Multi-Omic Analysis of Esophageal Adenocarcinoma Uncovers Candidate Therapeutic Targets and Cancer-Selective Posttranscriptional Regulation.

Efforts to address the poor prognosis associated with esophageal adenocarcinoma (EAC) have been hampered by a lack of biomarkers to identify early disease and therapeutic targets. Despite extensive efforts to understand the somatic mutations associated with EAC over the past decade, a gap remains in understanding how the atlas of genomic aberrations in this cancer impacts the proteome and which somatic variants are of importance for the disease phenotype. We performed a quantitative proteomic analysis of 23 EACs and matched adjacent normal esophageal and gastric tissues. We explored the correlation of transcript and protein abundance using tissue-matched RNA-seq and proteomic data from seven patients and further integrated these data with a cohort of EAC RNA-seq data (n = 264 patients), EAC whole-genome sequencing (n = 454 patients), and external published datasets. We quantified protein expression from 5879 genes in EAC and patient-matched normal tissues. Several biomarker candidates with EAC-selective expression were identified, including the transmembrane protein GPA33. We further verified the EAC-enriched expression of GPA33 in an external cohort of 115 patients and confirm this as an attractive diagnostic and therapeutic target. To further extend the insights gained from our proteomic data, an integrated analysis of protein and RNA expression in EAC and normal tissues revealed several genes with poorly correlated protein and RNA abundance, suggesting posttranscriptional regulation of protein expression. These outlier genes, including SLC25A30, TAOK2, and AGMAT, only rarely demonstrated somatic mutation, suggesting post-transcriptional drivers for this EAC-specific phenotype. AGMAT was demonstrated to be overexpressed at the protein level in EAC compared to adjacent normal tissues with an EAC-selective, post-transcriptional mechanism of regulation of protein abundance proposed. Integrated analysis of proteome, transcriptome, and genome in EAC has revealed several genes with tumor-selective, posttranscriptional regulation of protein expression, which may be an exploitable vulnerability.

Biochemical evidence for conformational variants in the anti-viral and pro-metastatic protein IFITM1.

Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.

Hydrogen deuterium exchange mass spectrometry identifies the dominant paratope in CD20 antigen binding to the NCD1.2 monoclonal antibody.

A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab.

The MDM2 ligand Nutlin-3 differentially alters expression of the immune blockade receptors PD-L1 and CD276.

BACKGROUND: The links between the p53/MDM2 pathway and the expression of pro-oncogenic immune inhibitory receptors in tumor cells are undefined. In this report, we evaluate whether there is p53 and/or MDM2 dependence in the expression of two key immune receptors, CD276 and PD-L1. METHODS: Proximity ligation assays were used to quantify protein-protein interactions in situ in response to Nutlin-3. A panel of p53-null melanoma cells was created using CRISPR-Cas9 guide RNA mediated genetic ablation. Flow cytometric analyses were used to assess the impact of TP53 or ATG5 gene ablation, as well as the effects of Nutlin-3 and an ATM inhibitor on cell surface PD-L1 and CD276. Targeted siRNA was used to deplete CD276 to assess changes in cell cycle parameters by flow cytometry. A T-cell proliferation assay was used to assess activity of CD4+ T-cells as a function of ATG5 genotype. RESULTS: CD276 forms protein-protein interactions with MDM2 in response to Nutlin-3, similar to the known MDM2 interactors p53 and HSP70. Isogenic HCT116 p53-wt/null cancer cells demonstrated that CD276 is induced on the cell surface by Nutlin-3 in a p53-dependent manner. PD-L1 was also unexpectedly induced by Nutlin-3, but PD-L1 does not bind MDM2. The ATM inhibitor KU55993 reduced the levels of PD-L1 under conditions where Nutlin-3 induces PD-L1, indicating that MDM2 and ATM have opposing effects on PD-L1 steady-state levels. PD-L1 is also up-regulated in response to genetic ablation of TP53 in A375 melanoma cell clones under conditions in which CD276 remains unaffected. A549 cells with a deletion in the ATG5 gene up-regulated only PD-L1, further indicating that PD-L1 and CD276 are under distinct genetic control. CONCLUSION: Genetic inactivation of TP53, or the use of the MDM2 ligand Nutlin-3, alters the expression of the immune blockade receptors PD-L1 and CD276. The biological function of elevated CD276 is to promote altered cell cycle progression in response to Nutlin-3, whilst the major effect of elevated PD-L1 is T-cell suppression. These data indicate that TP53 gene status, ATM and MDM2 influence PD-L1 and CD276 paralogs on the cell surface. These data have implications for the use of drugs that target the p53 pathway as modifiers of immune checkpoint receptor expression.

