The orphan insulin receptor-related receptor (IRR), in contrast to its homologs from the insulin receptor family, is activated by a mildly alkaline extracellular medium. We have previously demonstrated that IRR activation is defined by two synergistic sites located in the dimeric extracellular domain. Here, we describe artificial mutations in the IRR transmembrane domain that promote receptor activation. First, using molecular modeling based on the NMR-derived structure, we proposed amino acid substitutions that could enhance non-covalent interactions between the transmembrane segments of the IRR dimer. These mutations were subsequently tested for effects on pH sensing by IRR. We showed that double-mutant A938E-A939R was highly phosphorylated at neutral pH and still sensitive to alkaline pH. Remarkably, the double substitution of V929E-G930R resulted in strong basal phosphorylation of the receptor over the pH titration range. Through site-directed mutagenesis, we demonstrated that the transmembrane domain plays a critical role in IRR activation, allowing for targeted control of functioning of the receptor, including its pH sensitivity.

Homogeneous antibody-drug conjugates (ADCs) exhibit significantly improved pharmacological properties compared to their heterogeneous counterparts. Site-specific conjugation of the payload to the IgG required for homogeneity can be achieved using enzymes. One example is microbial transglutaminase (MTGase), which can selectively perform transamidation on the Q295 residue of human Fc when N297 glycans are removed. As a result, two modifications can be introduced per IgG molecule; however, achieving higher drug-to-antibody ratios (DARs) requires the use of branched linkers. While several such linkers have been reported, little information is available on the relationship between linker structure and ADC properties. To address this gap, we synthesized two branched amino triazide linkers, differing by a PEG4 fragment inserted after the branching point, which were used to prepare two homogeneous trastuzumab-based DAR 6 ADCs (a "short" and a "long" one). This was achieved by a two-step process consisting of enzymatic linker conjugation followed by bioorthogonal coupling with a cleavable linker bearing monomethyl auristatin E (MMAE). Two other trastuzumab-MMAE conjugates were used as controls: a heterogeneous DAR 6 ADC, made using conventional thiol-maleimide chemistry, and a homogeneous DAR 2 ADC. We found that, while the four conjugates had identical affinity for HER2, their cytotoxicity differed significantly: the "long" homogeneous DAR 6 ADC was just as active as its heterogeneous counterpart, but the "short" DAR 6 ADC was an order of magnitude less potent, inferior even to the DAR 2 conjugate. Our findings indicate that the length of the branched linker critically affects the cytotoxic activity of ADCs, possibly due to steric hindrance influencing the rate of linker cleavage by lysosomal enzymes.

This review discusses the importance of homeostasis with a particular emphasis on the acid-base (AB) balance, a crucial aspect of pH regulation in living systems. Two primary organ systems correct deviations from the standard pH balance: the respiratory system via gas exchange and the kidneys via proton/bicarbonate secretion and reabsorption. Focusing on kidney functions, we describe the complexity of renal architecture and its challenges for experimental research. We address specific roles of different nephron segments (the proximal convoluted tubule, the loop of Henle and the distal convoluted tubule) in pH homeostasis, while explaining the physiological significance of ion exchange processes maintained by the kidneys, particularly the role of bicarbonate ions (HCO3−) as an essential buffer system of the body. The review will be of interest to researchers in the fields of physiology, biochemistry and molecular biology, which builds a strong foundation and critically evaluates existing studies. Our review helps identify the gaps of knowledge by thoroughly understanding the existing literature related to kidney acid-base homeostasis.

