Creative Biostructure Updated Its MagHelix™ Thermal Gravimetric Analysis (TGA) Service Recently

Creative Biostructure, a biotechnology company actively working on the characterization of proteins using various techniques, recently updated its MagHelix™ Thermal Gravimetric Analysis (TGA) service for protein thermodynamics analysis to get its customers’ needs met.

 

TGA is a thermal analysis method in which the sample mass is measured according to time or temperature while the sample temperature is programmed in a controlled atmosphere. This analytical technique is often used to determine the composition of a sample and to predict its thermal stability.

 

Basically, typical applications of TGA include: Determining thermal stability-if a material (such as ceramic or polymer) is thermally stable, there will be no observed mass change; Determining oxidative stability-oxidative mass losses are the most common observable losses in TGA. Thus, it is very important to analyze the resistance to oxidation in copper alloys; Compositional analysis-temperature and weight change of decomposition reactions can allow quantitative composition analysis; Determining the purity of a mineral, inorganic compound, or organic material; Measuring the weight of fiberglass and inorganic fill materials in laminates, plastics, paints and composite materials; Determining water/carbon content or other residual solvents in a material; Allowing analysis of reactions with air, oxygen, or other reactive gases; and enhancing product formulation processes or ensuring product safety.

 

Through TGA analysis, Creative Biostructure provides information about chemical phenomena and physical phenomena, including chemisorption, dehydration, decomposition, solid gas reactions (such as oxidation or reduction), vaporization, sublimation, absorption and desorption. Essentially, TGA can assess samples that have lost or increased weight due to decomposition, oxidation, or dehydration.

 

According to the information on Creative Biostructure’s official website, TGA instruments can be utilizied to ramp temperature from room temperature to 1000°C in different gas atmospheres such as nitrogen, air or other inert gases. In addition, sample weight can range from 1 mg to 150 mg, and the sensitivity of weight change is 0.01 mg.

 

Now, MagHelix™ analytical methods for biophysical characterization of biomolecules at Creative Biostructure include but are not limited to:

 

Protein Thermal Shift Assay

Surface Plasmon Resonance

Isothermal Titration Calorimetry

Differential Scanning Calorimetry

Saturation-Transfer Difference

Bio-Layer Interferometry (BLI) Technology

 

“During the past years, we have been working on the characterization of proteins using a variety of techniques, as well as the engineering of proteins for various applications. The extensive experience got from those area enables us to provide customized, cost-effective, and quality services timely.” Commented Joanna, the official speaker from Creative Biostructure.

 

More information about MagHelix™ Thermal Gravimetric Analysis (TGA) at Creative Biostructure can be viewed here: https://www.creative-biostructure.com/maghelix%E2%84%A2-thermal-gravimetric-analysis-tga-216.htm.

 

About Creative Biostructure

With our advanced platform and equipment, our coronavirus research team can help researchers perform basic research on coronavirus-related bioinformatics analysis, viral particle identification and characterization, protein structure analysis, and protein interaction analysis. We also offer structure-based antiviral drug discovery and pre-clinical vaccine development services.

Cancer Res: Tumor Cell–Derived IL1β Promotes Desmoplasia and Immune Suppression in Pancreatic Cancer

Recently, in a study published in Cancer Research, scientists from New York University and other institutions have found that a key immune signal may play a previously unknown role in turning off the immune system to attack pancreatic cancer. The researchers found that the immune signaling protein interleukin 1β (IL-1β) can be made and released by pancreatic tumor cells, while it reduces the body’s anti-cancer immune response, thereby promoting the growth of pancreatic ductal adenocarcinoma (PDA), a type of cancer that is usually fatal within two years.

 

The researchers say blocking IL-1β activity in mice with antibodies may reduce PDA tumor growth by 32%, while other experiments can combine anti-IL-1β antibodies (which can lock and neutralize their targets) with antibody therapies that have been approved to turn off the PD1 protein checkpoint. To protect normal cells from immune attack, the immune system uses checkpoints on immune cells to turn off when they receive normal signals; cancer cells intercept checkpoints to turn off the function of the immune system, triggering the immune knowledge of CD8 + T cells, which in turn kills cancer cells, a therapy called checkpoint inhibitors can effectively neutralize this effect.

 

Although effective against a variety of cancers, checkpoint inhibitors are helpless for the treatment of PDA. In some experiments, the tumor response rate to therapy is only about 3%, and poor CD8 + T cell infiltration and immunosuppression are the main reasons for the limitations. In the current study, the researchers found that adding anti-IL-1β antibody to anti-PD-1 antibody therapy doubled T cell infiltration into PDA and increased the anti-tumor activity of PD-1 blockers by 40%.

 

Dr. Dafna Bar-Sagi, a researcher, said that by engineering mice to lack the IL-1β gene, we found for the first time that pancreatic cancer cells can produce IL-1β, which is essential for the continued growth of PDA tumors, and blocking IL-1β using antibody therapy may provide a novel strategy to make pancreatic tumors on the host immune system, thus potentially increasing the therapeutic potential of checkpoint inhibitors.

