De Novo Proteome Analysis of Genetically Modified Tumor Cells By a Metabolic Labeling/Azide-alkyne Cycloaddition Approach

Activin receptor type II (ACVR2) is a member of the transforming growth factor type II receptor family and controls cell growth and differentiation, thereby acting as a tumor suppressor. ACVR2 inactivation is known to drive colorectal tumorigenesis. We used an ACVR2-deficient microsatellite unstable colon cancer cell line (HCT116) to set up a novel experimental design for comprehensive analysis of proteomic changes associated with such functional loss of a tumor suppressor. To this end we combined two existing technologies. First, the ACVR2 gene was reconstituted in an ACVR2-deficient colorectal cancer (CRC) cell line by means of recombinase-mediated cassette exchange, resulting in the generation of an inducible expression system that allowed the regulation of ACVR2 gene expression in a doxycycline-dependent manner. Functional expression in the induced cells was explicitly proven. Second, we used the methionine analog azidohomoalanine for metabolic labeling of newly synthesized proteins in our cell line model. Labeled proteins were tagged with biotin via a Click-iT chemistry approach enabling specific extraction of labeled proteins by streptavidin-coated beads. Tryptic on-bead digestion of captured proteins and subsequent ultra-high-performance LC coupled to LTQ Orbitrap XL mass spectrometry identified 513 proteins, with 25 of them differentially expressed between ACVR2-deficient and -proficient cells. Among these, several candidates that had already been linked to colorectal cancer or were known to play a key role in cell growth or apoptosis control were identified, proving the utility of the presented experimental approach. In principle, this strategy can be adapted to analyze any gene of interest and its effect on the cellular de novo proteome.Human tumors acquire a large number of genetic and epigenetic alterations that arise during progression from preneoplastic lesions to metastatic disease. However, the diversity of these alterations reflects the intratumoral heterogeneity and represents the genomic landscape of tumors. Among a high background number of irrelevant passenger alterations, only a limited number of genetic alterations are considered to be driving events that confer a selective advantage to tumor cells. Major signaling pathways affected by such driver mutations include the TGFβ, BMP, Activin, Wnt, and Notch pathways, abrogating normal regulation of key cellular processes such as cell fate, cell survival, and genome maintenance.Both tumor-relevant driver mutations in a major signaling receptor and tumor-irrelevant passenger mutations can cause changes at the proteomic level. Passenger-mutation-associated proteomic patterns are propagated randomly and do not represent generic tumor-associated changes (1). Therefore, a focus on proteome alterations associated with single driver mutations is necessary in order for specific changes that underlie tumor development to be identified. However, such analyses encounter two major limitations at different levels.At the molecular level, the genetic heterogeneity of tumors—especially those of the microsatellite unstable and mutator phenotype—poses a significant problem in determining mutation-specific effects. Two principal strategies for detecting cellular consequences of a single mutation have been applied. First, targeted gene knock-out in target-gene-proficient cell lines by means of homologous recombination, adeno-associated viral delivery, or zinc finger nucleases has been used successfully (2–4). However, these approaches are often limited by their low efficiency, are laborious and time-consuming, and bear the potential for confounding off-target effects. Second, transfer of the target gene into deficient cell lines via gene insertion or gene targeting methods has been extensively applied. Unfortunately, insertion methods are often affected by random insertion, a variable number of integrated gene copies per cell, and inconsistent integration sites, eventually resulting in unpredictable expression patterns (5). However, many non-integrating vectors, such as adenoviral DNA, are not often replicated during cell division, which limits their use in basic research.At the protein level, sample complexity is a major limiting factor. In addition to