The Sequence-specific Peptide-binding Activity of the Protein Sulfide Isomerase AGR2 Directs Its Stable Binding to the Oncogenic Receptor EpCAM.

AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway.

Allosteric changes in HDM2 by the ATM phosphomimetic S395D mutation: implications on HDM2 function.

Allosteric changes imposed by post-translational modifications regulate and differentiate the functions of proteins with intrinsic disorder regions. HDM2 is a hub protein with a large interactome and with different cellular functions. It is best known for its regulation of the p53 tumour suppressor. Under normal cellular conditions, HDM2 ubiquitinates and degrades p53 by the 26S proteasome but after DNA damage, HDM2 switches from a negative to a positive regulator of p53 by binding to p53 mRNA to promote translation of the p53 mRNA. This change in activity is governed by the ataxia telangiectasia mutated kinase via phosphorylation on serine 395 and is mimicked by the S395D phosphomimetic mutant. Here we have used different approaches to show that this event is accompanied by a specific change in the HDM2 structure that affects the HDM2 interactome, such as the N-termini HDM2-p53 protein-protein interaction. These data will give a better understanding of how HDM2 switches from a negative to a positive regulator of p53 and gain new insights into the control of the HDM2 structure and its interactome under different cellular conditions and help identify interphases as potential targets for new drug developments.

Microgradient separation technique for purification and fractionation of permethylated N-glycans before mass spectrometric analyses.

Analysis of N-glycans released enzymatically from patients' sera or other clinical samples may provide diagnostically and prognostically important information on human disease. Permethylation of these biomolecules simultaneously increases their hydrophobicity and substantially improves their detection parameters in the following mass spectrometric analyses. The overall procedure, from the glycan cleavage to the final mass spectrometric determinations, includes several steps involving extraction, derivatization, and purification. During these steps, certain polymeric contaminants that may have been coincidentally introduced could hamper the final measurements. To understand and counter these interferences and further fractionate or preconcentrate these glycans, we introduce here an effective microgradient chromatographic technique that employs a small reversed-phase microcolumn connected to a gas-tight microsyringe delivering a mobile-phase gradient. After loading the glycan fraction onto the microcolumn, three elution steps are recommended: (1) remove polar contaminants; (2) recover permethylated glycans for either liquid chromatography with electrospray ionization mass spectrometry or matrix-assisted laser desorption/ionization mass spectrometry; and (3) remove larger polymeric contaminants and regenerate the precolumn. We further demonstrate that the trapped second fraction can be beneficially preconcentrated and further separated to achieve matrix-assisted laser desorption/ionization mass spectrometric detection of the derivatized N-glycans up to 6300 Da. The enhanced detection capabilities for tetra-antennary N-glycans are of increasing interest in disease biomarker discovery.

Novel Entropically Driven Conformation-specific Interactions with Tomm34 Protein Modulate Hsp70 Protein Folding and ATPase Activities.

Co-chaperones containing tetratricopeptide repeat (TPR) domains enable cooperation between Hsp70 and Hsp90 to maintain cellular proteostasis. Although the details of the molecular interactions between some TPR domains and heat shock proteins are known, we describe a novel mechanism by which Tomm34 interacts with and coordinates Hsp70 activities. In contrast to the previously defined Hsp70/Hsp90-organizing protein (Hop), Tomm34 interaction is dependent on the Hsp70 chaperone cycle. Tomm34 binds Hsp70 in a complex process; anchorage of the Hsp70 C terminus by the TPR1 domain is accompanied by additional contacts formed exclusively in the ATP-bound state of Hsp70 resulting in a high affinity entropically driven interaction. Tomm34 induces structural changes in determinants within the Hsp70-lid subdomain and modulates Hsp70/Hsp40-mediated refolding and Hsp40-stimulated Hsp70 ATPase activity. Because Tomm34 recruits Hsp90 through its TPR2 domain, we propose a model in which Tomm34 enables Hsp70/Hsp90 scaffolding and influences the Hsp70 chaperone cycle, providing an additional role for co-chaperones that contain multiple TPR domains in regulating protein homeostasis.