 

The findings of this study are consistent with previous research work by other researchers, which describe that the body’s microbiome changes when PDA is present, and that the microbiome is a key factor in cancer growth, an area that traditionally allocates IL-1β production to immune cells, but this study found that pancreatic tumor cells can also respond to proteins released by specific bacteria. The researchers say bacterial products can activate a protein on the cell surface called toll-like receptors, which can turn on the chain reaction needed for IL-1β production in cancer cells.

 

In addition, the researchers found that high levels of IL-1β promoted increased production of high-density proteins such as collagen in nearby pancreatic stellate cells, an overgrowth of fibrous tissue that often occurs near pancreatic tumors and is not associated with treatment resistance. Active pancreatic stellate cells are able to induce the production of signaling proteins, which can attract macrophages into tumors and reprogram them to become M2 macrophages that can effectively inhibit immune responses; now researchers have confirmed that high levels of IL-1β and M2 macrophages and fibroblast-driven connective tissue formation may reduce the ability of CD8 + T cells that kill cancer cells to enter tumors.

 

Finally, researcher Shipra Das said, this study provides strong evidence that blocking IL-1β activity may promote better penetration of T cells into tumors and kill cancer cells, which may hopefully overcome the dilemmas and limitations of current immunotherapy in the treatment of pancreatic cancer.

Bimekizumab Double Neutralizes IL-17A and IL-17F in Patients with Psoriasis

Psoriasis is a chronic, immune-mediated inflammatory skin disease that affects up to 3% of the population and is associated with diseases including chronic lung disease, diabetes, liver disease, cardiovascular disease, chronic kidney disease, and rheumatic diseases There is a significant correlation. The burden of disease is not just a physical manifestation, it has significant social and psychological factors that lead to a negative impact on the quality of life. Advances in basic immunology have enhanced our understanding of the pathogenesis of psoriasis, and promoted the development of targeted therapies for key proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin (IL) -17A And IL-23. Although targeting the TNF-dependent pathway has already shown positive reactions in patients with psoriasis, newly developed drugs targeting IL-23 / T-helper 17 cells have achieved greater efficacy. T-helper 17 cells are a key driver of psoriasis. Despite these advances, achieving complete removal of skin lesions and maintaining this level is still challenging; in addition, in these therapies, several safety issues have been noted, including injection site reactions, fungal infections (especially Candida) Infection) and inflammatory bowel disease (IBD) aggravation and new onset. Therefore, treatments that show more efficient and less safety issues can provide patients with additional benefits.

 

In the IL-17 cytokine family, the most extensive research has focused on IL-17A. Among the currently available treatments that target the IL-17 pathway, 2 drugs specifically inhibit IL-17A, and the third targets IL-17RA. IL-17RA is IL-17A, IL-17C, IL-25 and IL The receptor subunit used by 17F. Although IL-17A and IL-17F are the closest relatives in the IL-17 family, in history, the role of IL-17F has not been widely studied. IL-17F and IL-17A have more than 50% structural homology and overlapping biological pro-inflammatory functions, suggesting that IL-17F plays an important role in psoriasis. Evidence from preclinical studies indicates that both IL-17A and IL-17F are expressed in skin lesions and inflammatory synovium in patients with psoriatic arthritis (PsA). Compared with simply blocking IL-17A, double neutralization of IL-17A and IL-17F leads to a decrease in the expression levels of inflammation-related genes and cytokines, and an increase in the suppression of disease-related gene expression. Immune cell migration. These data support the theoretical basis for targeting IL-17A and IL-17F. To this end, bimelizumab, a humanized monoclonal IgG1 antibody, is designed to effectively and selectively neutralize the biological functions of IL-17A and IL-17F. In addition, in preclinical models, both IL-17A and IL-17F have been shown to cooperate with TNF to stimulate the production of key pro-inflammatory cytokines and amplify tissue inflammation. Compared with IL-17A blocking alone, the double neutralization of IL-17A and IL-17F leads to a reduction in the expression levels of inflammation-related genes and cytokines, and greater inhibition of disease-related immune cell migration. These data support the theoretical basis for targeting IL-17A and IL-17F. To this end, the humanized IgG1 monoclonal antibody bimekizumab was designed to effectively and selectively neutralize the biological functions of IL-17A and IL-17F.

 

method

 

This is a randomized, double-blind, placebo-controlled, parallel group, and dose range study (NCT02905006), recruiting 6 countries (Canada, Czech Republic, Hungary, Japan, Poland and the United States). All patients who have stopped or did not participate in the extended study (NCT03010527) will need to complete a 20-week safety follow-up after taking the final dose of study drug. The study was conducted in accordance with the principles of the Helsinki Declaration and the International Conference on Good Clinical Practice Coordination and Guidance. Obtained approval from the Independent Agency Review Committee.

