CNKSR2 interactome analysis indicates its association with the centrosome/microtubule system

The protein connector enhancer of kinase suppressor of Ras 2(CNKSR2),present in both the postsynaptic density and cytoplasm of neurons,is a scaffolding protein with several protein-binding domains.Variants of the CNKSR2 gene have been implicated in neurodevelopmental disorders,particularly intellectual disability,although the precise mechanism involved has not yet been fully understood.Research has demonstrated that CNKSR2 plays a role in facilitating the localization of postsynaptic density protein complexes to the membrane,thereby influencing synaptic signaling and the morphogenesis of dendritic spines.However,the function of CNKSR2 in the cytoplasm remains to be elucidated.In this study,we used immunoprecipitation and high-resolution liquid chromatography-mass spectrometry to identify the interactors of CNKSR2.Through a combination of bioinformatic analysis and cytological experiments,we found that the CNKSR2 interactors were significantly enriched in the proteome of the centrosome.We also showed that CNKSR2 interacted with the microtubule protein DYNC1H1 and with the centrosome marker CEP290.Subsequent colocalization analysis confirmed the centrosomal localization of CNKSR2.When we downregulated CNKSR2 expression in mouse neuroblastoma cells(Neuro 2A),we observed significant changes in the expression of numerous centrosomal genes.This manipulation also affected centrosome-related functions,including cell size and shape,cell proliferation,and motility.Furthermore,we found that CNKSR2 interactors were highly enriched in de novo variants associated with intellectual disability and autism spectrum disorder.Our findings establish a connection between CNKSR2 and the centrosome,and offer new insights into the underlying mechanisms of neurodevelopmental disorders.

Spastin and alsin protein interactome analyses begin to reveal key canonical pathways and suggest novel druggable targets

Developing effective and long-term treatment strategies for rare and complex neurodegenerative diseases is challenging. One of the major roadblocks is the extensive heterogeneity among patients. This hinders understanding the underlying disease-causing mechanisms and building solutions that have implications for a broad spectrum of patients. One potential solution is to develop personalized medicine approaches based on strategies that target the most prevalent cellular events that are perturbed in patients. Especially in patients with a known genetic mutation, it may be possible to understand how these mutations contribute to problems that lead to neurodegeneration. Protein–protein interaction analyses offer great advantages for revealing how proteins interact, which cellular events are primarily involved in these interactions, and how they become affected when key genes are mutated in patients. This line of investigation also suggests novel druggable targets for patients with different mutations. Here, we focus on alsin and spastin, two proteins that are identified as “causative” for amyotrophic lateral sclerosis and hereditary spastic paraplegia, respectively, when mutated. Our review analyzes the protein interactome for alsin and spastin, the canonical pathways that are primarily important for each protein domain, as well as compounds that are either Food and Drug Administration–approved or are in active clinical trials concerning the affected cellular pathways. This line of research begins to pave the way for personalized medicine approaches that are desperately needed for rare neurodegenerative diseases that are complex and heterogeneous.

Strategies for translating proteomics discoveries into drug discovery for dementia

Tauopathies,diseases characterized by neuropathological aggregates of tau including Alzheimer's disease and subtypes of fro ntotemporal dementia,make up the vast majority of dementia cases.Although there have been recent developments in tauopathy biomarkers and disease-modifying treatments,ongoing progress is required to ensure these are effective,economical,and accessible for the globally ageing population.As such,continued identification of new potential drug targets and biomarkers is critical."Big data"studies,such as proteomics,can generate information on thousands of possible new targets for dementia diagnostics and therapeutics,but currently remain underutilized due to the lack of a clear process by which targets are selected for future drug development.In this review,we discuss current tauopathy biomarkers and therapeutics,and highlight areas in need of improvement,particularly when addressing the needs of frail,comorbid and cognitively impaired populations.We highlight biomarkers which have been developed from proteomic data,and outline possible future directions in this field.We propose new criteria by which potential targets in proteomics studies can be objectively ranked as favorable for drug development,and demonstrate its application to our group's recent tau interactome dataset as an example.