ΔNp63 activates EGFR signaling to induce loss of adhesion in triple-negative basal-like breast cancer cells.

PURPOSE: The basal-A subtype of triple-negative breast cancer is characterized by high levels of ΔNp63. Various functions have been proposed for p63 in breast cancer initiation and growth, and p63 mediates chemotherapeutic response in a subset of triple-negative breast cancers. We investigated the signaling pathways that are controlled by ΔNp63 in basal-A triple-negative breast cancer. METHODS: Human basal-A triple-negative breast cancer cell lines with ΔNp63α induction or inhibition were studied, along with primary human triple-negative breast cancer tissues. Proteomic, phospho-kinase array, mRNA measurements, and immunohistochemistry were employed. RESULTS: Global phosphoproteomics identified increased EGFR phosphorylation in MDA-MB-468 cells expressing ΔNp63α. ΔNp63α expression increased EGFR mRNA, total EGFR protein, and phospho-EGFR(Y1086), whereas silencing endogenous ΔNp63 in HCC1806 cells reduced both total and phospho-EGFR levels and inhibited the ability of EGF to activate EGFR. EGFR pathway gene expression analysis indicated that ΔNp63 alters EGFR-regulated genes involved in cell adhesion, migration, and angiogenesis. Addition of EGF or neutralizing EGFR antibodies demonstrated that EGFR activation is responsible for ΔNp63-mediated loss of cellular adhesion. Finally, immunohistochemical staining showed that p63-positive triple-negative breast cancers were more likely to express high levels of EGFR than p63-negative cancers, corroborated by in silico analysis of gene expression profiling data. CONCLUSIONS: These data identify EGFR as a major target for ΔNp63 regulation that influences cancer cell adhesion in basal-like triple-negative breast cancer.

Protein-Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP).

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control.

The assembly and intermolecular properties of the Hsp70-Tomm34-Hsp90 molecular chaperone complex.

Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.

Quantitative proteomic profiling of pleomorphic human sarcoma identifies CLIC1 as a dominant pro-oncogenic receptor expressed in diverse sarcoma types.

Sarcomas are rare forms of cancer with a high unmet clinical need that develop in connective tissue, such as muscle, bone, nerves, cartilage, and fat. The outcome for patients is poor, with surgery and postoperative radiotherapy the standard treatment for patients. A better understanding of the molecular pathology of sarcoma may allow for the development of novel therapeutics. There are dozens of sarcoma subtypes where there is a need for targetted therapeutics, with the most commonly studied including Ewing's sarcoma and osteosarcoma. Here we initiate a proteomics-based target-discovery program to define "dominant" pro-oncogenic signaling targets in the most common sarcoma in adults: high-grade pleiomorphic soft tissue sarcoma. We have carried out a proteome screen using tandem mass tag isobaric labeling on three high-grade undifferentiated pleomorphic sarcoma biopsies from different tissue sites. We identified the commonly dysregulated proteins within the three sarcomas and further validated the most penetrant receptor as CLIC1, using immunohistochemistry arising from two different population cohorts representing over 300 patients. The dominant expression of CLIC1 in a broad range of human sarcomas suggests that studying this relatively unexplored signaling pathway might provide new insights into disease mechanism and facilitate the development of new CLIC1 targeted therapeutics.

Characterization of protein glycosylation in Francisella tularensis subsp. holarctica: identification of a novel glycosylated lipoprotein required for virulence.

FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways.

N-glycan profiling of tissue samples to aid breast cancer subtyping.

