Peptides and metal ions: A successful marriage for developing artificial metalloproteins.

The mutual relationship between peptides and metal ions enables metalloproteins to have crucial roles in biological systems, including structural, sensing, electron transport, and catalytic functions. The effort to reproduce or/and enhance these roles, or even to create unprecedented functions, is the focus of protein design, the first step toward the comprehension of the complex machinery of nature. Nowadays, protein design allows the building of sophisticated scaffolds, with novel functions and exceptional stability. Recent progress in metalloprotein design has led to the building of peptides/proteins capable of orchestrating the desired functions of different metal cofactors. The structural diversity of peptides allows proper selection of first- and second-shell ligands, as well as long-range electrostatic and hydrophobic interactions, which represent precious tools for tuning metal properties. The scope of this review is to discuss the construction of metal sites in de novo designed and miniaturized scaffolds. Selected examples of mono-, di-, and multi-nuclear binding sites, from the last 20 years will be described in an effort to highlight key artificial models of catalytic or electron-transfer metalloproteins. The authors' goal is to make readers feel like guests at the marriage between peptides and metal ions while offering sources of inspiration for future architects of innovative, artificial metalloproteins.

Evaluation of Major Pathologic Response and Pathologic Complete Response as Surrogate End Points for Survival in Randomized Controlled Trials of Neoadjuvant Immune Checkpoint Blockade in Resectable in NSCLC.

INTRODUCTION: Controversy remains as to whether pathologic complete response (pCR) and major pathologic response (MPR) represent surrogate end points for event-free survival (EFS) and overall survival (OS) in neoadjuvant trials for resectable NSCLC. METHODS: A search of PubMed and archives of international conference abstracts was performed from June 2017 through October 31, 2023. Studies incorporating a neoadjuvant arm with immune checkpoint blockade alone or in combination with chemotherapy were included. Those not providing information regarding pCR, MPR, EFS, or OS were excluded. For trial-level surrogacy, log ORs for pCR and MPR and log hazard ratios for EFS and OS were analyzed using a linear regression model weighted by sample size. The regression coefficient and R2 with 95% confidence interval were calculated by the bootstrapping approach. RESULTS: Seven randomized clinical trials were identified for a total of 2385 patients. At the patient level, the R2 of pCR and MPR with 2-year EFS were 0.82 (0.66-0.94) and 0.81 (0.63-0.93), respectively. The OR of 2-year EFS rates by response status was 0.12 (0.07-0.19) and 0.11 (0.05-0.22), respectively. For the 2-year OS, the R2 of pCR and MPR were 0.55 (0.09-0.98) and 0.52 (0.10-0.96), respectively. At the trial level, the R2 for the association of OR for response and HR for EFS was 0.58 (0.00-0.97) and 0.61 (0.00-0.97), respectively. CONCLUSIONS: Our analyses reveal a robust correlation between pCR and MPR with 2-year EFS but not OS. Trial-level surrogacy was moderate but imprecise. More mature follow-up and data to assess the impact of study crossover are needed.

A SPLICS reporter reveals [Formula: see text]-synuclein regulation of lysosome-mitochondria contacts which affects TFEB nuclear translocation.

Mitochondrial and lysosomal activities are crucial to maintain cellular homeostasis: optimal coordination is achieved at their membrane contact sites where distinct protein machineries regulate organelle network dynamics, ions and metabolites exchange. Here we describe a genetically encoded SPLICS reporter for short- and long- juxtapositions between mitochondria and lysosomes. We report the existence of narrow and wide lysosome-mitochondria contacts differently modulated by mitophagy, autophagy and genetic manipulation of tethering factors. The overexpression of α-synuclein (α-syn) reduces the apposition of mitochondria/lysosomes membranes and affects their privileged Ca2+ transfer, impinging on TFEB nuclear translocation. We observe enhanced TFEB nuclear translocation in α-syn-overexpressing cells. We propose that α-syn, by interfering with mitochondria/lysosomes tethering impacts on local Ca2+ regulated pathways, among which TFEB mediated signaling, and in turn mitochondrial and lysosomal function. Defects in mitochondria and lysosome represent a common hallmark of neurodegenerative diseases: targeting their communication could open therapeutic avenues.