prefractionation methods, metabolic labeling is a versatile tool in work focusing on proteomic changes induced by gene activation. Because the activation of tumor suppressor pathways directly regulates target gene expression, analysis of tumor-suppressor-dependent alterations of newly synthesized proteins via metabolic labeling is a reasonable approach for restricting proteomic complexity. Conventional methods for metabolic labeling usually rely on amino acids containing either radioactive or stable isotopes. Although radioactive labeling enables extremely sensitive detection methods, its use for proteomic analysis is limited because of the need for special handling and precautions against contamination of the analytical instrumentation. Stable isotopic labeling, in particular the SILAC methodology, is currently the preferred method for most metabolic labeling approaches in proteomic analyses, and especially for cell lines (6). However, when applying the SILAC technology, mass spectrometric detection of labeled peptides has to be conducted in the presence of numerous irrelevant, unlabeled peptides, which hampers the detection of labeled low-abundance peptides. A relatively new method, termed Click-iT labeling, that enables labeling of nascent proteins comparable to that by a radioactive compound can overcome this problem, because upon incorporation of the labeled compound a handle for specific extraction of the labeled protein is worked in. The click reaction makes use of a methionine derivative that is functionalized with an azide (l-azidohomoalanine (AHA)).¹ During protein synthesis AHA is incorporated into proteins, which can then be tagged with a biotin alkyne (PEG4 carboxamide-propargyl biotin), resulting in the specific biotinylation of the metabolically labeled proteins (7). The final step is extraction of the labeled proteins by streptavidin beads.In the present study we pursued a completely new strategy to determine proteomic changes caused by a tumor-specific mutation in a major signaling pathway exemplified by the activin receptor type II (ACVR2) tumor suppressor. Disruption of activin signaling is a frequent event during colorectal tumorigenesis and can be caused by different molecular mechanisms. In colon tumors with chromosomal instability but microsatellite stability, ACVR2 inactivation mainly occurs via epigenetic mechanisms, whereas in colon cancers with a high level of microsatellite instability (MSI) loss of ACVR2 emerges from frameshift mutation (8). MSI has been defined as a change in length (insertions/deletions) of small repetitive DNA sequences (microsatellites) that arises specifically in tumor cells with impaired DNA mismatch repair functions (mutator phenotype) but not in corresponding normal tissue (9, 10). The A8 coding microsatellite in exon 10 of the ACVR2 gene has been identified as one of the most frequent mutation targets in high-MSI colorectal tumors that abrogates normal ACVR2 protein expression (11–13). As a consequence, phosphorylation of downstream signaling mediators such as Smad2/3, as well as regulation of several target genes such as SERPINE, SMAD7, and MYC, is significantly impaired, and this contributes to MSI colorectal tumorigenesis.Our strategy relied on a combination of two established technologies. First, we employed a recombination-mediated genomic targeting approach (14–16) to generate a genetically modified MSI colorectal tumor cell line that enabled doxycycline-inducible and reversible expression of an ACVR2 transgene and activation of ligand-dependent signaling in an isogenic background. Second, we performed metabolic labeling via a click-chemistry approach (7) in this MSI cell line to uncover alterations in the pattern of newly synthesized proteins (de novo proteome). In particular, we determined the constituents of the total cellular de novo proteome and subsets derived thereof that were regulated in an ACVR2-dependent manner. Our combinatorial strategy represents a versatile approach allowing one to overcome the limitations inherent in the genetic heterogeneity and proteomic complexity of MSI tumor cells. Moreover, this model system allows one to address any tumor target gene and thereby possesses broad applicability for studies of cancer proteome complexity, which is the logical starting point for identifying diagnostic biomarkers and therapeutic targets for cancer.     

