Systematic Analysis of Post-Translational Modifications for Increased Longevity of Biotherapeutic Proteins

Protein-based therapeutics (PPTs) are drugs used to treat a variety of different conditions in the human body by alleviating enzymatic deficiencies, augmenting other proteins and drugs, modulating signal pathways, and more. However, many PPTs struggle from a short half-life due to degradation caused by irreversible protein aggregation in the bloodstream. Currently, the most researched strategies for improving the efficiency and longevity of PPTs are post-translational modifications (PTMs). The goal of our research was to determine which type of PTM increases longevity the most for each of three commonly-used therapeutic proteins by comparing the docking scores (DS) and binding free energies (BFE) from protein aggregation and reception simulations. DS and BFE values were used to create a quantitative index that outputs a relative number from −1 to 1 to show reduced performance, no change, or increased performance. Results showed that methylation was the most beneficial for insulin (p < 0.1) and human growth hormone (p < 0.0001), and both phosphorylation and methylation were somewhat optimal for erythropoietin (p < 0.1 and p < 0.0001, respectively). Acetylation consistently provided the worst benefits with the most negative indices, while methylation had the most positive indices throughout. However, PTM efficacy varied between PPTs, supporting previous studies regarding how each PTM can confer different benefits based on the unique structures of recipient proteins.

Post-translational modifications of prostaglandin-endoperoxide synthase 2 in colorectal cancer:An update

The biosynthesis of prostanoids is involved in both physiological and pathological processes. The expression of prostaglandin-endoperoxide synthase 2(PTGS2; also known as COX-2) has been traditionally associated to the onset of several pathologies, from inflammation to cardiovascular, gastrointestinal and oncologic events. For this reason, the search of selective PTGS2 inhibitors has been a focus for therapeutic interventions. In addition to the classic non-steroidal anti-inflammatory drugs, selective and specific PTGS2 inhibitors, termed coxibs, have been generated and widely used. PTGS2 activity is less restrictive in terms of substrate specificity than the homeostatic counterpart PTGS1, and it accounts for the elevated prostanoid synthesis that accompanies several pathologies. The main regulation of PTGS2 occurs at the transcription level. In addition to this, the stability of the mRNA is finely regulated through the interaction with several cytoplasmic elements, ranging from specificmicroR NAs to proteins that control mR NA degradation. Moreover, the protein has been recognized to be the substrate for several post-translational modifications that affect both the enzyme activity and the targeting for degradation via proteasomal and non-proteasomal mechanisms. Among these modifications, phosphorylation, glycosylation and covalent modifications by reactive lipidic intermediates and by free radicals associated to the proinflammatory condition appear to be the main changes. Identification of these post-translational modifications is relevant to better understand the role of PTGS2 in several pathologies and to establish a correct analysis of the potential function of this protein in diseases progress. Finally, these modifications can be used as biomarkers to establish correlations with other parameters, including the immunomodulation dependent on molecular pathological epidemiology determinants, which may provide a better frame for potential therapeutic interventions.

Aberrant post-translational protein modifications in the pathogenesis of alcohol-induced liver injury

It is likely that the majority of proteins will undergo post-translational modification, be it enzymatic or non-enzymatic. These modified protein(s) regulate activity, localization and interaction with other cellular molecules thereby maintaining cellular hemostasis. Alcohol exposure significantly alters several of these post-translational modifications leading to impairments of many essential physiological processes. Here, we present new insights into novel modifications following ethanol exposure and their role in the initiation and progression of liver injury. This critical review condenses the proceedings of a symposium at the European Society for the Biomedical Research on Alcoholism Meeting held September 12-15, 2015, in Valencia, Spain.

Protein post-translational modifications after spinal cord injury

Deficits in intrinsic neuronal capacities in the spinal cord,a lack of growth support,and suppression of axonal outgrowth by inhibitory molecules mean that spinal cord injury almost always has devastating consequences.As such,one of the primary targets for the treatment of spinal cord injury is to develop strategies to antagonize extrinsic or intrinsic axonal growth-inhibitory factors or enhance the factors that support axonal growth.Among these factors,a series of individual protein level disorders have been identified during the generation of axons following spinal cord injury.Moreover,an increasing number of studies have indicated that post-translational modifications of these proteins have important implications for axonal growth.Some researchers have discovered a variety of post-translational modifications after spinal cord injury,such as tyrosination,acetylation,and phosphorylation.In this review,we reviewed the post-translational modifications for axonal growth,functional recovery,and neuropathic pain after spinal cord injury,a better understanding of which may elucidate the dynamic change of spinal cord injury-related molecules and facilitate the development of a new therapeutic strategy for spinal cord injury.

