin knockdown. To note, the occurrence of a cytostatic survival response in settings of single β -catenin-depleted CRC cells was abrogated by combining NHERF1 inhibition via small hairpin RNA (shRNA) or RS5517, a novel PDZ1-domain ligand of NHERF1 that prevented its ectopic nuclear entry. Mechanistically, dual NHERF1/ β -catenin targeting promoted an autophagy-to-apoptosis switch consistent with the activation of Caspase-3, the cleavage of PARP and reduced levels of phospho-ERK1/2, Beclin-1, and Rab7 autophagic proteins compared with β -catenin knockdown alone. Collectively, our data unveil novel β -catenin/TCF-dependent mechanisms of CRC carcinogenesis, also offering preclinical proof of concept for combining β -catenin and NHERF1 pharmacological inhibitors as a mechanism-based strategy to augment apoptotic death of CRC cells refractory to current Wnt/ β -catenin-targeted therapeutics.

PDZ domains are the most prominent biological structural domains involved in protein-protein interactions in the human cell. The second PDZ domain of the protein tyrosine phosphatase BL (PDZ2) interacts and binds the C-termini of the tumour suppressor protein APC and of the LIM domain-containing protein RIL. One isoform of PDZ2 (PDZ2as) involves an alternative spliced form that exhibits an insertion of 5 residues in a loop. PDZ2as abrogates binding to its partners, even if the insertion is directly located in its binding pocket. Here, we investigate the folding and function of PDZ2as, in comparison to the previously characterized PDZ2 domain. Data reveal that, whilst the thermodynamic stability of PDZ2as appears as nearly identical to that of PDZ2, the insertion of 5 amino acids induces formation of some weak transient non-native interactions in the folding transition state, as mirrored by a concomitant increase of both the folding and unfolding rate constants. From a functional perspective, we show that the decrease in affinity is caused by a pronounced decrease of the association rate constants (by nearly ten fold), with no effect on the microscopic dissociation rate constants. The results are briefly discussed in the context of previous work on PDZ domains.

Nuclear activated [beta]-catenin plays a causative role in colorectal cancers (CRC) but remains an elusive therapeutic target. Using human CRC cells harboring different Wnt/[beta]-catenin pathway mutations in APC/KRAS or [beta]-catenin/KRAS genes, and both genetic and pharmacological knockdown approaches, we show that oncogenic [beta]-catenin signaling negatively regulates the expression of NHERF1 (Na.sup.+/H.sup.+ exchanger 3 regulating factor 1), a PDZ-adaptor protein that is usually lost or downregulated in early dysplastic adenomas to exacerbate nuclear [beta]-catenin activity. Chromatin immunoprecipitation (ChIP) assays demonstrated that [beta]-catenin represses NHERF1 via TCF4 directly, while the association between TCF1 and the Nherf1 promoter increased upon [beta]-catenin knockdown. To note, the occurrence of a cytostatic survival response in settings of single [beta]-catenin-depleted CRC cells was abrogated by combining NHERF1 inhibition via small hairpin RNA (shRNA) or RS5517, a novel PDZ1-domain ligand of NHERF1 that prevented its ectopic nuclear entry. Mechanistically, dual NHERF1/[beta]-catenin targeting promoted an autophagy-to-apoptosis switch consistent with the activation of Caspase-3, the cleavage of PARP and reduced levels of phospho-ERK1/2, Beclin-1, and Rab7 autophagic proteins compared with [beta]-catenin knockdown alone. Collectively, our data unveil novel [beta]-catenin/TCF-dependent mechanisms of CRC carcinogenesis, also offering preclinical proof of concept for combining [beta]-catenin and NHERF1 pharmacological inhibitors as a mechanism-based strategy to augment apoptotic death of CRC cells refractory to current Wnt/[beta]-catenin-targeted therapeutics.

The perivascular epithelioid cell (PEC) is a cell type constantly present in a group of tumors called PEComas. PEC expresses myogenic and melanocytic markers, such as HMB45 and actin. Recently, recurrent chromosomal alterations have been demonstrated in PEC. At present, PEComa is a widely accepted entity. In the past 10 years, the use of this term has allowed to report and describe numerous cases permitting to start highlighting the biology of this group of lesions. PEComas are related to the genetic alterations of tuberous sclerosis complex (TSC), an autosomal dominant genetic disease due to losses of TSC1 (9q34) or TSC2 (16p13.3) genes which seem to have a role in the regulation of the Rheb/mTOR/p70S6K pathway. There are some open questions about PEComas regarding its histogenesis, the definition of epithelioid angiomyolipoma and the identification of the histological criteria of malignancy. An innovative therapeutic trial using rapamycin is under way for tumors occurring in TSC such as renal angiomyolipoma and lymphangioleiomyomatosis. Its success could provide the rationale for the use of the same drug in other lesions composed of PECs, especially in the malignant ones.