Structure based discovery of small molecules to regulate the activity of human insulin degrading enzyme

Background

Insulin-degrading enzyme (IDE) is an allosteric Zn⁺² metalloprotease involved in the degradation of many peptides including amyloid-β, and insulin that play key roles in Alzheimer''s disease (AD) and type 2 diabetes mellitus (T2DM), respectively. Therefore, the use of therapeutic agents that regulate the activity of IDE would be a viable approach towards generating pharmaceutical treatments for these diseases. Crystal structure of IDE revealed that N-terminal has an exosite which is ∼30 Å away from the catalytic region and serves as a regulation site by orientation of the substrates of IDE to the catalytic site. It is possible to find small molecules that bind to the exosite of IDE and enhance its proteolytic activity towards different substrates.

Methodology/Principal Findings

In this study, we applied structure based drug design method combined with experimental methods to discover four novel molecules that enhance the activity of human IDE. The novel compounds, designated as D3, D4, D6, and D10 enhanced IDE mediated proteolysis of substrate V, insulin and amyloid-β, while enhanced degradation profiles were obtained towards substrate V and insulin in the presence of D10 only.

Conclusion/Significance

This paper describes the first examples of a computer-aided discovery of IDE regulators, showing that in vitro and in vivo activation of this important enzyme with small molecules is possible.     

Biogenesis of cbb3-type cytochrome c oxidase in Rhodobacter capsulatus

The cbb₃-type cytochrome c oxidases (cbb₃-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa₃-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixation. In addition, they are prevalent in many pathogenic bacteria, and important for colonizing low oxygen tissues. Studies related to cbb₃-Cox provide a fascinating paradigm for the biogenesis of sophisticated oligomeric membrane proteins. Complex subunit maturation and assembly machineries, producing the c-type cytochromes and the binuclear heme b₃-Cu_B center, have to be coordinated precisely both temporally and spatially to yield a functional cbb₃-Cox enzyme. In this review we summarize our current knowledge on the structure, regulation and assembly of cbb₃-Cox, and provide a highly tentative model for cbb₃-Cox assembly and formation of its heme b₃-Cu_B binuclear center. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Daphnia hybridization along ecological gradients in pelagic environments: the potential for the presence of hybrid zones in plankton

The relative homogeneity of pelagic environments has been regarded as the reason for the absence of hybrid zones for hybridizing planktonic Daphnia (Crustacea: Cladocera); occasional dominance of interspecific hybrids over parental species was explained by their temporal superiority in fluctuating environments. However, water bodies with spatially varying environmental conditions might facilitate the formation of hybrid zones in plankton. We studied the distribution of species and hybrids of the Daphnia longispina complex in 11 canyon-shaped reservoirs, localities characterized by horizontal environmental gradients (particularly of food supply and size-selective predation); we also analysed patterns of carapace size and fecundity among coexisting taxa. Spatial distribution of taxa agreed with their ecological characteristics; those showing different affinities along longitudinal reservoir profiles differed in size according to the presumed fish predation gradient. Only hybrids of Daphnia galeata with Daphnia cucullata and D. longispina (=hyalina) were recorded. The latter two species preferred opposite ends of gradients, such spatial segregation probably explaining the absence of their hybrids. Distributional patterns were relatively stable in two consecutive summers, apart from a substantial decline of D. galeata X cucullata in the second year. The observed pattern of a hybrid-dominated zone in intermediate conditions suggests that local Daphnia hybrid zones may indeed form within reservoirs.     

Immobilization of tyrosinase on chitosan-clay composite beads

Tyrosinase was immobilized on glutaraldehyde crosslinked chitosan-clay composite beads and used for phenol removal. Immobilization yield, loading efficiency and activity of tyrosinase immobilized beads were found as 67%, 25% and 1400 U/g beads respectively. Optimum pH of the free and immobilized enzyme was found as pH 7.0. Optimum temperature of the free and immobilized enzyme was determined as 25-30 °C and 25 °C respectively. The kinetic parameters of free and immobilized tyrosinase were calculated using l-catechol as a substrate and K(m) value for free and immobilized tyrosinase were found as 0.93 mM and 1.7 mM respectively. After seven times of repeated tests, each over 150 min, the efficiency of phenol removal using same immobilized tyrosinase beads were decreased to 43%.     