Breast cancer is a highly heterogeneous disease. Its intrinsic subtype classification for diagnosis and choice of therapy traditionally relies on the presence of characteristic receptors. Unfortunately, this classification is often not sufficient for precise prediction of disease prognosis and treatment efficacy. The N-glycan profiles of 145 tumors and 10 healthy breast tissues were determined using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. The tumor samples were classified into Mucinous, Lobular, No-Special-Type, Human Epidermal Growth Factor 2 + , and Triple-Negative Breast Cancer subtypes. Statistical analysis was conducted using the reproducibility-optimized test statistic software package in R, and the Wilcoxon rank sum test with continuity correction. In total, 92 N-glycans were detected and quantified, with 59 consistently observed in over half of the samples. Significant variations in N-glycan signals were found among subtypes. Mucinous tumor samples exhibited the most distinct changes, with 28 significantly altered N-glycan signals. Increased levels of tri- and tetra-antennary N-glycans were notably present in this subtype. Triple-Negative Breast Cancer showed more N-glycans with additional mannose units, a factor associated with cancer progression. Individual N-glycans differentiated Human Epidermal Growth Factor 2 + , No-Special-Type, and Lobular cancers, whereas lower fucosylation and branching levels were found in N-glycans significantly increased in Luminal subtypes (Lobular and No-Special-Type tumors). Clinically normal breast tissues featured a higher abundance of signals corresponding to N-glycans with bisecting moiety. This research confirms that histologically distinct breast cancer subtypes have a quantitatively unique set of N-glycans linked to clinical parameters like tumor size, proliferative rate, lymphovascular invasion, and metastases to lymph nodes. The presented results provide novel information that N-glycan profiling could accurately classify human breast cancer samples, offer stratification of patients, and ongoing disease monitoring.

Metaproteomic analysis from cervical biopsies and cytologies identifies proteinaceous biomarkers representing both human and microbial species.

The detection of HPV infection and microbial colonization in cervical lesions is currently done through PCR-based viral or bacterial DNA amplification. Our objective was to develop a methodology to expand the metaproteomic landscape of cervical disease and determine if protein biomarkers from both human and microbes could be detected in distinct cervical samples. This would lead to the development of multi-species proteomics, which includes protein-based lateral flow diagnostics that can define patterns of microbes and/or human proteins relevant to disease status. In this study, we collected both non-frozen tissue biopsy and exfoliative non-fixed cytology samples to assess the consistency of detecting human proteomic signatures between the cytology and biopsy samples. Our results show that proteomics using biopsies or cytologies can detect both human and microbial organisms. Across patients, Lumican and Galectin-1 were most highly expressed human proteins in the tissue biopsy, whilst IL-36 and IL-1RA were most highly expressed human proteins in the cytology. We also used mass spectrometry to assess microbial proteomes known to reside based on prior 16S rRNA gene signatures. Lactobacillus spp. was the most highly expressed proteome in patient samples and specific abundant Lactobacillus proteins were identified. These methodological approaches can be used in future metaproteomic clinical studies to interrogate the vaginal human and microbiome structure and metabolic diversity in cytologies or biopsies from the same patients who have pre-invasive cervical intraepithelial neoplasia, invasive cervical cancer, as well as in healthy controls to assess how human and pathogenic proteins may correlate with disease presence and severity.

Identification of a second Nutlin-3 responsive interaction site in the N-terminal domain of MDM2 using hydrogen/deuterium exchange mass spectrometry.

MDM2 is a multidomain protein that functions as an E3 ubiquitin ligase, transcription repressor, mRNA-binding protein, translation factor, and molecular chaperone. The small molecule Nutlin-3 has been engineered to bind to the N-terminal hydrophobic pocket domain of MDM2. This binding of Nutlin-3 has two consequences: (i) antagonistic effects through competitive disruption of the MDM2-p53 complex and (ii) agonist effects that allosterically stabilize MDM2 protein-protein interactions that increase p53 ubiquitination as well as nucleophosmin deoligomerization. We present a methodology using a hydrogen/deuterium (H/D) exchange platform that measures Nutlin-3 binding to the N-terminal domain of MDM2 (MDM2(1-126)) in order to begin to develop dynamic assays that evaluate MDM2 allostery. In order to localize the regions in MDM2 being suppressed by Nutlin-3, MDM2 was incubated with the ligand and H/D amide exchange was measured after pepsin digestion. One dynamic segment containing amino acids 55-60 exhibited slower deuterium exchange after Nutlin-3 binding, reflecting ligand binding within the hydrophobic pocket. However, another dominant suppression of H/D exchange was observed in a motif from amino acids 103-107 that reflects surface hydrophobic residues surrounding the hydrophobic pocket of MDM2. In order to explore the consequences of this latter Nutlin-3 interaction site on MDM2, the Y104G and L107G mutant series was constructed. The MDM2(Y104G) and MDM2(L107G) mutants were fully active in p53 binding. However, the authentic p53-derived peptide:MDM2(Y104G) complex exhibited partial resistance to Nutlin-3 inhibition, while the p53-mimetic 12.1 peptide:MDM2(Y104G) complex retained normal Nutlin-3 responsiveness. These data reveal the existence of a second functional Nutlin-3-binding site in a surface hydrophobic patch of MDM2, flanking the hydrophobic pocket. This reveals two modes of peptide binding by MDM2 and highlights the utility of H/D exchange as an assay for measuring allosteric effects in MDM2.

