Abstract
Chemoproteomics is a key technology to characterize the mode of action of drugs, as it directly identifies the protein targets of bioactive compounds and aids in the development of optimized ...small-molecule compounds. Current approaches cannot identify the protein targets of a compound and also detect the interaction surfaces between ligands and protein targets without prior labeling or modification. To address this limitation, we here develop LiP-Quant, a drug target deconvolution pipeline based on limited proteolysis coupled with mass spectrometry that works across species, including in human cells. We use machine learning to discern features indicative of drug binding and integrate them into a single score to identify protein targets of small molecules and approximate their binding sites. We demonstrate drug target identification across compound classes, including drugs targeting kinases, phosphatases and membrane proteins. LiP-Quant estimates the half maximal effective concentration of compound binding sites in whole cell lysates, correctly discriminating drug binding to homologous proteins and identifying the so far unknown targets of a fungicide research compound.
The rise of proteome‐wide biophysics Mateus, Andre; Savitski, Mikhail M; Piazza, Ilaria
Molecular systems biology,
July 2021, Letnik:
17, Številka:
7
Journal Article
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Odprti dostop
While informative, protein amounts and physical protein associations do not provide a full picture of protein function. This Commentary highlights the potential of structural and stability proteomic ...technologies to derive new insights in biology and medicine.
Cells respond to stress by blocking translation, rewiring metabolism and forming transient messenger ribonucleoprotein assemblies called stress granules (SGs). After stress release, re-establishing ...homeostasis and disassembling SGs requires ATP-consuming processes. However, the molecular mechanisms whereby cells restore ATP production and disassemble SGs after stress remain poorly understood. Here we show that upon stress, the ATP-producing enzyme Cdc19 forms inactive amyloids, and that their rapid re-solubilization is essential to restore ATP production and disassemble SGs in glucose-containing media. Cdc19 re-solubilization is initiated by the glycolytic metabolite fructose-1,6-bisphosphate, which directly binds Cdc19 amyloids, allowing Hsp104 and Ssa2 chaperone recruitment and aggregate re-solubilization. Fructose-1,6-bisphosphate then promotes Cdc19 tetramerization, which boosts its activity to further enhance ATP production and SG disassembly. Together, these results describe a molecular mechanism that is critical for stress recovery and directly couples cellular metabolism with SG dynamics via the regulation of reversible Cdc19 amyloids.
Obesity is associated with increased risk and worse prognosis of many tumours including those of the breast and of the esophagus. Adipokines released from the peritumoural adipose tissue promote the ...metastatic potential of cancer cells, suggesting the existence of a crosstalk between the adipose tissue and the surrounding tumour. Mitochondrial Ca2+ signaling contributes to the progression of carcinoma of different origins. However, whether adipocyte-derived factors modulate mitochondrial Ca2+ signaling in tumours is unknown. Here, we show that conditioned media derived from adipose tissue cultures (ADCM) enriched in precursor cells impinge on mitochondrial Ca2+ homeostasis of target cells. Moreover, in modulating mitochondrial Ca2+ responses, a univocal crosstalk exists between visceral adipose tissue-derived preadipocytes and esophageal cancer cells, and between subcutaneous adipose tissue-derived preadipocytes and triple-negative breast cancer cells. An unbiased metabolomic analysis of ADCM identified creatine and creatinine for their ability to modulate mitochondrial Ca2+ uptake, migration and proliferation of esophageal and breast tumour cells, respectively.
Homologous recombination (HR) is essential for high-fidelity DNA repair during mitotic proliferation and meiosis. Yet, context-specific modifications must tailor the recombination machinery to avoid ...(mitosis) or enforce (meiosis) the formation of reciprocal exchanges—crossovers—between recombining chromosomes. To obtain molecular insight into how crossover control is achieved, we affinity purified 7 DNA-processing enzymes that channel HR intermediates into crossovers or noncrossovers from vegetative cells or cells undergoing meiosis. Using mass spectrometry, we provide a global characterization of their composition and reveal mitosis- and meiosis-specific modules in the interaction networks. Functional analyses of meiosis-specific interactors of MutLγ-Exo1 identified Rtk1, Caf120, and Chd1 as regulators of crossing-over. Chd1, which transiently associates with Exo1 at the prophase-to-metaphase I transition, enables the formation of MutLγ-dependent crossovers through its conserved ability to bind and displace nucleosomes. Thus, rewiring of the HR network, coupled to chromatin remodeling, promotes context-specific control of the recombination outcome.
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•Affinity proteomics reveals the composition and interaction landscape of HR enzymes•Interaction network rewiring of HR enzymes during mitotic proliferation and meiosis•Chd1, Rtk1, and Caf120 regulate meiotic crossing-over•Chd1 remodels chromatin to enable formation of MutLγ-Exo1-dependent crossovers
Wild et al. used affinity proteomics to characterize the composition and interaction landscape of 7 DNA repair enzymes during mitotic proliferation and meiosis. They report a concerted and context-specific rewiring of the interactomes and reveal meiosis-specific network components with roles in crossing-over.