Candidate diagnostic biomarkers for neurodevelopmental disorders in children and adolescents: a systematic review

Neurodevelopmental disorders – including attention-deficit/hyperactivity disorder (ADHD), autism spectrum disorder, communication disorders, intellectual disability, motor disorders, specific learning disorders, and tic disorders – manifest themselves early in development. Valid, reliable and broadly usable biomarkers supporting a timely diagnosis of these disorders would be highly relevant from a clinical and public health standpoint. We conducted the first systematic review of studies on candidate diagnostic biomarkers for these disorders in children and adolescents. We searched Medline and Embase + Embase Classic with terms relating to biomarkers until April 6, 2022, and conducted additional targeted searches for genome-wide association studies (GWAS) and neuroimaging or neurophysiological studies carried out by international consortia. We considered a candidate biomarker as promising if it was reported in at least two independent studies providing evidence of sensitivity and specificity of at least 80%. After screening 10,625 references, we retained 780 studies (374 biochemical, 203 neuroimaging, 133 neurophysiological and 65 neuropsychological studies, and five GWAS), including a total of approximately 120,000 cases and 176,000 controls. While the majority of the studies focused simply on associations, we could not find any biomarker for which there was evidence – from two or more studies from independent research groups, with results going into the same direction – of specificity and sensitivity of at least 80%. Other important metrics to assess the validity of a candidate biomarker, such as positive predictive value and negative predictive value, were infrequently reported. Limitations of the currently available studies include mostly small sample size, heterogeneous approaches and candidate biomarker targets, undue focus on single instead of joint biomarker signatures, and incomplete accounting for potential confounding factors. Future multivariable and multi-level approaches may be best suited to find valid candidate biomarkers, which will then need to be validated in external, independent samples and then, importantly, tested in terms of feasibility and cost-effectiveness, before they can be implemented in daily clinical practice.     

Analysis of Volatile Constituent by Hydrodistillation and Solid-Phase Microextraction Techniques and Antimicrobial and Scolicidal Activities of Essential Oil and Soxhlet Extracts of Ulva rigida grown in Turkey

In the present study, the volatile composition of Ulva rigida (U. rigida) was elucidated by two different methods. As a result of the identification process of volatile components using the GC/MS-FID instrument, 31 compounds were identified by hydrodistillation (HD) method, and 15 compounds were identified by solid-phase microextraction (SPME) method, elucidating the structure of 99.86 % and 92.65 %, respectively. The most abundant compounds in the essential oil of U. rigida were n-hexadecanoic acid and pentadecanal, while the most abundant compound according to the SPME analysis was heptadecyne, a hydrocarbon compound. In the next step, hexane, dichloromethane, chloroform and methanol solvent extracts of U. rigida were prepared and the antimicrobial activities of the extracts and the essential oil obtained by hydro-distillation as well as the scolicidal activities of the solvent extracts were determined. The results of the antimicrobial activity test of the essential oil showed a high level of activity against Bacillus cereus ATCC 10876 and MRSA. The highest activity was found on the microorganism of Pseudomonas aeruginosa ATCC 9027 in chloroform and methanol extracts of U. rigida. Furthermore, viability detection was performed and the scolicidal effects of the extracts on protoscoleces were assessed. The values of lethal concentration doses (LD₅₀, LD₇₅ and LD₉₀) were calculated using probit analysis.     

Molecular dynamics,thermodynamic, and mutational binding studies for tumor-specific LyP-1 in complex with p32

Recent studies in tumor homing peptides have shown the specificity of LyP-1 (CGNKRTRGC) to tumor lymphatics. In this present work, we evaluated the possible interactions between cyclic LyP-1 and its receptor, p32, with molecular dynamics and docking studies in order to lead the design of novel LyP-1 derivatives, which could bind to p32 more effectively and perform enhanced antitumor effect. The total binding enthalpy energies have been obtained by MM-PBSA thermodynamic computations and the favorability of p32.LyP-1 complex in water has been shown by explicit water MD computations. The last 30 ns of molecular dynamics trajectory have shown the strong interaction of LyP-1 with the inner surface chains of p32, especially with chains B and C. ALA-SCAN mutagenesis studies have indicated the considerable influence of Asn3, Lys4, Arg5, and Arg7 amino acid residues on the specific binding of LyP-1. Within the knowledge of the critical role of p32 receptor in cancer cell metabolism, this study can lead to further developments in anticancer therapy by targeting p32 with LyP-1 derivatives as active targeting moiety. This data can also be applied for the development of new drug delivery systems in which LyP-1 can be used for its targeting and anticancer properties.