 

The criteria for eligible patients were age ≥18 years, diagnosed with moderate-severe plaque psoriasis over 6 months, PASI score ≥12, psoriatic lesions accounted for more than 10% of body surface area, the investigator ’s global assessment (IGA ) With a score of more than 3 (5-point scale), and patients who are going to receive systemic psoriasis treatment or phototherapy. Psoriasis treatment or phototherapy. Patients who had received anti-IL-17 treatment within 6 months or had been exposed to psoriasis or other biological therapies of PsA, apparently uncontrolled neuropsychiatric disorders, history of suicide attempts, or suicidal thoughts were excluded (severe use of electronic suicide) Degree evaluation scale). For other appendices and exclusion criteria, see the supplementary appendix (available at http://www-jad.org). All patients provided written informed consent in accordance with local requirements.

 

Patients were randomly divided into 6 groups (1: 1: 1: 1: 1: 1) and received bimekizumab every 4 weeks at doses of 64 mg, 160 mg, 160 mg (baseline loading dose of 320 mg), 320 mg, 480 mg or placebo. An interactive voice or network response system is used to assign eligible patients to a treatment plan based on a randomized schedule established by an independent biostatistician who is not involved in the design or analysis of the study. Treatment allocation is stratified by geographic area and previous biological exposure.

 

Bimekizumab is provided in single-use vials containing 160 mg / mL. Due to the different presentation methods, in order to ensure that the study is blinded, bimekizumab and placebo injections are prepared and administered by non-blind, dedicated researchers at the study site (Supplementary Appendix).

 

Process

 

Treatment was performed at baseline, weeks 4 and 8, with 3 subcutaneous injections (see supplementary materials). Efficacy and safety were evaluated at baseline and weeks 1, 2, 4, 6, 8, and 12 Security is monitored by an external data monitoring committee.

 

Result

 

The primary efficacy endpoint was PASI90 at week 12. The secondary efficacy endpoints were PASI90 at week 8, PASI75 and PASI100 at week 12, and IGA at weeks 8 and 12 considered effective (defined as complete improvement or most improvement. Compared with the baseline level, there were 2 or more aspects. Improvement). Safety monitoring during the study included recording the frequency and severity of adverse events (AE) (based on the common terminology standard for adverse events), serious AEs, suicidal ideation and behavior (through the Columbia Suicide Severity Rating Electronic Scale), and depression and anxiety ( (Assessed by the Hospital Anxiety and Depression Scale). In addition, we use electrocardiogram to assess the patient’s heart function, measure vital signs, and perform physical examinations and laboratory tests.

SIMCom Agents and Partners Conference 2020 Is Held Successfully

On June 24, SIMCom held the 2020 Agents and Partners Conference in Shenzhen Successfully.

 

 

At the conference, SIMCom CEO Mr. Yang Tao thanked all the agents for their presence and shared his ideas and strategic moves for SIMCom. Mr. Yang Tao says that the success of a brand is inseparable from its products, channels and marketing. Good products are the foundation of all success. As a research-based manufacturer, SIMCom attaches importance to and invests heavily in research. Starting from 2G technology to today’s 5G technology, SIMCom has always been at the forefront of the module industry. Every year, more than 10% of its operating revenue is spent on research. Since its 5G project was launched, nearly¥1 billion has been invested in 5G product research, development, testing, certification and so on. SIMCom continuously enriches the functions of its modules, integrating such complex functions as perception, edge data analysis and processing, and transmission into its modules, hoping to provide customers with a good user experience of one-point access and seamless switching.

 

 

SIMCom has full-scale product lines; products can meet the needs of different customers. NB-IoT, 4G, CAT. 1, 5G, vehicle-mounted and smart modules all adopt multi-standard, multi-platform compatible design, so that customers can upgrade their products flexibly and meet the requirements of different scenarios with the optimal cost performance. Its 5G modules have gone deep into the industry and become mature for mass production. With the advent of the 5G era and the sound of the bugle for new capital construction, modules will usher in new opportunities for development. There will be explosive demand in such markets as mobile payment, new retail, energy meters, GPS trackers, IoV, gateways. SIMCom is ready for it.

 

 

 

Mr. Yang Tao also thanked the agents for their support over the years. Mr. Yang Tao says that since its establishment 18 years ago, SIMCom has achieved a lot of success, which is inseparable from the efforts and support of its agents. As a sales channel, agents are the ground forces of SIMCom in the war of modules. In the future, SIMCom will strengthen cooperation with agents, strive to launch products that are in line with the market demand and cost-effective, to expand the sales radius and depth with agents. Respecting the market, revering the market, holding fast to the bottom line and doing the right thing and are the fundamentals of business success. A fair, open and transparent channel mechanism is the root of SIMCom’s corporate culture.

 

Good products and good channels need equally good marketing. Mr. Yang Tao says that as an 18-year old brand, SIMCom has its own history and accumulation. In the future, the company will continue to inject new blood. This means that SIMCom will strengthen its innovation and professional service, pay more attention to product quality and supply while constantly meeting the needs of IoT customers of from all walks of life, and constantly improve its supply process to provide customers with the best products. 2020 will be a year for rebranding SIMCom. The company will strengthen brand exposure and promotion globally, so that more young companies recognize the SIMCom brand. Mr. Yang Tao also hopes to work with agents to renew the SIMCom brand.

 

At the end of the conference, Mr. Yang Tao commended agents with outstanding sales achievements in 2019, and presented certificates of honor and trophies on the spot.