The BLOC Interactomes Form a Network in Endosomal Transport

With the identification of more than a dozen novel Hermansky-Pudlak Syndrome （HPS） proteins in vesicle trafficking in higher eukaryotes, a new class of trafficking pathways has been described. It mainly consists of three newly-defined protein com- plexes, BLOC-l, -2, and -3. Compelling evidence indicates that these complexes together with two other well-known complexes, AP3 and HOPS, play important roles in endosomal transport. The interactions between these complexes form a network in protein trafficking via endosomes and cytoskeleton. Each node of this network has intra-complex and extra-complex interactions. These complexes are connected by direct interactions between the subunits from different complexes or by indirect interactions through coupling nodes that interact with two or more subunits from different complexes. The dissection of this network facilitates the understanding of a dynamic but elaborate transport machinery in protein/membrane trafficking. The disruption of this network may lead to abnormal trafficking or defective organellar development as described in patients with Hermansky-Pudlak syndrome.

Fasciculation and elongation zeta proteins 1 and 2: From structural flexibility to functional diversity

Fasciculation and elongation zeta/zygin(FEZ) proteins are a family of hub proteins and share many characteristics like high connectivity in interaction networks, they are involved in several cellular processes, evolve slowly and in general have intrinsically disordered regions. In 1985, unc-76 gene was firstly described and involved in axonal growth in C. elegans, and in 1997 Bloom and Horvitz enrolled also the human homologues genes, FEZ1 and FEZ2, in this process. While nematodes possess one gene(unc-76), mammalians have one more copy(FEZ1 and FEZ2). Several animal models have been used to study FEZ family functions like: C. elegans, D. melanogaster, R. novergicus and human cells.Complementation assays were performed and demonstrated the function conservation between paralogues. Human FEZ1 protein is more studied followed by UNC-76 and FEZ2 proteins, respectively. While FEZ1 and UNC-76 shared interaction partners, FEZ2 evolved and increased the number of protein-protein interactions(PPI) with cytoplasmatic partners. FEZ proteins are implicated in intracellular transport, acting as bivalent cargo transport adaptors in kinesinmediated movement. Especially in light of this cellular function, this family of proteins has been involved in several processes like neuronal development,neurological disorders, viral infection and autophagy. However, nuclear functions of FEZ proteins have been explored as well, due to high content of PPI with nuclear proteins, correlating FEZ1 expression to Sox2 and Hoxb4 gene regulation and retinoic acid signaling. These recent findings open new avenue to study FEZ proteins functions and its involvement in already described processes.This review intends to reunite aspects of evolution, structure, interaction partners and function of FEZ proteins and correlate them to physiological and pathological processes.

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Base excision repair （BER） is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged （oxidized or aikylated） or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species （ROS）. BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease （APE） to generate 3＇ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA iigase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APEI, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3＇ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and iigases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organeile targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

Mapping the human protein interactome

Interactions are the essence of all biomolecules because they cannot fulfill their roles without interacting with other molecules. Hence, mapping the interactions of biomolecules can be useful for understanding their roles and functions. Furthermore, the development of molecular based systems biology requires an understanding of the biomolecular interactions. In recent years, the mapping of protein-protein interactions in different species has been reported, but few reports have focused on the large-scale mapping of protein-protein interactions in human. Here, we review the developments in protein interaction mapping and we discuss issues and strategies for the mapping of the human protein interactome.