Study of Protein Conformational Dynamics Using Hydrogen/Deuterium Exchange Mass Spectrometry.

Intrinsic protein dynamics contribute to their biological functions. Rational engineering of protein dynamics is extremely challenging with only a handful of successful examples. Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) represents a powerful technique for quantitative analysis of protein dynamics. Here we provide a detailed description of the preparation of protein samples, collection of high-quality data, and their in-depth analysis using various computational tools. We illustrate the application of HDX-MS for the study of protein dynamics in the rational engineering of flexible loops in the reconstructed ancestor of haloalkane dehalogenase and Renilla luciferase. These experiments provided unique and valuable data rigorously describing the modification of protein dynamics upon grafting of the loop-helix element. Tips and tricks are provided to stimulate the wider use of HDX-MS to study and engineer protein dynamics.

Identification, characterization, and engineering of glycosylation in thrombolytics.

Cardiovascular diseases, such as myocardial infarction, ischemic stroke, and pulmonary embolism, are the most common causes of disability and death worldwide. Blood clot hydrolysis by thrombolytic enzymes and thrombectomy are key clinical interventions. The most widely used thrombolytic enzyme is alteplase, which has been used in clinical practice since 1986. Another clinically used thrombolytic protein is tenecteplase, which has modified epitopes and engineered glycosylation sites, suggesting that carbohydrate modification in thrombolytic enzymes is a viable strategy for their improvement. This comprehensive review summarizes current knowledge on computational and experimental identification of glycosylation sites and glycan identity, together with methods used for their reengineering. Practical examples from previous studies focus on modification of glycosylations in thrombolytics, e.g., alteplase, tenecteplase, reteplase, urokinase, saruplase, and desmoteplase. Collected clinical data on these glycoproteins demonstrate the great potential of this engineering strategy. Outstanding combinatorics originating from multiple glycosylation sites and the vast variety of covalently attached glycan species can be addressed by directed evolution or rational design. Directed evolution pipelines would benefit from more efficient cell-free expression and high-throughput screening assays, while rational design must employ structure prediction by machine learning and in silico characterization by supercomputing. Perspectives on challenges and opportunities for improvement of thrombolytic enzymes by engineering and evolution of protein glycosylation are provided.

The risk of second primary malignancies in colorectal cancer patients using calcium channel blockers.

Calcium channel blockers are among the most commonly used agents in the treatment of cardiovascular diseases. There are several known side-effects associated with their long-term use, whereas other potential adverse effects are yet to be proven. This study aims to evaluate the association between calcium channel blockers exposure and the incidence of second primary malignancy. We established a cohort of 1401 patients with colorectal cancer diagnosed in our institution between January 2003 and December 2016. Patients were followed-up until December 2020. The tumor characteristics and basic clinical data including medication information were obtained from the hospital information system database. Second malignancy was detected in 301 patients (21.5%), and occurred in 27.8% of patients who used calcium channel blockers compared to only 19.9% among non-users. Their use was associated with an increased incidence of bladder cancer in particular. Subanalysis of patients with second malignancy displayed a higher proportion of right-sided colon cancer compared to rectal carcinoma in non-users. Survival analysis revealed significantly better outcomes in early-stage colorectal cancer patients without a history of calcium channel blockers treatment or second primary malignancy.

Domino-like effect of C112R mutation on ApoE4 aggregation and its reduction by Alzheimer's Disease drug candidate.

BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most prevalent risk factor for late-onset Alzheimer's Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. METHODS: Here, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE ε3/ε3 and ε4/ε4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. RESULTS: We found that C112R substitution in ApoE4 induces long-distance (> 15 Å) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE ε4/ε4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. CONCLUSIONS: Our results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.