Optogenetics Services Now Available at Profacgen

As a world leading protein service provider, Profacgen offers a diverse range of analytical reagents as well as services to assure efficient and economical characterization of its customers’ proteins. Recently, Profacgen released a set of optogenetics services, providing its customers the precise analysis and regulation of neural circuits and get insight into the treatment of neuropsychiatric diseases.

 

Optogenetics is a multidisciplinary technology integrating optics, software control, gene operation technology, electrophysiology, etc. Its main principle is to first use gene operation technology to transfer light perception genes (such as ChR2, eBR, NaHR3.0, Arch or OptoXR, etc.) into specific types of cells in the nervous system for the expression of special ion channels or GPCRs. Photosensitive ion channels will have selectivity for the passage of cations or anions, such as Cl-, Na+, H+, K+, respectively, under light stimulation at different wavelengths, resulting in changes in the membrane potential on both sides of the cell membrane and achieving the purpose of selective excitation or inhibition of cells. In 2010, optogenetics was selected by Nature Methods as one of the “most concerned scientific and technological achievement technologies.” This technology can be applied to the treatment of a variety of neurological disorders, such as epilepsy, Parkinson’s disease, chronic pain, depression, and addiction.

 

At Profacgen, two types of photosensitive proteins can be provided: activated and inhibited, which can cause neurons to excite or inhibit.

 

“We have a team of highly skilled, aggressive employees with a highly specialized scientific background, and we focus on innovative technologies to provide customers with a range of optogenetic services. With our in-house optogenetic imaging system, we can provide customers with precise analysis and modulation of neural circuits and insight into treatments for neuropsychiatric disorders.”

 

More detailed information can be viewed here: https://www.profacgen.com/services/optogenetics-services.

 

About Profacgen

Profacgen provides process development and cGMP manufacturing services for biological active ingredients. We have ready access to state-of-the-art biopharmaceutical product development and manufacturing facilities and our staff scientists have demonstrable expertise in all aspects of biopharmaceutical manufacturing, from facility design and operation to the ability to provide best advice relating to product development.

The Workflow of PROTAC System (Part One)

In order to use the PROTAC system, it is necessary to develop a PROTAC that can target the target protein from scratch or modify the latter with tags (as described above) to degrade the molecules by HaloPROTAC or dTAG. Indeed, before developing PROTAC against endogenous proteins, it may be advantageous to conduct preliminary studies with labeled target proteins to establish a proof of concept. In view of the latest advances in genome editing, it is relatively easy to label target proteins at endogenous sites to enable ligand-induced degradation without the need for target overexpression or the development of new ligands (Brand and Winter, 2019).

Target selection

Some ideal PROTAC targets are (1) proteins without enzymatic function that cannot be regulated by traditional small molecules, (2) proteins with other functions besides enzyme activity (such as FAK or BCR-ABL as a scaffold), or (3) After the protein is inhibited, it will be compensated up-regulated, so it is difficult to achieve the maximum loss of protein function. It may be useful to look at previous knockout experiments (for example, phenotypic genome-wide CRISPR screening or RNAi) or to understand cell type-specific dependence during target selection to gather information about the target protein (Tsherniak et al., 2017).

For some proteins, the inhibitory effect is sufficient to eliminate all functions and there is almost no increase in degradation from a biological point of view, but these proteins may be few and far apart. In these cases, PROTAC may still be beneficial in terms of reducing dose and duration of action. What’s more exciting is that PROTAC can extend the drug-curable proteome to any ligandable protein. Of the approximately 17,470 observed proteins (Omenn et al., 2018), only 10% to 15% are considered pharmacologically (Hopkins and Groom, 2002), and many more proteins have been shown to have ligands features (Backus et al., 2016, Parker et al., 2017), but many of these small molecule binding events have no effect. The ability to impart biologically inert ligand activity through PROTAC transformation greatly expands the medicinal proteome.

PROTAC development

Subsequently, a comprehensive literature study should be conducted on the known target ligands. If a ligand is available, checking the co-crystal structure or the known structure-activity relationship may reveal a suitable location for the linker to connect. At this point, the PROTAC transformation can be started, using synthetic chemistry and medicinal chemistry methods to attach linkers and E3 recruitment elements to the targeting ligand. If no ligand has been reported before, or if the reported compound has a suspicious structure, then a ligand identification activity for the target protein can be initiated; or, it may be simpler to use HaloPROTAC or dTAG initially. We recommend using multiple connector lengths and compositions when developing PROTAC, especially if the synthesis method allows modularity. The length and composition of the joint can have a profound effect. For example, HaloPROTAC with the ethylene glycol unit removed cannot induce degradation (Buckley et al., 2015), and BCR-ABL PROTACs with ether instead of amide have higher cell permeability (Burslem et al., 2019), based on The PROTACs of lapatinib can be either dual EGFR/Her2 degraders or selective EGFR degraders. In terms of linker length (Burslem et al., 2018b).