Post-translational modifications of hepatitis C viral proteins and their biological significance

Replication of hepatitis C virus(HCV)depends on the interaction of viral proteins with various host cellular proteins and signalling pathways.Similar to cellular proteins,post-translational modifications(PTMs)of HCV proteins are essential for proper protein function and regulation,thus,directly affecting viral life cycle and the generation of infectious virus particles.Cleavage of the HCV polyprotein by cellular and viral proteases into more than 10 proteins represents an early protein modification step after translation of the HCV positivestranded RNA genome.The key modifications include the regulated intramembranous proteolytic cleavage of core protein,disulfide bond formation of core,glycosylation of HCV envelope proteins E1 and E2,methylation of nonstructural protein 3(NS3),biotinylation of NS4A,ubiquitination of NS5B and phosphorylation of core and NS5B.Other modifications like ubiquitination of core and palmitoylation of core and NS4B proteins have been reported as well.For some modifications such as phosphorylation of NS3 and NS5A and acetylation of NS3,we have limited understanding of their effects on HCV replication and pathogenesis while the impact of other modifications is far from clear.In this review,we summarize the available information on PTMs of HCV proteins and discuss their relevance to HCV replication and pathogenesis.

Complex interactomes and post-translational modifications of the regulatory proteins HABP4 and SERBP1 suggest pleiotropic cellular functions

The 57 kDa antigen recognized by the Ki-1 antibody,is also known as intracellular hyaluronic acid binding protein 4 and shares 40.7%identity and 67.4%similarity with serpin mRNA binding protein 1,which is also named CGI-55,or plasminogen activator inhibitor type-1-RNA binding protein-1,indicating that they might be paralog proteins,possibly with similar or redundant functions in human cells.Through the identification of their protein interactomes,both regulatory proteins have been functionally implicated in transcriptional regulation,mRNA metabolism,specifically RNA splicing,the regulation of mRNA stability,especially,in the context of the progesterone hormone response,and the DNA damage response.Both proteins also show a complex pattern of post-translational modifications,involving Ser/Thr phosphorylation,mainly through protein kinase C,arginine methylation and SUMOylation,suggesting that their functions and locations are highly regulated.Furthermore,they show a highly dynamic cellular localization pattern with localizations in both the cytoplasm and nucleus as well as punctuated localizations in both granular cytoplasmic protein bodies,upon stress,and nuclear splicing speckles.Several reports in the literature show altered expressions of both regulatory proteins in a series of cancers as well as mutations in their genes that may contribute to tumorigenesis.This review highlights important aspects of the structure,interactome,post-translational modifications,sub-cellular localization and function of both regulatory proteins and further discusses their possible functions and their potential as tumor markers in different cancer settings.

Computer-Assisted analysis of subcellular localization signals and post-translational modifications of human prion proteins

In the present work, computational analyses were applied to study the subcellular localiza-tion and posttranslational modifications of hu-man prion proteins (PrPs). The tentative location of prion protein was determined to be in the nu-cleolus inside the nucleus by the following bio-informatics tools: Hum-PLoc, Euk-PLoc and Nuc-PLoc. Based on our results signal peptides with average of 22 base pairs in N-terminal were identified in human PrPs. This theoretical study demonstrates that PrP is post-translationally modified by: 1) attachment of two N-linked complex carbohydrate moieties (N181 and N197), 2) attachmet of glycosylphosphatidylinositol (GPI) at serine 230 and 3) formation of two di-sulfide bonds between “6–22” and “179–214” cysteines. Furthermore, ten protein kinase phosphorylation sites were predicted in human PrP. The above-noted phosphorylation was car-ried out by PKC and CK2. By using bioinfor-matics tools, we have shown that computation-ally human PrPs locate particularly into the nu-cleolus.

Comprehensive analysis of the gut microbiome and posttranslational modifications elucidates the route involved in microbiota-host interactions