Systematic review and meta-analysis of immune checkpoint inhibitors as single agent or in combination with chemotherapy in early-stage non-small cell lung cancer: Impact of clinicopathological factors and indirect comparison between treatment strategies.

BACKGROUND: In non-small cell lung cancer (NSCLC), the immune checkpoint inhibitors (ICI) revolution is rapidly moving from metastatic to early-stage, however, the impact of clinicopathological variables and optimal treatment sequencing remain unclear. METHODS: Randomized controlled trials (RCTs) in patients with early-stage NSCLC treated with ICI as single agent or in combination with platinum-based chemotherapy (PCT) were included. Primary outcomes were pathological complete response (pCR), event free survival (EFS) (neoadjuvant/perioperative), and disease-free survival (DFS) (adjuvant). Secondary outcomes were major pathological response (MPR), overall survival (OS), toxicity, surgical outcomes (neoadjuvant/perioperative); OS and toxicity (adjuvant). An additional secondary endpoint was to compare EFS and OS between neoadjuvant and perioperative strategies. RESULTS: 8 RCTs (2 neoadjuvant, 4 perioperative, 2 adjuvant) (4661 participants) were included. Neoadjuvant/perioperative ICI+PCT significantly improved pCR, EFS, OS, MPR and R0 resection compared to PCT. Adjuvant ICI significantly improved DFS compared to placebo. There was a significant subgroup interaction by PD-L1 status (χ2 = 10.72, P = 0.005), pCR (χ2 = 17.80, P < 0.0001), and stage (χ2 = 4.46, P = 0.003) for EFS. No difference according to PD-L1 status was found for pCR, with 14% of patients having PD-L1 negative tumors still experiencing a pCR. No interaction by PD-L1 status was found for DFS upon adjuvant ICI. Indirect comparison showed no difference in EFS and OS between neoadjuvant and perioperative ICI+PCT. CONCLUSIONS: PD-L1 status, pCR and stage impact on survival upon neoadjuvant/perioperative ICI. The restriction of neoadjuvant/perioperative ICI to PD-L1 + patients could preclude pCR and long-term benefit in the PD-L1- subgroup. Neoadjuvant and perioperative could be equivalent strategies.

High Nutritional Risk Is Associated with Poor Functional Status and Prognostic Biomarkers in Stroke Patients at Admission to a Rehabilitation Unit.

Considering that malnutrition (undernutrition) is common in stroke patients and may negatively impact body function, the aim of this study was to determine the relationship between nutritional risk and functional status in stroke patients at admission to a rehabilitation unit. Nutritional risk was assessed using the Geriatric Nutritional Risk Index (GNRI), the Prognostic Nutritional Index (PNI) and the Controlling Nutritional Status (CONUT) score. Functional status was assessed using the Barthel Index, the modified Rankin Scale, the Trunk Control Test and the Sitting Balance Scale, and cognitive function was assessed using the Short Portable Mental Status Questionnaire. C-reactive protein, fibrinogen and D-dimer were also evaluated as established prognostic biomarkers. Stroke patients (n = 245; age 69.7 ± 12.8 years; 47%, women; 82% ischemic stroke) at admission to a rehabilitation unit were included in this study. A high prevalence of nutritional risk was detected with each tool and was found to be greater using the GNRI and in patients aged ≥75 years. Multiple logistic regression analysis showed that age and dysphagia were independent predictors of high nutritional risk. High risk groups performed worse on all functional tests compared to the low-risk groups (p < 0.05). Nutritional risk with each tool was associated with functional and cognitive statuses (with the highest correlation being with the Trunk Control Test). Significant associations were also found with C-reactive protein, fibrinogen and D-dimer. In conclusion, a high nutritional risk, as evaluated with the GNRI, the PNI and the CONUT score, was detected in stroke patients at admission to a rehabilitation unit. High nutritional risk was associated with functional status and with predictors of clinical outcomes (and specifically in older patients).

What is the ideal endpoint in early-stage immunotherapy neoadjuvant trials in lung cancer?