When PROTAC candidate molecules or cell lines expressing marker proteins are available, it is important to first confirm that they actually induce target degradation. Although mass spectrometry or flow cytometry can be used, it is usually achieved by immunoblotting, depending on the characteristics of the target protein (Buckley et al., 2015). Cyclic screening may be necessary to synthesize a sufficiently effective PROTAC, or it may be necessary to explore several labeling methods and/or to incorporate the label into the target for maximum degradation. We usually recommend 24 hours for the initial treatment time of PROTAC, but kinetic characterization usually indicates that degradation occurs faster. Recently, customized techniques for studying PROTAC in cells have been developed, and these techniques may prove to help guide its development (Riching et al., 2018). The synthesis and analysis of candidates is usually the rate-limiting step using PROTAC and depends on the quality of the chemicals available for the target. Starting with well-defined small molecules provides clear advantages, and as more advantages can be obtained through the Target 2035 program (Carter et al., 2019), the development speed and simplicity of PROTAC will undoubtedly increase.

PROTAC verification

When PROTAC is identified, it is critical to carefully characterize the degradation event through controlled experiments (for example, confirming by qPCR that protein loss occurs at post-translational levels rather than a decrease in mRNA) (Bondeson et al., 2015). Some small molecules induce significant degradation of their target proteins, but actually work at the transcription level (Field et al., 2017). According to the toxicity of the compound, it is usually necessary to use the shortest incubation time required for significant

Importantly, degradation by application of small molecules enables the use of inactive analogs as control compounds. When using PROTAC derived from inhibitors, this is essential for discovering new biology. Generally, the E3 ligase ligand structure can be discretely modified to disrupt its activity to produce inactive PROTAC. The resulting molecule cannot induce degradation, but is as effective as PROTAC in inhibiting the target. We are in favor of using VHL recruitment ligands to carefully study the relationship between inhibition and degradation, because a simple inversion of the stereocenter on the VHL ligand will produce compounds with the same inhibitory activity and pharmacological properties (such as cell permeability), thereby enabling it to As a true control (Burslem et al., 2018b). The inversion of the stereocenter can also be used to generate inactive compounds as a control for PROTAC recruited by MDM2 (Hines et al., 2019). It is possible to impair the IMiD ligand recruited by CRBN by omitting the carbonyl group or by methylation of the imide functional group. However, this does fine-tune its physical and chemical properties (Burslem et al., 2018a, Huang et al., 2018). Care must be taken when using IMiD analogs, as they may still induce the degradation of new zinc finger substrates related to the pathophysiology of IMiD (Ishoey et al., 2018).

Inactivated PROTAC is an important experimental control. It can confirm or deny the degradation mechanism of PROTAC, because the ligand alone can make the target unstable without recruiting E3 ligase (Huang et al., 2017), such as hydrophobic tag or SERD (Gustafson Et al., 2015; Neklesa et al., 2011; Wardell et al., 2011).

Another optional experiment performed at this time is proteomics to characterize the effects induced by PROTAC treatment. For example, mass spectrometry or reversed-phase protein arrays allow users to obtain PROTAC selectivity data. Quantitative proteomics is a powerful tool that can identify proteins other than target proteins that are down-regulated after PROTAC treatment. Of course, this may be the biological result of the loss of the target protein, such as the loss of c-Myc when BRD4 is degraded (Lu et al., 2015; Winter et al., 2015), or the pharmacological result, such as the accidental degradation of foretinib-based PROTAC P38α (Bondeson et al., 2018; Smith et al., 2019).

To be continued in Part Two…

Targeted Proteolytic Chimera (PROTAC) As A Therapeutic Tool (Part One)

The ubiquitin-proteasome system

The ubiquitin-proteasome system (UPS) is the main intracellular mechanism that destroys damaged or no longer needed proteins (Amm et al., 2014). This has been extensively reviewed elsewhere (Kleiger and Mayor, 2014); however, a brief description of PROTAC’s UPS is provided here. The 76-residue protein ubiquitin is linked to the protein as a post-translational modification (PTM) through a cascade of three enzymes through a lysine isopeptide bond: E1 activase, E2 conjugase and E3 ligase. Free ubiquitin is activated by E1 in an ATP-dependent process, during which it is converted into a C-terminal thioester. Trans-thioesterification transfers ubiquitin from El to E2. Finally, the E3 complex promotes the direct or indirect transfer of ubiquitin to the substrate protein lysine.

In turn, ubiquitin itself can be ubiquitinated on one or more of its seven surface lysine residues. These PTMs have a variety of biological functions and are still under study (Komander and Rape, 2012; Yau and Rape, 2016), but the typical role of K48 polyubiquitin is to send out a signal to destroy proteins through the proteasome (Grice and Nathan , 2016)). With the development of protein ubiquitination enzymes from E1 (only 2 to E3) and more than 600 members of the E3 family (Ardley and Robinson, 2005), more and more of them are present in the human genome. ). The E3 ligase is used to recruit the substrate and facilitate the transfer of ubiquitin from the E2 conjugate enzyme to the target protein. When a protein is multi-K48 ubiquitinated and recognized by the proteasome, the ubiquitin chain is removed and recycled by the proteasome-related deubiquitinating enzyme (DUB), while the protein substrate is folded and degraded.