The gut microbiome interacts with the host to maintain body homeostasis,with gut microbial dysbiosis implicated in many diseases.However,the underlying mechanisms of gut microbe regulation of host behavior and brain functions remain unclear.This study aimed to elucidate the influence of gut microbiota on brain functions via post-translational modification mechanisms in the presence or absence of bacteria without any stimulation.We conducted succinylome analysis of hippocampal proteins in germ-free(GF)and specific pathogen-free(SPF)mice and metagenomic analysis of feces from SPF mice.These results were integrated with previously reported hippocampal acetylome and phosphorylome data from the same batch of mice.Subsequent bioinformatics analyses revealed 584 succinylation sites on 455 proteins,including 54 up-regulated succinylation sites on 91 proteins and 99 down-regulated sites on 51 proteins in the GF mice compared to the SPF mice.We constructed a panoramic map of gut microbiota-regulated succinylation,acetylation,and phosphorylation,and identified cross-talk and relative independence between the different types of post-translational modifications in modulating complicated intracellular pathways.Pearson correlation analysis indicated that 13 taxa,predominantly belonging to the Bacteroidetes phylum,were correlated with the biological functions of post-translational modifications.Positive correlations between these taxa and succinylation and negative correlations between these taxa and acetylation were identified in the modulation of intracellular pathways.This study highlights the hippocampal physiological changes induced by the absence of gut microbiota,and proteomic quantification of succinylation,phosphorylation,and acetylation,contributing to our understanding of the role of the gut microbiome in brain function and behavioral phenotypes.

Human T-lymphotropic virus proteins and post-translational modification pathways

Cell life from the cell cycle to the signaling transduction and response to stimuli is finely tuned by protein post-translational modifications(PTMs).PTMs alter the conformation,the stability,the localization,and hence the pattern of interactions of the targeted protein.Cell pathways involve the activation of enzymes,like kinases,ligases and transferases,that,once activated,act on many proteins simultaneously,altering the state of the cell and triggering the processes they are involved in.Viruses enter a balanced system and hijack the cell,exploiting the potential of PTMs either to activate viral encoded proteins or to alter cellular pathways,with the ultimate consequence to perpetuate through their replication.Human T-lymphotropic virus type 1(HTLV-1)is known to be highly oncogenic and associates with adult T-cell leukemia/lymphoma,HTLV-1-associated myelopathy/tropical spastic paraparesis and other inflammatory pathological conditions.HTLV-1 protein activity is controlled by PTMs and,in turn,viral activity is associated with the modulation of cellular pathways based on PTMs.More knowledge is acquired about the PTMs involved in the activation of its proteins,like Tax,Rex,p12,p13,p30,HTLV-I basic leucine zipper factorand Gag.However,more has to be understood at the biochemical level in order to counteract the associated fatal outcomes.This review will focus on known PTMs that directly modify HTLV-1 components and on enzymes whose activity is modulated by viral proteins.

Microfluidic Immunoprecipitation for Post-Translational Modified Protein Purification

In this paper, we report an antibody functionalized microimmunopreci- pitation （IX IP） method used for enrich lowabundant post-translational modified （PT~ proteins. The device is fabricated by inert, nontoxic and disposable polydimethylsiloxane （PDMS） using a silane-based chemical modification protocol, which yield antibody- terminated PDMS surfaces. In this study, the IX IP device is specifically designed for the purification of carbonylated protein, a representative example here to illustrate the potential applications for any other PTMs, which could be immuno-tagged by specific antibodies. The test model in vitro oxidized bovine serum albumin （BSA） was first derivitized by dinitrophenylhydrazide （DNPH） and then captured by the anti-DNP immobilized on this Ix lP device. The surface functional group mapping was systematically analyzed and validated by fluorescence microscopy. Quantitative study of DNP-derivatized carbonylated protein capture recovery and elution efficiency of the device was also studied. We also envision that this proteome enrichment Ix IP device can be assembled with other lab-on-a-chip components, such as microelectrophoresis or micro-chromatographic devices for follow-up protein analysis. This selective enrichment of modified proteins greatly facilitates the study of low abundant protein biomarkers discovery.

Protein post-translational modifications in auxin signaling

Protein post-translational modifications(PTMs),such as ubiquitination,phosphorylation,and small ubiquitin-like modifier(SUMO)ylation,are crucial for regulating protein stability,activity,subcellular localization,and binding with cofactors.Such modifications remarkably increase the variety and complexity of proteomes,which are essential for regulating numerous cellular and physiological processes.The regulation of auxin signaling is finely tuned in time and space to guide various plant growth and development.Accumulating evidence indicates that PTMs play critical roles in auxin signaling regulations.Thus,a thorough and systematic review of the functions of PTMs in auxin signal transduction will improve our profound comprehension of the regulation mechanism of auxin signaling and auxin-mediated various processes.This review discusses the progress of protein ubiquitination,phosphorylation,histone acetylation and methylation,SUMOylation,and S-nitrosylation in the regulation of auxin signaling.