Numerous clinical trials investigating neoadjuvant immune checkpoint inhibitors (ICI) have been performed over the last 5 years. As the number of neoadjuvant trials increases, attention must be paid to identifying informative trial endpoints. Complete pathologic response has been shown to be an appropriate surrogate endpoint for clinical outcomes, such as event-free survival or overall survival, in breast cancer and bladder cancer, but it is less established for non-small-cell lung cancer (NSCLC). The simultaneous advances reported with adjuvant ICI make the optimal strategy for early-stage disease debatable. Considering the long time required to conduct trials, it is important to identify optimal endpoints and discover surrogate endpoints for survival that can help guide ongoing clinical research. Endpoints can be grouped into two categories: medical and surgical. Medical endpoints are measures of survival and drug activity; surgical endpoints describe the feasibility of neoadjuvant approaches at a surgical level as well as perioperative attrition and complications. There are also several exploratory endpoints, including circulating tumor DNA clearance and radiomics. In this review, we outline the advantages and disadvantages of commonly reported endpoints for clinical trials of neoadjuvant regimens in NSCLC.

TFEB and TFE3 control glucose homeostasis by regulating insulin gene expression.

To fulfill their function, pancreatic beta cells require precise nutrient-sensing mechanisms that control insulin production. Transcription factor EB (TFEB) and its homolog TFE3 have emerged as crucial regulators of the adaptive response of cell metabolism to environmental cues. Here, we show that TFEB and TFE3 regulate beta-cell function and insulin gene expression in response to variations in nutrient availability. We found that nutrient deprivation in beta cells promoted TFEB/TFE3 activation, which resulted in suppression of insulin gene expression. TFEB overexpression was sufficient to inhibit insulin transcription, whereas beta cells depleted of both TFEB and TFE3 failed to suppress insulin gene expression in response to amino acid deprivation. Interestingly, ChIP-seq analysis showed binding of TFEB to super-enhancer regions that regulate insulin transcription. Conditional, beta-cell-specific, Tfeb-overexpressing, and Tfeb/Tfe3 double-KO mice showed severe alteration of insulin transcription, secretion, and glucose tolerance, indicating that TFEB and TFE3 are important physiological mediators of pancreatic function. Our findings reveal a nutrient-controlled transcriptional mechanism that regulates insulin production, thus playing a key role in glucose homeostasis at both cellular and organismal levels.

Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation.

Batten disease, one of the most devastating types of neurodegenerative lysosomal storage disorders, is caused by mutations in CLN3. Here, we show that CLN3 is a vesicular trafficking hub connecting the Golgi and lysosome compartments. Proteomic analysis reveals that CLN3 interacts with several endo-lysosomal trafficking proteins, including the cation-independent mannose 6 phosphate receptor (CI-M6PR), which coordinates the targeting of lysosomal enzymes to lysosomes. CLN3 depletion results in mis-trafficking of CI-M6PR, mis-sorting of lysosomal enzymes, and defective autophagic lysosomal reformation. Conversely, CLN3 overexpression promotes the formation of multiple lysosomal tubules, which are autophagy and CI-M6PR-dependent, generating newly formed proto-lysosomes. Together, our findings reveal that CLN3 functions as a link between the M6P-dependent trafficking of lysosomal enzymes and lysosomal reformation pathway, explaining the global impairment of lysosomal function in Batten disease.

Targeted Toxicities: Protocols for Monitoring the Adverse Events of Targeted Therapies Used in the Treatment of Non-Small Cell Lung Cancer.

Targeted therapies have revolutionized the treatment for many patients with non-small cell lung cancer (NSCLC). Multiple new oral targeted therapies have been approved in the last decade; however, their overall efficacy may be reduced by poor adherence, treatment interruptions, or dose reductions due to adverse events. Most institutions lack standard monitoring protocols for toxicities from these targeted agents. This review describes important adverse events observed in clinical trials and reported by the U.S. Food and Drug Administration for both currently approved and upcoming promising therapies in the treatment of NSCLC. These agents cause a range of toxicities, including dermatologic, gastroenteric, pulmonary, and cardiac toxicities. This review proposes protocols for routine monitoring of these adverse events, both prior to initiation of therapy and while on treatment.