Hijacking the UPS system

PROTAC technology allows UPS systems to be chemically selected and is designed to degrade specific target proteins. This method uses the E3 ligase ligand to fuse with the target protein’s targeting element through a flexible chemical linker, causing heterotopic ubiquitination, leading to protein degradation. Beginning with proof-of-concept experiments in cell lysates with peptide ligands (Sakamoto et al., 2001), the technology has matured and has been routinely used in cultured cells and in vivo and even entered clinical trials. Importantly, the technology now consists of fully synthetic modular compounds (Bondeson et al., 2015; Winter et al., 2015) that work in a wide range of protein categories and function in different subcellular locations, including the cytoplasm, Nucleus and plasma membrane (Burslem et al., 2018b). As we better understand this new technology, it is clear that the protein-protein contact between the new substrate and the E3 complex is a key determinant of the success of PROTAC (Bondeson et al., 2018; Gadd Et al. (2017), Nowak et al., 2018, Smith et al., 2019, Zorba et al., 2018). Farnaby et al. (2019) and Roy et al. (2019) played an important role in the cooperative structure and biophysical characterization of this field.

Only a few E3 ligases are used in PROTAC technology, and most of the reported compounds use cereblon (CRBN) or Von Hippel-Lindau (VHL), because they can recruit small molecules similar to drugs (Buckley et al., 2012; Winter et al., 2015). Cell inhibitors of apoptosis (cIAP) ligands have also been used, but they usually cause the self-ubiquitination and degradation of E3 ligase itself, making it less attractive (Sekine et al., 2008). The use of nutlin compounds to recruit MDM2 played a key role in the development of the first all-small molecule PROTAC (Schneekloth et al., 2008), and has recently reappeared as an effective method to target protein degradation (Hines et al., 2019). Assuming that there are more than 600 members of the E3 superfamily, it is exciting to see the emergence of new E3 ligases and their applications in targeted protein degradation (Ottis et al., 2017; Spradlin et al., 2019). In fact, due to their limitations, some of these new ligands are only degraded in specific cell compartments (Zhang et al., 2019b). In view of the importance of E3/target protein-protein interaction in PROTAC-mediated degradation, more E3 available for recruitment provides greater opportunities for successful development of PROTAC.

The application of PROTAC

PROTAC technology has applications in biological discovery and drug discovery. In many ways, PROTAC represents the chemical equivalent of small interfering RNA (siRNA), although it allows the removal of proteins at the post-translational level, rather than silence at the post-transcriptional level. Therefore, they are useful tools for studying the role of proteins in laboratory biological systems. In addition, the small molecule nature of PROTAC avoids problems related to delivery and biodistribution, which hinder the clinical application of siRNA, which has aroused great interest in the pharmaceutical industry.

  1. PROTAC in drug discovery

Not surprisingly, given its small molecule nature, PROTAC technology is entering the clinic with many indications. The initial research focused on the degradation of hormone receptors (Flanagan and Neklesa, 2019), especially the androgen receptor (Neklesa et al., 2019) and estrogen receptor (Flanagan et al., 2019). It is worth noting that these trials are for oral bioavailable PROTAC, emphasizing that PROTAC is relative to therapeutic RNAi (Setten et al., 2019) or traditional selective estrogen downregulators (SERDs) (Patel and Bihani, 2018) benefit.

  1. Androgen receptor

Androgen receptor (AR) antagonists, such as enzalutamide (Rathkopf and Scher, 2013), have been used for treatment and have great benefits for prostate cancer patients. However, there is often resistance. It has been shown that AR PROTAC outperforms enzalutamide many times, especially in the context of resistance to elevated androgen levels or mutations in AR that convert antagonists to agonists (Han et al., 2019; Salami et al., 2018). AR PROTAC ARV-110 is currently in clinical trials for metastatic castration-resistant prostate cancer.

To be continued in Part Two…

Magical Peptides and Their Functions (I)

Peptides are compounds in which two or more amino acids are connected by peptide bonds. They play an important physiological role in the organism. It plays a key role in the growth and development, metabolism, disease, aging, and death of organisms.

Functional peptides are the most important active substances in the body. It is precisely because of the increase or decrease of its secretion in the body that biological species have a cycle of infancy, childhood, adulthood, old age and death.

Active peptides are called functional peptides, also called biologically active peptides. The molecular structure of functional peptides varies in complexity, from simple dipeptides to circular macromolecular polypeptides, and these polypeptides can be modified by phosphorylation, glycosylation or acylation. The peptide chains of functional peptides have special shapes, or it is composed of one or several peptide chains and active ingredients.

There are currently more than 1,000 functional peptides (such as soybean peptide, deep-sea fish skin peptide, egg protein peptide, milk protein peptide, etc.).

Peptides that do not have physiological functions to organisms are called small peptides.

What effect does the reduction of functional peptides have on the organism?