Serine β-lactamase TEM-1 is the first β-lactamase discovered and is still common in Gram-negative pathogens resistant to β-lactam antibiotics. It hydrolyzes penicillins and cephalosporins of early generations. Some of the emerging TEM-1 variants with one or several amino acid substitutions have even broader substrate specificity and resistance to known covalent inhibitors. Key amino acid substitutions affect catalytic properties of the enzyme, and secondary mutations accompany them. The occurrence of the secondary mutation M182T, called a “global suppressor”, has almost doubled over the last decade. Therefore, we performed saturating mutagenesis at position 182 of TEM-1 to determine the influence of this single amino acid substitution on the catalytic properties, thermal stability, and ability for thermoreactivation. Steady-state parameters for penicillin, cephalothin, and ceftazidime are similar for all TEM-1 M182X variants, whereas melting temperature and ability to reactivate after incubation at a higher temperature vary significantly. The effects are multidirectional and depend on the particular amino acid at position 182. The M182E variant of β-lactamase TEM-1 demonstrates the highest residual enzymatic activity, which is 1.5 times higher than for the wild-type enzyme. The 3D structure of the side chain of residue 182 is of particular importance as observed from the comparison of the M182I and M182L variants of TEM-1. Both of these amino acid residues have hydrophobic side chains of similar size, but their residual activity differs by three-fold. Molecular dynamic simulations add a mechanistic explanation for this phenomenon. The important structural element is the V159-R65-E177 triad that exists due to both electrostatic and hydrophobic interactions. Amino acid substitutions that disturb this triad lead to a decrease in the ability of the β-lactamase to be reactivated.

The pancreas plays a key role in the endocrine system of animals, as well as in the digestion and absorption of nutrients. The exocrine and endocrine portions of the pancreas are structurally separated from each other, however, multiple studies suggest the anatomical and functional unity between them. While previously, the interactions of these two portions received less attention, nowadays the pancreas is considered as a single organ consisting of functionally interlinked components, which coordinates endocrine and exocrine responses. The review addresses the latest data indicating the functional relationship and reciprocal influence of the endocrine and exocrine pancreatic portions. In addition, we consider the impact of SARS-CoV-2 infection on pancreatic function.

The pancreas plays a key role in the endocrine system of animals and in the digestion and absorption of nutrients. The exocrine and endocrine pancreas are structurally separate from each other, but numerous studies suggest anatomical and functional connections between these parts. Previously, less attention was paid to these interactions, but the pancreas is now viewed as a single organ consisting of functionally related components that coordinates endocrine and exocrine responses. Our review examines the latest data indicating the functional connection and mutual influence of the endocrine and exocrine parts of the pancreas. In addition, we will also look at the impact of SARS-CoV-2 infection on pancreatic function.

The maintenance of plasma pH is critical for life in all organisms. The kidney plays a critical role in acid–base regulation in vertebrates by controlling the plasma concentration of bicarbonate. The receptor tyrosine kinase IRR (insulin receptor-related receptor) is expressed in renal β-intercalated cells and is involved in alkali sensing due to its ability to autophosphorylate under alkalization of extracellular medium (pH > 7.9). In mice with a knockout of the insrr gene, which encodes for IRR, urinary bicarbonate secretion in response to alkali loading is impaired. The specific regulatory mechanisms in the kidney that are under the control of IRR remain unknown. To address this issue, we analyzed and compared the kidney transcriptomes of wild-type and insrr knockout mice under basal or bicarbonate-loaded conditions. Transcriptomic analyses revealed a differential regulation of a number of genes in the kidney. Using TaqMan real-time PCR, we confirmed different expressions of the slc26a4, rps7, slc5a2, aqp6, plcd1, gapdh, rny3, kcnk5, slc6a6 and atp6v1g3 genes in IRR knockout mice. Also, we found that the expression of the kcnk5 gene is increased in wild-type mice after bicarbonate loading but not in knockout mice. Gene set enrichment analysis between the IRR knockout and wild-type samples identified that insrr knockout causes alterations in expression of genes related mostly to the ATP metabolic and electron transport chain processes.

Human InsR, IGF1R, and IRR receptor tyrosine kinases (RTK) of the insulin receptor subfamily play an important role in signaling pathways for a wide range of physiological processes and are directly associated with many pathologies, including neurodegenerative diseases. The disulfide-linked dimeric structure of these receptors is unique among RTKs. Sharing high sequence and structure homology, the receptors differ dramatically in their localization, expression, and functions. In this work, using high-resolution NMR spectroscopy supported by atomistic computer modeling, conformational variability of the transmembrane domains and their interactions with surrounding lipids were found to differ significantly between representatives of the subfamily. Therefore, we suggest that the heterogeneous and highly dynamic membrane environment should be taken into account in the observed diversity of the structural/dynamic organization and mechanisms of activation of InsR, IGF1R, and IRR receptors. This membrane-mediated control of receptor signaling offers an attractive prospect for the development of new targeted therapies for diseases associated with dysfunction of insulin subfamily receptors.