Structure of the lysosomal mTORC1-TFEB-Rag-Ragulator megacomplex.

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy1. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1)2-5 is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN6,7. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates. We used cryogenic-electron microscopy to determine the structure of TFEB as presented to mTORC1 for phosphorylation, which we refer to as the 'megacomplex'. Two full Rag-Ragulator complexes present each molecule of TFEB to the mTOR active site. One Rag-Ragulator complex is bound to Raptor in the canonical mode seen previously in the absence of TFEB. A second Rag-Ragulator complex (non-canonical) docks onto the first through a RagC GDP-dependent contact with the second Ragulator complex. The non-canonical Rag dimer binds the first helix of TFEB with a RagCGDP-dependent aspartate clamp in the cleft between the Rag G domains. In cellulo mutation of the clamp drives TFEB constitutively into the nucleus while having no effect on mTORC1 localization. The remainder of the 108-amino acid TFEB docking domain winds around Raptor and then back to RagA. The double use of RagC GDP contacts in both Rag dimers explains the strong dependence of TFEB phosphorylation on FLCN and the RagC GDP state.

CXCR4 mediates the effects of IGF-1R signaling in rodent bone homeostasis and fracture repair.

Non-union fractures have considerable clinical and economic burdens and yet the underlying pathogenesis remains largely undetermined. The fracture healing process involves cellular differentiation, callus formation and remodeling, and implies the recruitment and differentiation of mesenchymal stem cells that are not fully characterized. C-X-C chemokine receptor 4 (CXCR4) and Insulin-like growth factor 1 receptor (IGF-1R) are expressed in the fracture callus, but their interactions still remain elusive. We hypothesized that the regulation of CXCR4 by IGF-1R signaling is essential to maintain the bone homeostasis and to promote fracture repair. By using a combination of in vivo and in vitro approaches, we found that conditional ablation of IGF-1R in osteochondroprogenitors led to defects in bone formation and mineralization that associated with altered expression of CXCR4 by a discrete population of endosteal cells. These defects were corrected by AMD3100 (a CXCR4 antagonist). Furthermore, we found that the inducible ablation of IGF-1R in osteochondroprogenitors led to fracture healing failure, that associated with an altered expression of CXCR4. In vivo AMD3100 treatment improved fracture healing and normalized CXCR4 expression. Moreover, we determined that these effects were mediated through the IGF-1R/Insulin receptor substrate 1 (IRS-1) signaling pathway. Taken together, our studies identified a novel population of endosteal cells that is functionally regulated through the modulation of CXCR4 by IGF-1R signaling, and such control is essential in bone homeostasis and fracture healing. Knowledge gained from these studies has the potential to accelerate the development of novel therapeutic interventions by targeting CXCR4 signaling to treat non-unions.

Current methods to analyze lysosome morphology, positioning, motility and function.

Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.

Fluoxetine ameliorates mucopolysaccharidosis type IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is an autosomal recessive disorder caused by mutations in SGSH involved in the degradation of heparan sulfate. MPS-IIIA presents severe neurological symptoms such as progressive developmental delay and cognitive decline, for which there is currently no treatment. Brain targeting represents the main challenge for therapeutics to treat MPS-IIIA, and the development of small-molecule-based treatments able to reach the CNS could be a relevant advance for therapy. Using cell-based high content imaging to survey clinically approved drugs in MPS-IIIA cells, we identified fluoxetine, a selective serotonin reuptake inhibitor. Fluoxetine increases lysosomal and autophagic functions via TFEB activation through a RagC-dependent mechanism. Mechanistically, fluoxetine increases lysosomal exocytosis in mouse embryonic fibroblasts from MPS-IIIA mice, suggesting that this process may be responsible for heparan sulfate clearance. In vivo, fluoxetine ameliorates somatic and brain pathology in a mouse model of MPS-IIIA by decreasing the accumulation of glycosaminoglycans and aggregated autophagic substrates, reducing inflammation, and slowing down cognitive deterioration. We repurposed fluoxetine for potential therapeutics to treat human MPS-IIIA disease.

Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype.

Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent-conjugated bacterial toxins to label Gb3 to develop a cell-based high content imaging (HCI) screening assay for the repurposing of FDA-approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient-derived induced pluripotent stem cells (iPSC). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease.