Functional peptides mainly control the growth, development, immune regulation and metabolism of organisms. It is in a balanced state in the organism. If the functional peptides are reduced, the function of the organism will undergo important changes. For children, lack of functional peptides, their growth and development becomes slow, or even stops. For adult or elderly organisms, lack of functional peptides, their own immunity will decline, metabolic disorders, endocrine disorders, and cause various diseases, such as insomnia, body weight loss or swelling. Since functional peptides also act on the nervous system, the organism will become slow and the mind will no longer be intelligent. The more important thing is the reduction of functional peptides, which directly causes the gradual aging of various parts of the biological body and causes various diseases.

Manufacturing method of functional peptide

There are two main methods for extracting peptides, one is chemical extraction and the other is physical extraction. Both have their own advantages and disadvantages. The “chemical extraction method” is mainly the “chemical enzyme reaction extraction method.” The reaction is fast and the production volume is large, but the disadvantage is that the activity of the extracted peptides is low. This method is the main source of peptide products on the market; “physical extraction method” mainly refers to the “dielectric capacitance method functional peptide extraction technology”. It is characterized by slow reaction and small production volume, but the peptides produced have high activity.

Secretory cycle

At different ages, the secretion of various functional peptides is also very different. According to the secretion, the life of organisms can generally be divided into:

  1. Adequate secretion period (adolescence before the age of 25). During this period, the endocrine volume is balanced, the immune function is strong, and the organism is generally not prone to disease;
  2. Insufficient secretion period (imbalance period) (30-50 years old and middle-aged) If the functional peptide secretion is insufficient or imbalanced, various related sub-health states and mild disease symptoms will appear (common in biological groups over 40 years old) );
  3. The secretory deficiency period (severe insufficiency period) (middle-aged and elderly over 50 years old). This period is severe. If the functional peptide is severely insufficient and severely imbalanced, very prominent symptoms of aging may occur, or various related diseases may occur;
  4. The secretion termination period (senescence period), this period is very short, because the “commander” functional peptide that controls the secretion of the organism is not secreted or secreted, it leads to the decline of cell function, leads to organ failure and loss, and finally leads to the end of life.

Physiological function

At present, it has become a research hotspot in the world. A large number of research results indicate that biological functional peptides are biologically active substances involved in a variety of cell functions in organisms. Hundreds of species have been found in organisms. Different biological peptides have different structural and physiological functions, such as anti-virus, anti-cancer, anti-thrombosis, anti-hypertension, immune regulation, hormone regulation, antibacterial, and cholesterol-lowering effects.

The physiological functions of functional peptides are as follows:

1. Regulate the body’s water and electrolyte balance;

2. Manufacture antibodies against bacteria and infections for the immune system to improve immune function;

3. Promote wound healing; quickly repair epithelial tissue damage.

4. Produce enzymes in the body, which help convert food into energy;

5. Repair cells, improve cell metabolism, prevent cell degeneration, and play a role in preventing cancer;

6. Promote the synthesis and regulation of proteins and enzymes;

7. An important chemical messenger that communicates information between cells and organs;

8. Prevent cardiovascular and cerebrovascular diseases;

9. Regulate the endocrine and nervous system;

10. Improve the digestive system and treat chronic gastrointestinal diseases;

11. Improve diabetes, rheumatism, rheumatoid and other diseases;

12. Anti-viral infection, anti-aging, eliminate excess free radicals in the body;

13. Promote hematopoietic function, treat anemia, prevent platelet aggregation, and improve the oxygen carrying capacity of red blood cells.

14. Directly fight against DNA viruses and have targeting properties to viruses and bacteria.

Through the research of functional peptides, the application of peptides in biology has been promoted. Nutritionists and biomedical scientists have continuously developed various peptide products to meet the needs of biological health.

To be continued in Part II…

Be Alert! Ketogenic Diets May Cause Temporary Flu-like Symptoms!

According to a new study, the first weeks of a ketogenic diet may have a lot of flu-like symptoms, including nausea, fatigue and dizziness.

 

The study was recently published in the journal Frontiers in Nutrition. The researchers extracted data from the experiences of 101 people who started on a ketogenic diet and described their symptoms in 43 forums. As with anecdotal reports of ketogenic influenza—the symptoms of influenza that began with such a high-fat, medium-protein, low-carbohydrate diet gradually worsened in the first seven days of starting a new ketogenic diet, but gradually decreased over time and eventually disappeared spontaneously after about 4 weeks.

 

Dr. Emmanuelle Bostock, one of the authors of the study and a researcher at the Menzies Institute for Medical Research at the University of Tasmania in Australia, said: “We sought to describe the nature of ketogenic influenza because the incidence in media reports is inconsistent with that in the academic literature. We found that some users of online forums self-reported flu-like symptoms, which usually occurred in the first few weeks of starting a diet.”

 

In the 1920s, ketogenic foods first emerged as drugs for the treatment of epilepsy. But recently, the ketogenic diet has become a health boom because it is thought to lose weight by forcing the body into a state called ketogenesis. When the human body is in this state, cells take energy from compounds called ketones, which are extracted from fatty acids. Experts are still unsure why it leads to weight loss, and studies have generally failed to demonstrate that a ketogenic diet is more effective than other weight loss strategies. The ketogenic diet seems to have an unexpected difference from other diets, that is, it predisposes to flu-like symptoms, at least in the first few weeks of starting this diet.