At the moment, a lot of research is being conducted to study the mechanisms of activation of the insulin receptor family, but the exact mechanisms of conformational changes in receptors during activation are still being investigated. It is assumed that in the inactive state, the TM domains of the insulin receptor are in a conformation that prevents the interaction of kinase domains. Binding to the ligand changes the conformation of the receptor, as a result, intracellular tyrosine kinase domains converge and cause a cellular response.

TrkA and TrkB knockout HT-22 cell lines were used to prove that the neuroprotective properties of GK-2 [ bis ( N -monosuccinyl- L -glutamyl- L -lysine hexamethylenediamide], a low-molecular-weight dipeptide mimetic of the NGF 4 th loop, are definitely mediated by selective interaction with TrkA receptor upon oxidative stress-induced cellular damage in vitro .

The orphan insulin receptor-related receptor (IRR) encoded by insrr gene is the third member of the insulin receptor family, also including the insulin receptor (IR) and the insulin-like growth factor receptor (IGF-1R). IRR is the extracellular alkaline medium sensor. In mice, insrr is expressed only in small populations of cells in specific tissues, which contain extracorporeal liquids of extreme pH. In particular, IRR regulates the metabolic bicarbonate excess in the kidney. In contrast, the role of IRR during Xenopus laevis embryogenesis is unknown, although insrr is highly expressed in frog embryos. Here, we examined the insrr function during the Xenopus laevis early development by the morpholino-induced knockdown. We demonstrated that insrr downregulation leads to development retardation, which can be restored by the incubation of embryos in an alkaline medium. Using bulk RNA-seq of embryos at the middle neurula stage, we showed that insrr downregulation elicited a general shift of expression towards genes specifically expressed before and at the onset of gastrulation. At the same time, alkali treatment partially restored the expression of the neurula-specific genes. Thus, our results demonstrate the critical role of insrr in the regulation of the early development rate in Xenopus laevis.

The most important property of a living organism is the maintenance of optimal acid–base balance and the ionic composition of the internal environment. The kidneys are one of the main pH-regulating organs in the body. Receptor tyrosine kinase IRR (an insulin receptor-related receptor) is an alkaline pH-sensor. In mice (Mus Musculus) with a knockout of the insrr gene encoding the IRR receptor, bicarbonate secretion is impaired under the conditions of alkaline loading, which indicates the role of the receptor tyrosine kinase IRR in the regulation of acid–base balance in the body. In order to search for proteins functionally associated with the receptor tyrosine kinase IRR, we performed a large-scale sequencing of the mouse kidney transcriptome of wild type and insrr knockout mice kept under normal conditions and under alkaline conditions. As a result, we found a decrease in the gapdh gene expression in the kidneys of insrr knockout mice compared to wild type mice. RNA sequencing data were confirmed by TaqMan real-time PCR and Western blotting. Using the TaqMan real-time PCR method, we revealed a decrease in the level of gapdh expression not only in the kidneys, but also in the liver and brain of insrr knockout mice. Thus, the changes in the gapdh gene expression in the kidneys of insrr knockout mice may indicate a functional relationship between genes and a possible role of GAPDH in previously undescribed molecular mechanisms of regulation of acid–base balance in the body.