Transcriptional memory, by which cells respond faster to repeated stimuli, is key for cellular adaptation and organism survival. Chromatin organization has been shown to play a role in the faster ...response of primed cells. However, the contribution of post-transcriptional regulation is not yet explored. Here we perform a genome-wide screen to identify novel factors modulating transcriptional memory in S. cerevisiae in response to galactose. We find that depletion of the nuclear RNA exosome increases GAL1 expression in primed cells. Our work shows that gene-specific differences in intrinsic nuclear surveillance factor association can enhance both gene induction and repression in primed cells. Finally, we show that primed cells present altered levels of RNA degradation machinery and that both nuclear and cytoplasmic mRNA decay modulate transcriptional memory. Our results demonstrate that mRNA post-transcriptional regulation, and not only transcription regulation, should be considered when investigating gene expression memory.
MitoK
is a channel of the inner mitochondrial membrane that controls mitochondrial K
influx according to ATP availability. Recently, the genes encoding the pore-forming (MITOK) and the regulatory ...ATP-sensitive (MITOSUR) subunits of mitoK
were identified, allowing the genetic manipulation of the channel. Here, we analyzed the role of mitoK
in determining skeletal muscle structure and activity. Mitok
muscles were characterized by mitochondrial cristae remodeling and defective oxidative metabolism, with consequent impairment of exercise performance and altered response to damaging muscle contractions. On the other hand, constitutive mitochondrial K
influx by MITOK overexpression in the skeletal muscle triggered overt mitochondrial dysfunction and energy default, increased protein polyubiquitination, aberrant autophagy flux, and induction of a stress response program. MITOK overexpressing muscles were therefore severely atrophic. Thus, the proper modulation of mitoK
activity is required for the maintenance of skeletal muscle homeostasis and function.
Few studies have compared the efficacy of switching from etanercept to adalimumab in the real-life setting in rheumatoid arthritis (RA) and psoriatic arthritis (PsA). This study evaluated the 2-year ...retention rate and 12-month efficacy of adalimumab in RA and PsA patients, previously treated with etanercept. RA and PsA patients from 11 Italian Rheumatology Units received adalimumab after first-line etanercept failure. Two-year adalimumab retention rate was calculated by the Kaplan–Meier method and Cox proportional hazard models were developed to examine predictors of drug persistence. Univariate and multivariate logistic regression analyses were developed to examine potential predictors of 12-month DAS-28 remission. The study population included 117 RA (disease duration of 10.1 ± 7.7 years and baseline DAS28-ESR of 4.97 ± 1.3) and 102 PsA (disease duration of 7.1 ± 5.1 years and baseline DAPSA of 24.6 ± 11.8). The 2-year retention rate was 48.2% in RA and 56.5% in PsA patients. Concomitant methotrexate treatment was not associated with increased drug survival in both groups. Similarly, cause of etanercept discontinuation or treatment duration was not associated with retention rate. 12-month remission and low disease activity were achieved, respectively, in 27.3% and 23.9% of RA patients and 27.4% and 23.5% PsA of patients. In multivariate models, etanercept discontinuation due to inefficacy (OR 0.27, 95% CI 1.03–0.73;
p
= 0.009) and baseline DAS-28 (OR 0.45, 95% CI 0.29–0.69;
p
< 0.001) remained significant negative predictors of remission in RA patients. No variable was associated with remission in PsA patients. Adalimumab after etanercept failure was highly effective and safe in both RA and PsA patients.
Anakinra (ANA) is an effective treatment choice in patients with adult onset Still's disease (AOSD). Variables affecting treatment survival include loss of efficacy or adverse events, but also the ...decision to discontinue treatment after long-term clinical remission.
Aims of this study were: (i) to assess the drug retention rate (DRR) of ANA during a long-term follow-up looking for any difference related to the line of biologic treatment, the concomitant use of conventional disease modifying anti-rheumatic drugs (cDMARDs) and the different type of AOSD (systemic versus chronic articular); (ii) to identify predictive factors of lack of efficacy, loss of efficacy, and ANA withdrawal owing to long-term remission.
AOSD patients classified according with Yamaguchi criteria and treated with ANA were retrospectively enrolled in 18 Italian tertiary Centers. Demographic, laboratory, clinical and therapeutic data related to the start of ANA (
), the 3-month assessment and the last follow-up visit while on ANA treatment were retrospectively collected and statistically analyzed.
One hundred and forty-one AOSD patients (48 males, 93 females) treated with ANA for a mean period of 35.96 ± 36.05 months were enrolled. The overall DRR of ANA was 44.6 and 30.5% at the 60- and 120-month assessments, respectively, with no significant differences between: (i) biologic naïve patients and those previously treated with other biologics (log-rank
= 0.97); (ii) monotherapy and concomitant use of cDMARDs (log-rank
= 0.45); (iii) systemic and chronic articular types of AOSD (log-rank
= 0.67). No variables collected at
could predict primary inefficacy, while the number of swollen joints at baseline was significantly associated with secondary inefficacy (
= 0.01, OR = 1.194, C.I. 1.043-1.367). The typical AOSD skin rash was negatively related with ANA withdrawal owing to long-term remission (
= 0.03, OR = 0.224, C.I. 0.058-0.863).
Long-term DRR of ANA has been found excellent and is not affected by different lines of biologic treatment, concomitant use of cDMARDs, or type of AOSD. The risk of losing ANA efficacy increases along with the number of swollen joints at the start of therapy, while the typical skin rash is a negative predictor of ANA withdrawal related to sustained remission.
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