Background Approaches based on expression signatures of prostate cancer (PCa) have been proposed to predict patient outcomes and response to treatments. The transcription factor NF-Y participates to the progression from benign epithelium to both localized and metastatic PCa and is associated with aggressive transcriptional profile. The gene encoding for NF-YA, the DNA-binding subunit of NF-Y, produces two alternatively spliced transcripts, NF-YAs and NF-YAl. Bioinformatic analyses pointed at NF-YA splicing as a key transcriptional signature to discriminate between different tumor molecular subtypes. In this study, we aimed to determine the pathophysiological role of NF-YA splice variants in PCa and their association with aggressive subtypes. Methods Data on the expression of NF-YA isoforms were extracted from the TCGA (The Cancer Genome Atlas) database of tumor prostate tissues and validated in prostate cell lines. Lentiviral transduction and CRISPR-Cas9 technology allowed the modulation of the expression of NF-YA splice variants in PCa cells. We characterized 3D cell cultures through in vitro assays and RNA-seq profilings. We used the rank-rank hypergeometric overlap approach to identify concordant/discordant gene expression signatures of NF-YAs/NF-YAl-overexpressing cells and human PCa patients. We performed in vivo studies in SHO-SCID mice to determine pathological and molecular phenotypes of NF-YAs/NF-YAl xenograft tumors. Results NF-YA depletion affects the tumorigenic potential of PCa cells in vitro and in vivo. Elevated NF-YAs levels are associated to aggressive PCa specimens, defined by Gleason Score and TNM classification. NF-YAl overexpression increases cell motility, while NF-YAs enhances cell proliferation in PCa 3D spheroids and xenograft tumors. The transcriptome of NF-YAs-spheroids has an extensive overlap with localized and metastatic human PCa signatures. According to PCa PAM50 classification, NF-YAs transcript levels are higher in LumB, characterized by poor prognosis compared to LumA and basal subtypes. A significant decrease in NF-YAs/NF-YAl ratio distinguishes PCa circulating tumor cells from cancer cells in metastatic sites, consistently with pro-migratory function of NF-YAl. Stratification of patients based on NF-YAs expression is predictive of clinical outcome. Conclusions Altogether, our results indicate that the modulation of NF-YA isoforms affects prostate pathophysiological processes and contributes to cancer-relevant phenotype, in vitro and in vivo. Evaluation of NF-YA splicing may represent a new molecular strategy for risk assessment of PCa patients.

Immune mechanisms contribute to the neuropathology of Alzheimer’s disease (AD) but the role of adaptive immune cells is unclear. Here we show that the brain CD8 + T cell compartment is dysregulated in AD patients and in the 3xTg-AD mouse model, accumulating activated CD103 – tissue-resident memory T cells that produce large amounts of granzyme K (GrK). These CD103 – CD8 + T cells originate from the circulation and migrate into the brain using LFA-1 integrin. Ablation of brain CD103 – CD8 + T cells in 3xTg-AD mice ameliorates cognitive decline and reduces neuropathology. GrK induces neuronal dysfunction and tau hyperphosphorylation in human and mouse cells via protease-activated receptor-1 (PAR-1), which is expressed at higher levels in the AD brain, revealing a key immune-mediated neurotoxic axis. We conclude that communication between CD8 + T cells and the nervous system is altered in AD, paving the way for therapies targeting T cell-dependent neurotoxic inflammation. Neuroimmune communication in Alzheimer’s disease (AD) is poorly understood. Here, the authors show that CD103–CD8+ resident memory T cells induce neurotoxicity and neuroinflammation in AD via the granzyme K–PAR-1 axis.

Background: Different antiangiogenics are currently indicated in the second-line treatment of metastatic colorectal cancer (mCRC), following a first-line bevacizumab-containing treatment. The magnitude of benefit is limited, but no predictors of benefit have been identified. Methods: A total of 184 mCRC patients progressing to a first-line bevacizumab-containing treatment were randomised in the BEBYP study to continue or not the antiangiogenic in combination with a second-line chemotherapy. A subgroup analysis according to baseline serum lactate dehydrogenase (LDH) levels was carried out. Results: A significant interaction effect between LDH levels and treatment was found in terms of progression-free survival (PFS; P =0.002). Although patients with low LDH levels achieved significant PFS benefit from the continuation of bevacizumab (HR: 0.39 (95% CI: 0.23–0.65)), patients with high levels did not (HR: 1.10 (95% CI: 0.74–1.64)). Consistent results were reported in overall survival (OS; P =0.075). Conclusions: As preclinical evidence suggests that serum LDH may be a marker of tumour angiogenesis activation, low levels may indicate that bevacizumab is still efficacious in inhibiting angiogenesis. Validation of present results in subgroup analyses of other randomised trials of second-line angiogenesis inhibitors is warranted.