— Neurexins are a family of synaptic adhesion proteins that play a key role in synapse formation and maintenance. Neurexins undergo extensive alternative splicing at six sites (SS1–SS6) resulting in expression of multiplicity of different isoforms. Alternative splicing regulates the functional activity of neurexins in different types of tissues and cells and presumably plays a key role in determining the specificity of the interaction of various neurons. In this study, we have investigated the pattern of tissue expression of neurexin-1α mRNA isoforms including an insert in the recently discovered splice site SS6 using TaqMan Real-Time PCR in different organs of Wistar rats. The isoform containing the insert in the SS6 site was found only in neural tissues suggesting its potential functional importance. Position of the SS6 insert in the hinge region between the LNS5 and LNS6 domains increases variability of possible conformations of the molecule which may represent an additional mechanism for regulating functional activity of the neurexin-1α in the brain.

Maintaining an optimal acid-base balance of the organism has a pivotal role in the regulation of metabolism. It is provided by the functioning of endogenous pH-sensors, the molecules with abilities to change their activity with the changes in the pH of the medium. Receptor tyrosine kinase IRR (insulin receptor-related receptor) is an alkaline pH sensor that is activated when the pH of the extracellular medium rises above 7.9. The expression of IRR is specific; the receptor is found in some organs, in certain types of cells. It has been established that the IRR receptor is involved in the excretion of bicarbonate by kidneys. The mechanism of action and function of the IRR receptor as an alkaline pH sensor in other organs is not understood yet. To reveal the role of the IRR receptor in the embryogenesis, we carried out experiments to evaluate the development of preimplantation embryos of wild-type and insrr knockout mice using the MEA (Mouse Embryo Assay) test. The development was assessed using the blastocyst yield index, the percentage of blastocyst formation from the total number of obtained zygotes. The yield of blastocysts in knockout animals was lower than in wild-type animals; it was 6.7% of the total number of extracted cells for knockout animals and 43.8% for wild-type animals. The number of obtained zygotes in case of wild-type and insrr knockout mice also differed. The average number of extracted zygotes from one female was 18.9 zygotes for wild-type mice and 11.8 zygotes for insrr knockout mice. Our results reveal a possible role for the receptor tyrosine kinase IRR in the development of preimplantation embryos.

The determination of pH in live cells and tissues is of high importance in physiology and cell biology. In this report, we outline the process of the creation of SypHerExtra, a genetically encoded fluorescent sensor that is capable of measuring extracellular media pH in a mildly alkaline range. SypHerExtra is a protein created by fusing the previously described pH sensor SypHer3s with the neurexin transmembrane domain that targets its expression to the cytoplasmic membrane. We showed that with excitation at 445 nm, the fluorescence lifetime of both SypHer3s and SypHerExtra strongly depend on pH. Using FLIM microscopy in live eukaryotic cells, we demonstrated that SypHerExtra can be successfully used to determine extracellular pH, while SypHer3s can be applied to measure intracellular pH. Thus, these two sensors are suitable for quantitative measurements using the FLIM method, to determine intracellular and extracellular pH in a range from pH 7.5 to 9.5 in different biological systems.