Cytokinome profile evaluation in patients with hepatitis C virus infection

The ‘‘omics sciences’’ (genomics, transcriptomics, proteomics) are often used to study living organisms as a whole system by evaluating the complex expression patterns of genes, miRNA, proteins, and metabolites. This study aimed, through bioinformatics and systems biology, to decipher the cytokinome profile in the evolution of inflammatory processes leading to cancer. The cytokinome was defined as the totality of cytokines and their interactions in and around biological cells. The system biology approach would provide a better understanding of the complex interaction network of cytokines, especially in cancer patients. Acquired knowledge would enable health providers with tools to evaluate disease onset through progression as well as identifying innovative therapeutic strategies. Understanding the role each cytokine plays in the metabolic network is of great importance. This paper reviews our group’s ‘‘omics’’ work. In particular, it addresses the role cytokines play in liver disease in six different scenarios. The first is the role the cytokines play in chronic inflammatory diseases and cancers. The second is the significance of the cytokinome profile. The third is the role of liver cirrhosis as an inflammatory disease. The fourth is the comparison of cytokine levels evaluated in patients with chronic hepatitis C virus (HCV) or with HCV-related cirrhosis. The fifth is the comparison of cytokine levels evaluated in patients with HCV-related cirrhosis in the presence and absence of type 2 diabetes. And lastly, we present a comparison of cytokine levels evaluated in patients with HCV-related cirrhosis in the presence and absence of hepatocellular carcinoma.

TurboID Proximity Labeling of a Protocadherin Protein to Characterize Interacting Protein Complex

The study of the neuron has always been a fundamental aspect when it came to studying mental illnesses such as autism and depression. The protein protocadherin-9 (PCDH9) is an important transmembrane protein in the development of the neuron synapse. Hence, research on its protein interactome is key to understanding its functionality and specific properties. A newly discovered biotin ligase, TurboID, is a proximity labeler that is designed to be able to label and observe transmembrane proteins, something that previous methods struggled with. The TurboID method is verified in HEK293T cells and primary cultured mouse cortical neurons. Results have proven the validity of the TurboID method in observing PCDH9-interacting proteins.

The cellular interactome for glycoprotein 5 of the Chinese highly pathogenic porcine reproductive and respiratory syndrome virus

supported by the Earmarked Fund for Modern Agro-industry Technology Research System of China （CARS-36） from the Ministry of Agriculture of China;the National Natural Science Foundation of China （31572549）;the National Key Technology R＆D Program of China （2015BAD12B01-2） from the Ministry of Science and Technology of China

The sperm-interacting proteome in the bovine isthmus and ampulla during the periovulatory period

Background Spermatozoa interact with oviduct secretions before fertilization in vivo but the molecular players of this dialog and underlying dynamics remain largely unknown.Our objectives were to identify an exhaustive list of sperm-interacting proteins(SIPs)in the bovine oviduct fluid and to evaluate the impact of the oviduct anatomical region(isthmus vs.ampulla)and time relative to ovulation(pre-ovulatory vs.post-ovulatory)on SIPs number and abundance.Methods Pools of oviduct fluid(OF)from the pre-ovulatory ampulla,pre-ovulatory isthmus,post-ovulatory ampulla,and post-ovulatory isthmus in the side of ovulation were collected from the slaughterhouse.Frozen-thawed bull sperm were incubated with OF or phosphate-buffered saline(control)for 60 min at 38.5℃.After protein extraction and digestion,sperm and OF samples were analyzed by nanoLC-MS/MS and label-free protein quantification.Results A quantitative comparison between proteins identified in sperm and OF samples(2333 and 2471 proteins,respectively)allowed for the identification of 245 SIPs.The highest number(187)were found in the pre-ovulatory isthmus,i.e.,time and place of the sperm reservoir.In total,41 SIPs(17%)were differentially abundant between stages in a given region or between regions at a given stage and 76 SIPs(31%)were identified in only one region×stage condition.Functional analysis of SIPs predicted roles in cell response to stress,regulation of cell motility,fertilization,and early embryo development.Conclusion This study provides a comprehensive list of SIPs in the bovine oviduct and evidences dynamic spatiotemporal changes in sperm-oviduct interactions around ovulation time.Moreover,these data provide protein candidates to improve sperm conservation and in vitro fertilization media.