LC3 lipidation is essential for TFEB activation during the lysosomal damage response to kidney injury.

Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)-a master transcriptional regulator of lysosomal biogenesis and autophagy-is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation. Furthermore, we demonstrate the presence and importance of this TFEB activation mechanism in kidneys in a mouse model of oxalate nephropathy accompanying lysosomal damage. A proximal tubule-specific TFEB-knockout mouse exhibited progression of kidney injury induced by oxalate crystals. Together, our results reveal unexpected mechanisms of TFEB activation by LC3 lipidation and their physiological relevance during the lysosomal damage response.

A substrate-specific mTORC1 pathway underlies Birt-Hogg-Dubé syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1-3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB-unlike other substrates of mTORC1, such as S6K and 4E-BP1- is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt-Hogg-Dubé syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt-Hogg-Dubé syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer.

Role of Prx1-expressing skeletal cells and Prx1-expression in fracture repair.

The healing capacity of bones after fracture implies the existence of adult regenerative cells. However, information on identification and functional role of fracture-induced progenitors is still lacking. Paired-related homeobox 1 (Prx1) is expressed during skeletogenesis. We hypothesize that fracture recapitulates Prx1's expression, and Prx1 expressing cells are critical to induce repair. To address our hypothesis, we used a combination of in vivo and in vitro approaches, short and long-term cell tracking analyses of progenies and actively expressing cells, cell ablation studies, and rodent animal models for normal and defective fracture healing. We found that fracture elicits a periosteal and endosteal response of perivascular Prx1+ cells that participate in fracture healing and showed that Prx1-expressing cells have a functional role in the repair process. While Prx1-derived cells contribute to the callus, Prx1's expression decreases concurrently with differentiation into cartilaginous and bone cells, similarly to when Prx1+ cells are cultured in differentiating conditions. We determined that bone morphogenic protein 2 (BMP2), through C-X-C motif-ligand-12 (CXCL12) signaling, modulates the downregulation of Prx1. We demonstrated that fracture elicits an early increase in BMP2 expression, followed by a decrease in CXCL12 that in turn down-regulates Prx1, allowing cells to commit to osteochondrogenesis. In vivo and in vitro treatment with CXCR4 antagonist AMD3100 restored Prx1 expression by modulating the BMP2-CXCL12 axis. Our studies represent a shift in the current research that has primarily focused on the identification of markers for postnatal skeletal progenitors, and instead we characterized the function of a specific population (Prx1+ cells) and their expression marker (Prx1) as a crossroad in fracture repair. The identification of fracture-induced perivascular Prx1+ cells and regulation of Prx1's expression by BMP2 and in turn by CXCL12 in the orchestration of fracture repair, highlights a pathway in which to investigate defective mechanisms and therapeutic targets for fracture non-union.

TGF-ß type 2 receptor-mediated modulation of the IL-36 family can be therapeutically targeted in osteoarthritis.

Mechanisms that govern the shift from joint homeostasis to osteoarthritis (OA) remain unknown. Here, we identify a pathway used for joint development and homeostasis, and its role in OA. Using a combination of transgenic, pharmacological, and surgical conditions in mouse and human tissues, we found that TGF-ß signaling promotes joint homeostasis through regulation of the IL-36 family. We identified IL-36 receptor antagonist (IL-36 in mice and IL-36RN in humans) as a potential disease-modifying OA drug. Specifically, OA development was associated with IL-36α up-regulation and IL-36Ra down-regulation in mice with tissue-specific postnatally induced ablation of Tgfbr2, mice treated with a TGF-ß signaling inhibitor, mice with posttraumatic OA, and aging mice with naturally occurring OA. In human cartilage, OA severity was associated with decreased TGFBR2 and IL-36RN, whereas IL-36α increased. Functionally, intra-articular treatment with IL-36Ra attenuated OA development in mice, and IL-36RN reduced MMP13 in human OA chondrocytes. These findings highlight the relevance of TGFBR2-IL-36 interplay in joint homeostasis and IL-36RN as a potential therapeutic agent for OA.