The orphan insulin receptor‐related receptor (IRR), in contrast to its close homologs, the insulin receptor (IR) and insulin‐like growth factor receptor (IGF‐IR) can be activated by mildly alkaline extracellular medium. Unlike ubiquitously expressed IR and IGF‐IR, IRR is found in specific sets of cells in only some tissues, most of them being exposed to extracorporal liquids of extreme pH. In the evolution, IRR is highly conserved since its divergence from the insulin and insulin‐like growth factor receptors in amphibia. IR and IGF‐IR signaling are important in growth and development in zebrafish or frog embryogenesis. In contrast, the role of IRR activation during embryogenesis is unknown although Xenopus embryo have strong IRR expression. To address this, we examined the function of the IRR during Xenopus laevis development by morpholino‐mediated selective knockdown of IRR. We demonstrated that inhibition of IRR expression in Xenopus laevis leads to delayed development, but this phenotype can be restored by incubation of embryos in alkaline medium. Also, using RNA‐seq of total RNA we showed that IRR inhibition dramatically changed the embryo transcriptome, and partially restored after alkali treatment. We have identified several hundred genes (eomes, frzb, pax6 etc.) whose expression changes after IRR knockdown and restores by alkali exposure. Our results clearly demonstrate that IRR plays previously unidentified important role in frog embryogenesis and growth.Support or Funding InformationThis work was financially supported by the Russian Foundation for Basic Research (grants No.19‐04‐00815, 17‐00‐00486)To understand which role IRR could play in the embryonic development, we downregulated it by injecting anti‐sense morpholino oligonucleotide to IRR mRNA (MO1 xIRR) into the two‐cells embryos (Fig. 1A). As a result, we observed significant retardation of development of these embryos comparing to their siblings injected by the control MO (Fig. 1B,C). As IRR in mouse is known as a receptor which activity depends on pH, we decided to verify if pH different from the neutral one could interfere with the effects observed in embryos with downregulated pH. To this end, we incubated embryos injected with IRR MO and by the control one in solutions with pH 5.5, 7.2 and 8.4.Whereas clear retardation of development was seen in the MO1 xIRR injected embryos incubated in pH 5.2 and 7.2 (Fig, 1B,C,D,E). Surprisingly, the embryos incubated at pH 8.4 developed approximately synchronously with the control ones (Fig. 1F,G). In other words, unexpectedly alkaline pH “rescues” embryos with downregulated IRR from retardation of development.We represented in table 1 top of 15 up‐regulated and 15 down‐regulated genes after MO injection in embryos at pH 7.2, which included several key transcription factors such as eomes, pou5f3.2 (or oct25), hhex and pax6. Also, we see that genes with the largest modified expression are actin or myosin coding genes.Figure 1 gene Fold change MO xIRR 7.2/control 7.2 Fold change MO xIRR 8.4/MO xIRR 7.2 mylpf.L 0,15 2,95 myosin light chain, phosphorylatable, fast skeletal muscle S homeolog(mylpf.S) act3.L 0,15 1,89 actin, alpha skeletal muscle MGC64484 0,15 1,89 Actin, alpha cardiac muscle 1 act2 0,20 2,32 actin, alpha sarcomeric/cardiac myl1.S 0,25 1,93 myosin light chain 1 S homeolog MGC53823 0,25 1,99 Actin, alpha cardiac muscle 2‐like col2a1.L 0,26 2,07 collagen, type II, alpha 1 L homeolog des.1.L 0,26 2,11 desmin, gene 1 L homeolog fbxl22.S 0,30 1,94 F‐box and leucine‐rich repeat protein 22 S homeolog tnnc2.L 0,30 1,68 troponin C type 2 (fast) L homeolog pax6.S 0,30 2,43 paired box 6 S homeolog des.1.S 0,30 1,95 desmin, gene 1 S homeolog smyd1.L 0,31 2,21 SET and MYND domain containing 1 L homeolog tnnt3.L 0,32 2,26 troponin T3, fast skeletal type L homeolog nr2f5.S 0,32 1,87 nuclear receptor subfamily 2, group F, member 5 S homeolog dctn2.L 1,92 0,58 dynactin subunit 2 L homeolog pou5f3.2.L 1,93 0,51 POU class 5 homeobox 3, gene 2 L homeolog ywhag.L 1,93 0,50 tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein, gamma L homeolog ppp2r2b.L 1,93 0,52 protein phosphatase 2 regulatory subunit B, beta L homeolog chm.L 1,97 0,56 choroideremia (Rab escort protein 1) L homeolog hhex.L 1,97 0,62 hematopoietically expressed homeobox L homeolog slc22a15.2.S 1,97 0,63 solute carrier family 22, member 15, gene 2 S homeolog gramd1c.L 1,97 0,70 GRAM domain containing 1C L homeolog ccna1.L 1,99 0,48 cyclin A1 L homeolog neu1.L 2,00 0,56 neuraminidase 1 (lysosomal sialidase) L homeolog cdk5r2.S 2,03 0,57 cyclin‐dependent kinase 5, regulatory subunit 2 (p39) S homeolog cer1.S 2,11 0,54 cerberus 1, DAN family BMP antagonist S homeolog xpo6.S 2,19 0,67 exportin 6 S homeolog eomes.S 2,42 0,36 eomesodermin S homeolog frzb.S 2,71 0,39 frizzled‐related protein S homeolog