Shared(epi)genomic background connecting neurodegenerative diseases and type 2 diabetes

The progressive aging of populations has resulted in an increased prevalence of chronic pathologies,especially of metabolic,neurodegenerative and movement disorders.In particular,type 2 diabetes(T2D),Alzheimer’s disease(AD)and Parkinson’s disease(PD)are among the most prevalent age-related,multifactorial pathologies that deserve particular attention,given their dramatic impact on patient quality of life,their economic and social burden as well the etiopathogenetic mechanisms,which may overlap in some cases.Indeed,the existence of common triggering factors reflects the contribution of mutual genetic,epigenetic and environmental features in the etiopathogenetic mechanisms underlying T2D and AD/PD.On this subject,this review will summarize the shared(epi)genomic features that characterize these complex pathologies.In particular,genetic variants and gene expression profiles associated with T2D and AD/PD will be discussed as possible contributors to determine the susceptibility and progression to these disorders.Moreover,potential shared epigenetic modifications and factors among T2D,AD and PD will also be illustrated.Overall,this review shows that findings from genomic studies still deserves further research to evaluate and identify genetic factors that directly contribute to the shared etiopathogenesis.Moreover,a common epigenetic background still needs to be investigated and characterized.The evidences discussed in this review underline the importance of integrating largescale(epi)genomic data with additional molecular information and clinical and social background in order to finely dissect the complex etiopathogenic networks that build up the“disease interactome”characterizing T2D,AD and PD.

Amylotrophic Lateral Sclerosis-Like Motor Impairment in Prion Diseases

Neurodegenerative diseases are collective diseases that affect different parts of the brain with common or distinct disease phenotype. In almost all of the Prion diseases, motor impairments that are characterized by motor derangement, apathy, ataxia, and myoclonus are documented and again are shared by motor neuron diseases (MND). Proteins such as;B-Cell lymphoma 2 (BCL2), Copper chaperone for superoxide dismutase (CCS), Amyloid beta precursor protein (APP), Amyloid Precursor-Like Protein1/2 (APLP1/2), Catalase (CAT), and Stress induced phosphoprotein 1 (STIP1), are common interactomes of Prion and superoxide dismutase 1 (SOD1). Although there is no strong evidence to show the interaction of SOD1 and Prion, the implicated common interacting proteins indicate the potential bilateral interaction of those proteins in health and disease. For example, down-regulation of Heat shock protein A (HSPA5), a Prion interactome, increases accumulation of misfolded SOD1 leading to MND. Loss of Cu uptake function disturbs normal function of CCS. Over-expressed proteasome subunit alpha 3 (PSMA3) could fatigue its normal function of removing misfolded proteins. Studies showed the increase in CAT and lipid oxidation both in Prion-knocked out animal and in catalase deficiency cases. Up regulation, down regulation or direct interaction with their interactomes are predicted molecular mechanisms by which Prion and SOD exert their effect. The loss of protective function or the gain of a novel toxic property by the principal proteins is shared in Prion and MND. Thus, it might be possible to conclude that the interplay of proteins displayed in both diseases could be a key phenomenon in motor dysfunction development.

eRNA-IDO:A One-stop Platform for Identification,Interactome Discovery,and Functional Annotation of Enhancer RNAs

Growing evidence supports the transcription of enhancer RNAs(eRNAs)and their important roles in gene regulation.However,their interactions with other biomolecules and their corresponding functionality remain poorly understood.In an attempt to facilitate mechanistic research,this study presents eRNA-IDO,the first integrative computational platform for the identification,interactome discovery,and functional annotation of human eRNAs.eRNA-IDO comprises two modules:eRNA-ID and eRNA-Anno.Functionally,eRNA-ID can identify eRNAs from de novo assembled transcriptomes.eRNA-ID includes eight kinds of enhancer makers,enabling users to customize enhancer regions flexibly and conveniently.In addition,eRNA-Anno provides cell-/tissue-specific functional annotation for both new and known eRNAs by analyzing the eRNA interactome from prebuilt or user-defined networks between eRNAs and protein-coding genes.The prebuilt networks include the Genotype-Tissue Expression(GTEx)-based co-expression networks in normal tissues,The Cancer Genome Atlas(TCGA)-based co-expression networks in cancer tissues,and omics-based eRNA-centric regulatory networks.eRNA-IDO can facilitate research on the biogenesis and functions of eRNAs.The eRNA-IDO server is freely available at http://bioinfo.szbl.ac.cn/eRNA_IDO/.