PTMD: A Database of Human Disease-associated Post-translational Modifications

Various posttranslational modifications （PTMs） participate in nearly all aspects of biological processes by regulating protein functions, and aberrant states of PTMs are frequently implicated in human diseases. Therefore, an integral resource of PTM–disease associations （PDAs）would be a great help for both academic research and clinical use. In this work, we reported PTMD,a well-curated database containing PTMs that are associated with human diseases. We manually collected 1950 known PDAs in 749 proteins for 23 types of PTMs and 275 types of diseases from the literature. Database analyses show that phosphorylation has the largest number of disease associations, whereas neurologic diseases have the largest number of PTM associations. We classified all known PDAs into six classes according to the PTM status in diseases and demonstrated that the upregulation and presence of PTM events account for a predominant proportion of diseaseassociated PTM events. By reconstructing a disease–gene network, we observed that breast cancers have the largest number of associated PTMs and AKT1 has the largest number of PTMs connected to diseases. Finally, the PTMD database was developed with detailed annotations and can be a useful resource for further analyzing the relations between PTMs and human diseases. PTMD is freely accessible at http：//ptmd.biocuckoo.org.

Applications of post-translational modifications of FoxO family proteins in biological functions

The functions of the FoxO family proteins,in particular their transcriptional activities,are modulated by post-translational modifi-cations(PTMs),including phosphorylation,acetylation,ubiquitination,methylation and glycosylation.These PTMs occur in response to different cellular stresses,which in turn regulate the subcellular localization of FoxO family proteins,as well as their half-life,DNA binding,transcriptional activity and ability to interact with other cellular proteins.In this review,we summarize the role of PTMs of FoxO family proteins in linking their biological and functional relevance with various diseases.

Diagnostic and Prognostic Value of Protein Post-translational Modifications in Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is a common malignant tumor with high incidence and cancer mortality worldwide.Post-translational modifications(PTMs)of proteins have a great impact on protein function.Almost all proteins can undergo PTMs,including phosphorylation,acetylation,methylation,glycosylation,ubiquitination,and so on.Many studies have shown that PTMs are related to the occurrence and development of cancers.The findings provide novel therapeutic targets for cancers,such as glypican-3 and mucin-1.Other clinical implications are also found in the studies of PTMs.Diagnostic or prognostic value,and response to therapy have been identified.In HCC,it has been shown that glycosylated alpha-fetoprotein(AFP)has a higher detection rate for early liver cancer than conventional AFP.In this review,we mainly focused on the diagnostic and prognostic value of PTM,in order to provide new insights into the clinical implication of PTM in HCC.

Mass Spectrometry Analysis of SARS-CoV-2 Nucleocapsid Protein Reveals Camouflaging Glycans and Unique Post-Translational Modifications

The devastating coronavirus disease 2019(COVID-19)pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and its viral components.Compared to the Spike protein,which is the primary target for currently available vaccines or antibodies,knowledge about other virion structural components is incomplete.Using high-resolution mass spectrometry,we report a comprehensive post-translational modification(PTM)analysis of nucleocapsid phosphoprotein(NCP),the most abundant structural component of the SARS-CoV-2 virion.In addition to phosphoryl groups,we show that the SARS-CoV-2 NCP is decorated with a variety of PTMs,including N-glycans and ubiquitin.Based on newly identified PTMs,refined protein structural models of SARS-CoV-2 NCP were proposed and potential immune recognition epitopes of NCP were aligned with PTMs.These data can facilitate the design of novel vaccines or therapeutics targeting NCP,as valuable alternatives to the current vaccination and treatment paradigm that is under threat of the ever-mutating SARS-CoV-2 Spike protein.

三个对“氧化应激”的重新认识

生命离不开氧,细胞的氧化还原状态直接调控蛋白质等生物大分子功能,介导细胞信号转导以及衰老、神经退行性疾病、代谢病、肿瘤等许多生理和病理过程。鉴于20世纪50年代提出的自由基衰老学说,长期以来氧化应激常与氧化损伤混为一谈,“抗氧化”一度成为“抗衰老”的代名词。本文结合本实验室相关研究工作及领域前沿,提出三个对“氧化应激”的重新认识:第一,氧化应激不等于氧化损伤,具有重要生理功能;第二,氧化应激不是“万金油”,氧化还原调控具有特异性,氧化还原修饰是其作用机制;第三,不能泛泛抗氧化,氧化还原平衡具有精准属性,抗氧化要考虑5R(Right species,Right place,Right time,Right level,Right target)原则,精准氧化还原医药时代已开启。未来挑战体现在氧化还原生物学与医学基础研究、氧化应激在生理和病理过程及环境胁迫中的特异分子机制研究及精准氧化还原干预三大方面,需要多学科交叉、基础与临床研究深度合作、国际协同攻关,实现对生命过程氧化还原认识的突破、机理的突破、精准干预的突破!