 

“I did see some clients getting ketogenic flu,” said nutritionist Ginger Hultin, a spokesman for the Nutrition and Dietetics Society. Hultin was not involved in the study. “Some people have few symptoms, some have these symptoms, and some have more severe symptoms.”

 

To quantify the phenomenon, Bostock and her colleagues identified consistent complaints from 43 online forums. The researchers found that within the first few weeks of starting the ketogenic diet, people reported sudden onset of headache, stomach pain, nausea, and other flu-like symptoms.

 

The researchers say it is not clear why a ketogenic diet produces this effect, but they suspect that this may be related to changes in bacteria and other microbes in the gut when people start consuming a ketogenic diet.

 

For example, the ketogenic diet excludes many foods that support gut bacteria, such as starchy vegetables and whole grains. Given that many studies have found a link between the brain and the gut, changes in gut bacteria may contribute to some of the symptoms of ketogenic influenza.

 

More importantly, many people start eating ketones in order to reduce carbohydrate intake, which requires major changes in the food they eat. Carbohydrate intake can cause symptoms similar to caffeine withdrawal.

 

However, an important limitation of this new study is that the data are entirely derived from online conversations, and there is no evidence that people who describe symptoms are consuming a ketogenic diet, let alone experiencing ketosis. “More clinical studies need to be done because this is only a review of online forums,” Hultin said. But Bostock hopes that this pilot study will lay the groundwork for future exploration of the side effects of the ketogenic diet in carefully designed clinical trials.

 

Author

As a biotechnology company, Lifeasible is specialized in agricultural science, offering a wide variety of agro-related services and products for environmental and energy solutions.

A New Methylation Site on P53 Found

Structure published online the research results of Chen Yong’s research group at the Center for Excellence in Molecular Cell Science (Institute of Biochemistry and Cell Biology) of the Chinese Academy of Sciences “Crystal Structure of MLL2 Complex Guides the Identification of a Methylation Site on P53 Catalyzed by KMT2 Family Methyltransferases”.

 

This work revealed that MLL family proteins can catalyze the methylation of P53 through structural biology studies of the MLL2-RBBP5-ASH2L methyltransferase complex, and identified a new lysine methylation site on P53.

 

MLL family proteins (MLL1/2/3/4, SET1A/B) play an important role in gene expression regulation, cell proliferation, differentiation and other life activities, and are closely related to the occurrence and development of leukemia and other diseases.

 

Previous studies have focused on the activity of catalyzing the methylation of lysine (H3K4) at position 4 of histone H3, and it is believed that diseases caused by abnormal function of MLL family proteins are closely related to abnormal H3K4 methylation levels. .

 

In recent years, it has been discovered that many histone lysine methyltransferases have non-histone substrates.

 

For example, SET1A, one of the members of the MLL family of proteins, was shown in 2018 to catalyze the methylation of YAP protein K342, a key effector molecule of the Hippo pathway, thereby increasing the residence time of YAP in the nucleus and promoting YAP transcription and tumorigenesis.

 

Are there other non-histone substrates for MLL family proteins? Is the life activity process they participate in is far more complicated than we know?

 

Chen Yong’s research group has long been committed to the structure and function research of MLL family protein complexes.

 

In this work, the researchers solved the crystal structure of the MLL2-RBBP5-ASH2L core complex, in which an asymmetric unit is composed of two M2RA complexes due to crystal packing, and one of the MLL2 SET domain N-terminal loop (NFL) can be inserted into the catalytic pocket of the other MLL2.

 

Interestingly, the conformation of this NFL is very consistent with the conformation of H3 in the M3RA-H3 complex, but the sequence is very different, indicating that MLL family proteins have the ability to recognize and bind to sequences other than H3.

 

According to the sequence characteristics of MLL2 NFL, the researchers used the ScanProsite database and found that there is a sequence in P53 that is very similar to MLL2 NFL.

 

Subsequently, through a series of biochemical experiments combined with mass spectrometry technology, they revealed that the MLL family protein complex can catalyze the methylation of P53 K305 in vitro. This is a brand new P53 methylation site.

 

The methylation sites of P53 reported in the past are mostly concentrated at the C-terminus of P53, and the methylation sites found in this article are located between the tetramerization domain of P53 and the DNA binding domain. It may affect the transcriptional activity of P53, and the specific function needs to be further explored later.

 

Lysine methylation modification of P53 is a new P53 post-translational modification discovered in recent years. Abnormal P53 methylation modification often affects the transcriptional activity of P53 and causes P53-related diseases.

 

The discovery of this site and the identification of the key enzyme responsible for the methylation modification of this site provide new ideas and research perspectives for people to further explore the physiological significance of P53 methylation and to understand the functional regulation of P53 in cells.

 

At the same time, the research ideas of this research content are also worthy of further promotion in other research directions, that is, the use of known structural biological information to predict unknown biological phenomena, and continuously enrich our relevant understanding of the dynamic regulation process of proteins in cells.