Potential role of the protein interactome in translating TCM theory and clinical practice into modern biomedical knowledge

With the awarding of the 2015 Nobel Prize in Physiology or Medicine to Chinese pharmacologist Tu Youyou,and the significant contributions of traditional Chinese medicine(TCM)for coronavirus disease 2019(COVID-19),TCM has garnered increasing attention and interest globally.Although advanced research progress has been made in the efficacy research,mechanism elucidation and target prediction of TCM in recent years[1].

Overlap in oncogenic and pro-inflammatory pathways associated with areca nut and nicotine exposure

Background:Betel nut/areca nut/Areca catechu is one of the most commonly used psychoactive substance,and is also a major preventable cause of cancer.Unlike other psychoactive substances,such as nicotine,the mechanisms underlying addiction to areca nuts and related oncogenesis remain elusive.Recent reports suggest a possible overlap in the mechanisms of action of nicotine and areca nuts in the human body.Thus,this study aimed to investigate the interactome of human proteins associated with areca nut exposure and the intricate similarities and differences in the effects of the two psychoactive substances on humans.Methods:A list of proteins associated with areca nut use was obtained from the available literature using terms from Medical Subject Headings(MeSH).Protein-protein interaction(PPI)networks and functional enrichment were analyzed.The results obtained for both psychoactive substances were compared.Results:Given the limited number of common proteins(36/226,16%)in the two sets,a substantial overlap(612/1176 nodes,52%)was observed in the PPI networks,as well as in Gene Ontology.Areca nuts mainly affect signaling pathways through three hub proteins(alpha serine/threonine-protein kinase,tumor protein 53,and interleukin-6),which are common to both psychoactive substances,as well as two unique hub proteins(epidermal growth factor receptor and master regulator of cell cycle entry and proliferative metabolism).Areca nut-related proteins are associated with unique pathways,such as extracellular matrix organization,lipid storage,and metabolism,which are not found in nicotine-associated proteins.Conclusions:Areca nuts affect regulatory mechanisms,leading to systemic toxicity and oncogenesis.Areca nuts also affect unique pathways that can be studied as potential markers of exposure,as well as targets for anticancer therapeutic agents.

Capturing the hierarchically assorted modules of protein–protein interactions in the organized nucleome

Nuclear proteins are major constituents and key regulators of nucleome topological organization and manipulators of nuclear events.To decipher the global connectivity of nuclear proteins and the hierarchically organized modules of their interactions,we conducted two rounds of cross-linking mass spectrometry(XL-MS)analysis,one of which followed a quantitative double chemical cross-linking mass spectrometry(in vivo qXL-MS)workflow,and identified 24,140 unique crosslinks in total from the nuclei of soybean seedlings.This in vivo quantitative interactomics enabled the identification of 5340 crosslinks that can be converted into 1297 nuclear protein–protein interactions(PPIs),1220(94%)of which were non-confirmative(or novel)nuclear PPIs compared with those in repositories.There were 250 and 26 novel interactors of histones and the nucleolar box C/D small nucleolar ribonucleoprotein complex,respectively.Modulomic analysis of orthologous Arabidopsis PPIs produced 27 and 24 master nuclear PPI modules(NPIMs)that contain the condensate-forming protein(s)and the intrinsically disordered region–containing proteins,respectively.These NPIMs successfully captured previously reported nuclear protein complexes and nuclear bodies in the nucleus.Surprisingly,these NPIMs were hierarchically assorted into four higher-order communities in a nucleomic graph,including genome and nucleolus communities.This combinatorial pipeline of 4C quantitative interactomics and PPI network modularization revealed 17 ethylene-specific module variants that participate in a broad range of nuclear events.The pipeline was able to capture both nuclear protein complexes and nuclear bodies,construct the topological architectures of PPI modules and module variants in the nucleome,and probably map the protein compositions of biomolecular condensates.