The pathogenic mechanism of TAR DNA-binding protein 43(TDP-43)in amyotrophic lateral sclerosis

The onset of amyotrophic lateral sclerosis is usually characterized by focal death of both upper and/or lower motor neurons occurring in the motor cortex,basal ganglia,brainstem,and spinal cord,and commonly involves the muscles of the upper and/or lower extremities,and the muscles of the bulbar and/or respiratory regions.However,as the disease progresses,it affects the adjacent body regions,leading to generalized muscle weakness,occasionally along with memory,cognitive,behavioral,and language impairments;respiratory dysfunction occurs at the final stage of the disease.The disease has a complicated pathophysiology and currently,only riluzole,edaravone,and phenylbutyrate/taurursodiol are licensed to treat amyotrophic lateral sclerosis in many industrialized countries.The TAR DNA-binding protein 43 inclusions are observed in 97%of those diagnosed with amyotrophic lateral sclerosis.This review provides a preliminary overview of the potential effects of TAR DNAbinding protein 43 in the pathogenesis of amyotrophic lateral sclerosis,including the abnormalities in nucleoplasmic transport,RNA function,post-translational modification,liquid-liquid phase separation,stress granules,mitochondrial dysfunction,oxidative stress,axonal transport,protein quality control system,and non-cellular autonomous functions(e.g.,glial cell functions and prion-like propagation).

磷酸化或乙酰化修饰使Fascin的促癌功能翻转

大量研究结果表明,食管鳞癌等恶性肿瘤中,Fascin异常高表达并发挥促癌功能,其与癌症患者的不良预后密切相关。近年来,包括本实验室在内,多家实验室的研究结果一致表明,磷酸化或乙酰化修饰可使Fascin的促癌功能翻转,犹如川剧中的“变脸”。最近,本室进一步研究发现,Fascin的翻译后修饰明显影响Fascin小分子抑制剂的抑癌效果,提示以Fascin为靶点的抗肿瘤药物研究应该将Fascin翻译后修饰状态纳入是否适合用药的评价体系中。为此,本文在系统评述Fascin促癌功能的基础上,重点讨论了翻译后修饰对Fascin功能的调控作用,以及翻译后修饰对Fascin小分子抑制剂抑癌功效的影响;同时还对相关领域针对Fascin的未来研究趋势进行了展望。

Histone post-translational modification and the DNA damage response

DNA is highly vulnerable to spontaneous and environmental timely damage in living cells.DNA damage may cause genetic instability and increase cancer risk if the damages are not repaired timely and efficiently.Human cells possess several DNA damage response(DDR)mechanisms to protect the integrity of their genome.Clarification of the mechanisms under-lying the DNA damage response following lethal damage will facilitate the identification of therapeutic targets for cancers.Histone post-translational modifications(PTMs)have been indicated to play different roles in the repair of DNA damage.In this context,histone PTMs regulate recruitment of downstream effectors,and facilitate appropriate repair response.This review outlines the current understanding of different histone PTMs in response to DNA dam-age repair,besides,enumerates the role of new type PTMs such as histone succinylation and crotonylation in regulating DNA damage repair processes.

Regulations of m^(6)A and other RNA modifications and their roles in cancer

RNA modification is an essential component of the epitranscriptome,regulating RNA metabolism and cellular functions.Several types of RNA modifications have been identified to date;they include N^(6)-methyladenosine(m^(6)A),N^(1)-methyladenosine(m1A),5-methylcytosine(m5C),N^(7)-methylguanosine(m^(7)G),N^(6),2′-O-dimethyladenosine(m^(6)Am),N4-acetylcytidine(ac^(4)C),etc.RNA modifications,mediated by regulators including writers,erasers,and readers,are associated with carcinogenesis,tumor microenvironment,metabolic reprogramming,immunosuppression,immunotherapy,chemotherapy,etc.A novel perspective indicates that regulatory subunits and post-translational modifications(PTMs)are involved in the regulation of writer,eraser,and reader functions in mediating RNA modifications,tumorigenesis,and anticancer therapy.In this review,we summarize the advances made in the knowledge of different RNA modifications(especially m^(6)A)and focus on RNA modification regulators with functions modulated by a series of factors in cancer,including regulatory subunits(proteins,noncoding RNA or peptides encoded by long noncoding RNA)and PTMs(acetylation,SUMOylation,lactylation,phosphorylation,etc.).We also delineate the relationship between RNA modification regulator functions and carcinogenesis or cancer progression.Additionally,inhibitors that target RNA modification regulators for anticancer therapy and their synergistic effect combined with immunotherapy or chemotherapy are discussed.