Marking RNA:m^6A writers,readers,and functions in Arabidopsis

N^6-methyladenosine(m^6A)emerges as an important modification in eukaryotic mRNAs.m^6A has first been reported in 1974,and its functional significance in mammalian gene regulation and importance for proper development have been well established.An arsenal of writer,eraser,and reader proteins accomplish deposition,removal,and interpretation of the m^6A mark,resulting in dynamic function.This led to the concept of an epitranscriptome,the compendium of RNA species with chemical modification ofthe nucleobases in the cell,in analogy to the epigenome.While m^6A has long been known to also exist in plant mRNAs,proteins involved in m^6A metabolism have only recently been detected by mutant analysis,homology search,and mRNA interactome capture in the reference plant Arabidopsis thaliana.Dysregulation ofthe m^6A modification causes severe developmental abnormalities of leaves and roots and altered timing of reproductive development.Furthermore,m^6A modification affects viral infection.Here,we discuss recent progress in identifying m^6A sites transcriptome-wide,in identifying the molecular players involved in writing,removing,and reading the mark,and in assigning functions to this RNA modification in 4.thaliana.We highlight similarities and differences to m^6A modification in mammals and provide an outlook on important questions that remain to be addressed.

Using Interactome Big Data to Crack Genetic Mysteries and Enhance Future Crop Breeding

The functional genes underlying phenotypic variation and their interactions represent“genetic mysteries”.Understanding and utilizing these genetic mysteries are key solutions for mitigating the current threats to agriculture posed by population growth and individual food preferences.Due to advances in highthroughput multi-omics technologies,we are stepping into an Interactome Big Data era that is certain to revolutionize genetic research.In this article,we provide a brief overview of current strategies to explore genetic mysteries.We then introduce the methods for constructing and analyzing the Interactome Big Data and summarize currently available interactome resources.Next,we discuss how Interactome Big Data can be used as a versatile tool to dissect genetic mysteries.We propose an integrated strategy that could revolutionize genetic research by combining Interactome Big Data with machine learning,which involves mining information hidden in Big Data to identify the genetic models or networks that control various traits,and also provide a detailed procedure for systematic dissection of genetic mysteries,Finally,we discuss three promising future breeding strategies utilizing the Interactome Big Data to improve crop yields and quality.

IDH1 fine-tunes cap-dependent translation initiation

The metabolic enzyme isocitrate dehydrogenase 1(IDH1)catalyzes the oxidative decarboxylation of isocitrate to a-ketoglutarate(a-KG).Its mutation often leads to aberrant gene expression in cancer.IDH1 was reported to bind thousands of RNA transcripts in a sequence-dependent manner;yet,the functional significance of this RNA-binding activity remains elusive.Here,we report that IDH1 promotes mRNA translation via direct associations with polysome mRNA and translation machinery.Comprehensive proteomic analysis in embryonic stem cells(ESCs)revealed strikingenrichmentof ribosomal proteins and translation regulators in IDH1-bound protein interactomes.We performed ribosomal profiling and analyzed mRNA transcripts that are associated with actively translating polysomes.Interestingly,knockout of IDH1 in ESCs led to significant downregulation of polysome-bound mRNA in IDH1 targets and subtle upregulation of ribosome densities at the start codon,indicating inefficient translation initiation upon loss of IDH1.Tethering IDH1 to a luciferase mRNA via the MS2-MBP system promotes luciferase translation,independently of the catalytic activity of IDH1.Intriguingly,IDH1 fails to enhance luciferase translation driven by an internal ribosome entry site.Together,these results reveal an unforeseen role of IDH1 in fine-tuning cap-dependent translation via the initiation step.